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Zellbiologie

Dr. Stefan Hillmer

Behnke, H.-D., Hummel, E., Hillmer, S., Sauer-Gürth, H., Gonzalez, J., and Wink, M. (2013).A revision of African Velloziaceae based on leaf anatomy characters and rbcL nucleotide sequences Botanical Journal of the Linnean Society 172: 22–94.

 

Lerich A, Hillmer S, Langhans M, Scheuring D, van Bentum P, Robinson DG. (2012). ER Import Sites and Their Relationship to ER Exit Sites: A New Model for Bidirectional ER-Golgi Transport in Higher Plants. 1. Front Plant Sci. 2012;3:143.

 

Per definition, ER exit sites are COPII vesiculation events at the surface of the ER and in higher plants are only visualizable in the electron microscope through cryofixation techniques. Fluorescent COPII labeling moves with Golgi stacks and locates to the interface between the ER and the Golgi. In contrast, the domain of the ER where retrograde COPI vesicles fuse, i.e., ER import sites (ERIS), has remained unclear. To identify ERIS we have employed ER-located SNAREs and tethering factors. We screened several SNAREs (SYP81, the SYP7 family, and USE1) to find a SNARE whose overexpression did not disrupt ER-Golgi traffic and which gave rise to discrete fluorescent punctae when expressed with an XFP tag. Only the Qc-SNARE SYP72 fulfilled these criteria. When coexpressed with SYP72-YFP, both the type I-membrane protein RFP-p24δ5 and the luminal marker CFP-HDEL whose ER localization are due to an efficient COPI-mediated recycling, form nodules along the tubular ER network. SYP72-YFP colocalizes with these nodules which are not seen when RFP-p24δ5 or CFP-HDEL is expressed alone or when SYP72-YFP is coexpressed with a mutant form of RFP-p24δ5 that cannot exit the ER. SYP72-YFP does not colocalize with Golgi markers, except when the Golgi stacks are immobilized through actin depolymerization. Endogenous SYP7 SNAREs, also colocalize with immobilized COPII/Golgi. In contrast, XFP-tagged versions of plant homologs to TIP20 of the Dsl1 COPI-tethering factor complex, and the COPII-tethering factor p115 colocalize perfectly with Golgi stacks irrespective of the motile status. These data suggest that COPI vesicle fusion with the ER is restricted to periods when Golgi stacks are stationary, but that when moving both COPII and COPI vesicles are tethered and collect in the ER-Golgi interface. Thus, the Golgi stack and an associated domain of the ER thereby constitute a mobile secretory and recycling unit: a unique feature in eukaryotic cells.

 

Montesinos JC, Sturm S, Langhans M, Hillmer S, Marcote MJ, Robinson DG, Aniento F. (2012). Coupled transport of Arabidopsis p24 proteins at the ER-Golgi interface. J Exp Bot. 63(11):4243-61.
Abstract
p24 proteins are a family of type I membrane proteins localized to compartments of the early secretory pathway and to coat protein I (COPI)- and COPII-coated vesicles. They can be classified, by sequence homology, into four subfamilies, named p24α, p24β, p24γ, and p24δ. In contrast to animals and fungi, plants contain only members of the p24β and p24δ subfamilies. It has previously been shown that transiently expressed red fluorescent protein (RFP)-p24δ5 localizes to the endoplasmic reticulum (ER) as a consequence of highly efficient COPI-based recycling from the Golgi apparatus. Using specific antibodies, endogenous p24δ5 has now been localized to the ER and p24β2 to the Golgi apparatus in Arabidopsis root tip cells by immunogold electron microscopy. The relative contributions of the cytosolic tail and the luminal domains to p24δ5 trafficking have also been characterized. It is demonstrated that whereas the dilysine motif in the cytoplasmic tail determines the location of p24δ5 in the early secretory pathway, the luminal domain may contribute to its distribution downstream of the Golgi apparatus. By using knock-out mutants and co-immunoprecipitation experiments, it is shown that p24δ5 and p24β2 interact with each other. Finally, it is shown that p24δ5 and p24β2 exhibit coupled trafficking at the ER-Golgi interface. It is proposed that p24δ5 and p24β2 interact with each other at ER export sites for ER exit and coupled transport to the Golgi apparatus. Once in the Golgi, p24δ5 interacts very efficiently with the COPI machinery for retrograde transport back to the ER.
Haydon MJ, Kawachi M, Wirtz M, Hillmer S, Hell R, Krämer U. (2012). Vacuolar nicotianamine has critical and distinct roles under iron deficiency and for zinc sequestration in Arabidopsis. Plant Cell. 24(2):724-37.
Abstract
Comment in Plant Cell. 2012 Feb;24(2):373.
Pubmed 
Hillmer S, Viotti C, Robinson DG. (2012). An improved procedure for low-temperature embedding of high-pressure frozen and freeze-substituted plant tissues resulting in excellent structural preservation and contrast. J Microsc. 247(1):43-7.
Abstract
Here we describe refinements in the processing of high-pressure frozen samples of delicate plant tissues for immuno-electron microscopy. These involve: shortened freeze-substitution schedules, lower temperatures during processing and polymerisation, the avoidance of temperature fluctuations and the optimisation of heat transfer from the specimens using small disposable aluminium containers. The application of these modifications leads to very good structural preservation and selective membrane contrast. As a result, the versatility of the method is increased since not only immuno-electron microscopical studies can be performed but often the quality is also quite suitable for structural investigations.
Pubmed 
Takatsuka C, Inoue Y, Higuchi T, Hillmer S, Robinson DG, Moriyasu Y. (2011). Autophagy in tobacco BY-2 cells cultured under sucrose starvation conditions: isolation of the autolysosome and its characterization. Plant Cell Physiol. 52(12):2074-87.
Abstract
Tobacco culture cells carry out a large-scale degradation of intracellular proteins in order to survive under sucrose starvation conditions. We have previously suggested that this bulk degradation of cellular proteins is performed by autophagy, where autolysosomes formed de novo act as the major lytic compartments. The digestion process in autolysosomes can be retarded by addition of the cysteine protease inhibitor E-64c to the culture medium, resulting in the accumulation of autolysosomes. In the present study, we have investigated several properties of autolysosomes in tobacco cells. Electron microscopy showed that the autolysosomes contain osmiophilic particles, some of which resemble partially degraded mitochondria. It also revealed the presence of two kinds of autolysosome precursor structures; one resembled the isolation membrane and the other the autophagosome of mammalian cells. Immunofluorescence microscopy showed that autolysosomes contain acid phosphatase, in accordance with cytochemical enzyme analyses by light and electron microscopy in a previous study. Autolysosomes isolated by cell fractionation on Percoll gradients showed the localization of acid phosphatase, vacuolar H(+)-ATPase and cysteine protease. These results show that starvation-induced autophagy in tobacco cells follows a macroautophagic-type response similar to that described for other eukaryotes. However, our results indicate that, although the plant vacuole is often described as being equivalent to the lysosome of the animal cell, a new low pH lytic compartment-the autolysosome-also contributes to proteolytic degradation when tobacco cells are subjected to sucrose deprivation.
Scheuring D, Viotti C, Krüger F, Künzl F, Sturm S, Bubeck J, Hillmer S, Frigerio L, Robinson DG, Pimpl P, Schumacher K. (2011). Multivesicular bodies mature from the trans-Golgi network/early endosome in Arabidopsis. Plant Cell. 23(9):3463-81.
Abstract
The plant trans-Golgi network/early endosome (TGN/EE) is a major hub for secretory and endocytic trafficking with complex molecular mechanisms controlling sorting and transport of cargo. Vacuolar transport from the TGN/EE to multivesicular bodies/late endosomes (MVBs/LEs) is assumed to occur via clathrin-coated vesicles, although direct proof for their participation is missing. Here, we present evidence that post-TGN transport toward lytic vacuoles occurs independently of clathrin and that MVBs/LEs are derived from the TGN/EE through maturation. We show that the V-ATPase inhibitor concanamycin A significantly reduces the number of MVBs and causes TGN and MVB markers to colocalize in Arabidopsis thaliana roots. Ultrastructural analysis reveals the formation of MVBs from the TGN/EE and their fusion with the vacuole. The localization of the ESCRT components VPS28, VPS22, and VPS2 at the TGN/EE and MVBs/LEs indicates that the formation of intraluminal vesicles starts already at the TGN/EE. Accordingly, a dominant-negative mutant of VPS2 causes TGN and MVB markers to colocalize and blocks vacuolar transport. RNA interference-mediated knockdown of the annexin ANNAT3 also yields the same phenotype. Together, these data indicate that MVBs originate from the TGN/EE in a process that requires the action of ESCRT for the formation of intraluminal vesicles and annexins for the final step of releasing MVBs as a transport carrier to the vacuole.
Sorieul M, Langhans M, Guetzoyan L, Hillmer S, Clarkson G, Lord JM, Roberts LM, Robinson DG, Spooner RA, Frigerio L. (2011). An Exo2 derivative affects ER and Golgi morphology and vacuolar sorting in a tissue-specific manner in arabidopsis. Traffic. 12(11):1552-62.
Abstract
We screened a panel of compounds derived from Exo2 - a drug that perturbs post-Golgi compartments and trafficking in mammalian cells - for their effect on the secretory pathway in Arabidopsis root epidermal cells. While Exo2 and most related compounds had no significant effect, one Exo2 derivative, named LG8, induced severe morphological alterations in both the Golgi (at high concentrations) and the endoplasmic reticulum (ER). LG8 causes the ER to form foci of interconnecting tubules, which at the ultrastructural level appear similar to those previously reported in Arabidopsis roots after treatment with the herbicide oryzalin. In cotyledonary leaves, LG8 causes redistribution of a trans Golgi network (TGN) marker to the vacuole. LG8 affects the anterograde secretory pathway by inducing secretion of vacuolar cargo and preventing the brassinosteroid receptor BRI1 from reaching the plasma membrane. Uptake and arrival at the TGN of the endocytic marker FM4-64 is not affected. Unlike the ADP ribosylation factor-GTP exchange factor (ARF-GEF) inhibitor brefeldin A (BFA), LG8 affects these post-Golgi events without causing the formation of BFA bodies. Up to concentrations of 50 µm, the effects of LG8 are reversible.
Pubmed 
Wang H, Zhuang XH, Hillmer S, Robinson DG, Jiang LW. (2011). Vacuolar sorting receptor (VSR) proteins reach the plasma membrane in germinating pollen tubes. Mol Plant. 4(5):845-53.
Abstract
Vacuolar sorting receptors (VSRs) are type I integral membrane proteins that mediate the vacuolar transport of soluble cargo proteins via prevacuolar compartments (PVCs) in plants. Confocal immunofluorescent and immunogold Electron Microscope (EM) studies have localized VSRs to PVCs or multivesicular bodies (MVBs) and trans-Golgi network (TGN) in various plant cell types, including suspension culture cells, root cells, developing and germinating seeds. Here, we provide evidence that VSRs reach plasma membrane (PM) in growing pollen tubes. Both immunofluorescent and immunogold EM studies with specific VSR antibodies show that, in addition to the previously demonstrated PVC/MVB localization, VSRs also localize to PM in lily and tobacco pollen tubes prepared from chemical fixation or high-pressure freezing/frozen substitution. Such a PM localization suggests an additional role of VSR proteins in mediating protein transport to PM and endocytosis in growing pollen tubes. Using a high-speed Spinning Disc Confocal Microscope, the possible fusion between VSR-positive PVC organelles and the PM was also observed in living tobacco pollen tubes transiently expressing the PVC reporter GFP-VSR. In contrast, the lack of a prominent PM localization of GFP-VSR in living pollen tubes may be due to the highly dynamic situation of vesicular transport in this fast-growing cell type.
Loos A, Van Droogenbroeck B, Hillmer S, Grass J, Pabst M, Castilho A, Kunert R, Liang M, Arcalis E, Robinson DG, Depicker A, Steinkellner H. (2011). Expression of antibody fragments with a controlled N-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of Arabidopsis. Plant Physiol. 155(4):2036-48.
Abstract
Intracellular trafficking and subcellular deposition are critical factors influencing the accumulation and posttranslational modifications of proteins. In seeds, these processes are not yet fully understood. In this study, we set out to investigate the intracellular transport, final destination, N-glycosylation status, and stability of the fusion of recombinant single-chain variable fragments to the crystallizing fragment of an antibody (scFv-Fc) of two antiviral monoclonal antibodies (2G12 and HA78). The scFv-Fcs were expressed in Arabidopsis (Arabidopsis thaliana) seeds and leaves both as secretory molecules and tagged with an endoplasmic reticulum (ER) retention signal. We demonstrate differential proteolytic degradation of scFv-Fcs in leaves versus seeds, with higher degradation in the latter organ. In seeds, we show that secretory versions of HA78 scFv-Fcs are targeted to the extracellular space but are deposited in newly formed ER-derived vesicles upon KDEL tagging. These results are in accordance with the obtained N-glycosylation profiles: complex-type and ER-typical oligomannosidic N-glycans, respectively. HA78 scFv-Fcs, expressed in seeds of an Arabidopsis glycosylation mutant lacking plant-specific N-glycans, exhibit custom-made human-type N-glycosylation. In contrast, 2G12 scFv-Fcs carry exclusively ER-typical oligomannosidic N-glycans and were deposited in newly formed ER-derived vesicles irrespective of the targeting signals. HA78 scFv-Fcs exhibited efficient virus neutralization activity, while 2G12 scFv-Fcs were inactive. We demonstrate the efficient generation of scFv-Fcs with a controlled N-glycosylation pattern. However, our results also reveal aberrant subcellular deposition and, as a consequence, unexpected N-glycosylation profiles. Our attempts to elucidate intracellular protein transport in seeds contributes to a better understanding of this basic cell biological mechanism and is a step toward the versatile use of Arabidopsis seeds as an alternative expression platform for pharmaceutically relevant proteins.
Loos A, Van Droogenbroeck B, Hillmer S, Grass J, Kunert R, Cao J, Robinson DG, Depicker A, Steinkellner H. (2011). Production of monoclonal antibodies with a controlled N-glycosylation pattern in seeds of Arabidopsis thaliana. Plant Biotechnol J. 9(2):179-92.
Abstract
Seed-specific expression is an appealing alternative technology for the production of recombinant proteins in transgenic plants. Whereas attractive yields of recombinant proteins have been achieved by this method, little attention has been paid to the intracellular deposition and the quality of such products. Here, we demonstrate a comparative study of two antiviral monoclonal antibodies (mAbs) (HA78 against Hepatitis A virus; 2G12 against HIV) expressed in seeds of Arabidopsis wild-type (wt) plants and glycosylation mutants lacking plant specific N-glycan residues. We demonstrate that 2G12 is produced with complex N-glycans at great uniformity in the wt as well as in the glycosylation mutant, carrying a single dominant glycosylation species, GnGnXF and GnGn, respectively. HA78 in contrast, contains additionally to complex N-glycans significant amounts of oligo-mannosidic structures, which are typical for endoplasmic reticulum (ER)-retained proteins. A detailed subcellular localization study demonstrated the deposition of both antibodies virtually exclusively in the extracellular space, illustrating their efficient secretion. In addition, although a KDEL-tagged version of 2G12 exhibited an ER-typical N-glycosylation pattern, it was surprisingly detected in protein storage vacuoles. The different antibody variants showed different levels of degradation with hardly any degradation products detectable for HA78 carrying GnGnXF glycans. Finally, we demonstrate functional integrity of the HA78 and 2G12 glycoforms using viral inhibition assays. Our data therefore demonstrate the usability of transgenic seeds for the generation of mAbs with a controlled N-glycosylation pattern, thus expanding the possibilities for the production of optimally glycosylated proteins with enhanced biological activities for the use as human therapeutics.
Pubmed 
Schott A, Ravaud S, Keller S, Radzimanowski J, Viotti C, Hillmer S, Sinning I, Strahl S. (2010). Arabidopsis stromal-derived Factor2 (SDF2) is a crucial target of the unfolded protein response in the endoplasmic reticulum. J Biol Chem. 285(23):18113-21.
Abstract

Stresses increasing the load of unfolded proteins that enter the endoplasmic reticulum (ER) trigger a protective response termed the unfolded protein response (UPR). Stromal cell-derived factor2 (SDF2)-type proteins are highly conserved throughout the plant and animal kingdoms. In this study we have characterized AtSDF2 as crucial component of the UPR in Arabidopsis thaliana. Using a combination of biochemical and cell biological methods, we demonstrate that SDF2 is induced in response to ER stress conditions causing the accumulation of unfolded proteins. Transgenic reporter plants confirmed induction of SDF2 during ER stress. Under normal growth conditions SDF2 is highly expressed in fast growing, differentiating cells and meristematic tissues. The increased production of SDF2 due to ER stress and in tissues that require enhanced protein biosynthesis and secretion, and its association with the ER membrane qualifies SDF2 as a downstream target of the UPR. Determination of the SDF2 three-dimensional crystal structure at 1.95 A resolution revealed the typical beta-trefoil fold with potential carbohydrate binding sites. Hence, SDF2 might be involved in the quality control of glycoproteins. Arabidopsis sdf2 mutants display strong defects and morphological phenotypes during seedling development specifically under ER stress conditions, thus establishing that SDF2-type proteins play a key role in the UPR.

Wang J, Ding Y, Wang J, Hillmer S, Miao Y, Lo SW, Wang X, Robinson DG, Jiang L. (2010). EXPO, an exocyst-positive organelle distinct from multivesicular endosomes and autophagosomes, mediates cytosol to cell wall exocytosis in Arabidopsis and tobacco cells. Plant Cell. 22(12):4009-30.

 

Abstract
The exocyst protein complex mediates vesicle fusion with the plasma membrane. By expressing an (X)FP-tagged Arabidopsis thaliana homolog of the exocyst protein Exo70 in suspension-cultured Arabidopsis and tobacco (Nicotiana tabacum) BY-2 cells, and using antibodies specific for Exo70, we detected a compartment, which we term EXPO (for exocyst positive organelles). Standard markers for the Golgi apparatus, the trans-Golgi network/early endosome, and the multivesicular body/late endosome in plants do not colocalize with EXPO. Inhibitors of the secretory and endocytic pathways also do not affect EXPO. Exo70E2-(X)FP also locates to the plasma membrane (PM) as discrete punctae and is secreted outside of the cells. Immunogold labeling of sections cut from high-pressure frozen samples reveal EXPO to be spherical double membrane structures resembling autophagosomes. However, unlike autophagosomes, EXPOs are not induced by starvation and do not fuse with the lytic compartment or with endosomes. Instead, they fuse with the PM, releasing a single membrane vesicle into the cell wall. EXPOs are also found in other cell types, including root tips, root hair cells, and pollen grains. EXPOs therefore represent a form of unconventional secretion unique to plants.

 

Grundstein AJ(1), Duzinski SV, Dolinak D, Null J, Iyer SS. (2014). Evaluating infant core temperature response in a hot car using a heat balance model. 1. Forensic Sci Med Pathol. 2014 Oct 21. [
Abstract
PURPOSE: Using a 1-year old male infant as the model subject, the objectives of this study were to measure increased body temperature of an infant inside an enclosed vehicle during the work day (8:00 am-4:00 pm) during four seasons and model the time to un-compensable heating, heat stroke [>40 °C (>104 °F)], and critical thermal maximum [>42 °C (>107.6 °F)]. METHODS: A human heat balance model was used to simulate a child's physiological response to extreme heat exposure within an enclosed vehicle. Environmental variables were obtained from the nearest National Weather Service automated surface observing weather station and from an observational vehicular temperature study conducted in Austin, Texas in 2012. RESULTS: In all four seasons, despite differences in starting temperature and solar radiation, the model infant reached heat stroke and demise before 2:00 pm. Time to heat stroke and demise occurred most rapidly in summer, at intermediate durations in fall and spring, and most slowly in the winter. In August, the model infant reached un-compensable heat within 20 min, heat stroke within 105 min, and demise within 125 min. The average rate of heating from un-compensable heat to heat stroke was 1.7 °C/h (3.0 °F/h) and from heat stroke to demise was 4.8 °C/h (8.5 °F/h). CONCLUSIONS: Infants left in vehicles during the workday can reach hazardous thermal thresholds quickly even with mild environmental temperatures. These results provide a seasonal analogue of infant heat stroke time course. Further effort is required to create a universally available forensic tool to predict vehicular hyperthermia time course to demise.
Macciotta NP(1), Dimauro C, Null DJ, Gaspa G, Cellesi M, Cole JB. (2014). Dissection of genomic correlation matrices of US Holsteins using multivariate factor analysis. 2. J Anim Breed Genet. 2014 Aug 6.
Abstract
The aim of this study was to compare correlation matrices between direct genomic predictions for 31 traits at the genomic and chromosomal levels in US Holstein bulls. Multivariate factor analysis carried out at the genome level identified seven factors associated with conformation, longevity, yield, feet and legs, fat and protein content traits. Some differences were found at the chromosome level; variations in covariance structure on BTA 6, 14, 18 and 20 were interpreted as evidence of segregating QTL for different groups of traits. For example, milk yield and composition tended to join in a single factor on BTA 14, which is known to harbour the DGAT1 locus that affects these traits. Another example was on BTA 18, where a factor strongly correlated with sire calving ease and conformation traits was identified. It is known that in US Holstein, there is a segregating QTL on BTA18 influencing these traits. Moreover, a possible candidate gene for daughter pregnancy rate was suggested for BTA28. The methodology proposed in this study could be used to identify individual chromosomes, which have covariance structures that differ from the overall (whole genome) covariance structure. Such differences can be difficult to detect when a large number of traits are evaluated, and covariances may be affected by QTL that do not have large allele substitution effects.
Pubmed 
Dinwiddie A(1), Null R(2), Pizzano M(2), Chuong L(2), Leigh Krup A(2), Ee Tan H(2), Patel NH(3). (2014). Dynamics of F-actin prefigure the structure of butterfly wing scales. Dev Biol. 392(2):404-18.
Abstract
The wings of butterflies and moths consist of dorsal and ventral epidermal surfaces that give rise to overlapping layers of scales and hairs (Lepidoptera, "scale wing"). Wing scales (average length ~200 µm) are homologous to insect bristles (macrochaetes), and their colors create the patterns that characterize lepidopteran wings. The topology and surface sculpture of wing scales vary widely, and this architectural complexity arises from variations in the developmental program of the individual scale cells of the wing epithelium. One of the more striking features of lepidopteran wing scales are the longitudinal ridges that run the length of the mature (dead) cell, gathering the cuticularized scale cell surface into pleats on the sides of each scale. While also present around the periphery of other insect bristles and hairs, longitudinal ridges in lepidopteran wing scales gain new significance for their creation of iridescent color through microribs and lamellae. Here we show the dynamics of the highly organized F-actin filaments during scale cell development, and present experimental manipulations of actin polymerization that reveal the essential role of this cytoskeletal component in wing scale elongation and the positioning of longitudinal ribs.
Pubmed 
Cooper TA(1), Wiggans GR(2), Null DJ(2), Hutchison JL(2), Cole JB(2). (2014). Genomic evaluation, breed identification, and discovery of a haplotype affecting fertility for Ayrshire dairy cattle. J Dairy Sci. 97(6):3878-82.
Abstract
Genomic evaluations of dairy cattle in the United States have been available for Brown Swiss, Holsteins, and Jerseys since 2009. As of January 2013, 1,023 Ayrshires had genotypes in the North American database. Evaluation accuracy was assessed using genomic evaluations based on 646 bulls with 2008 traditional evaluations to predict daughter performance of up to 180 bulls in 2012. Mean gain in reliability over parent average for all traits was 8.2 percentage points. The highest gains were for protein yield (16.9 percentage points), milk yield (16.6 percentage points), and stature (16.2 percentage points). Twelve single nucleotide polymorphisms were useful for Ayrshire breed determination. Fewer breed-determining SNP were available for Ayrshires than for Holsteins, Jerseys, and Brown Swiss because of the similarity of Ayrshires and Holsteins. A haplotype that affects fertility was identified on chromosome 17 and traces back in the genotyped population to the bull Selwood Betty's Commander (born in 1953). The haplotype carrier frequency for genotyped Ayrshires was 26.1%. Sire conception rate was decreased by 4.3 ± 2.5 percentage points for carriers of the haplotype as determined by 618 matings of carrier sire by carrier maternal grandsire. Genomic evaluations for Ayrshires were officially implemented in the United States in April 2013.
Pubmed 
McClure MC(1), Bickhart D(2), Null D(2), Vanraden P(2), Xu L(1), Wiggans G(2), Liu G(1), Schroeder S(1), Glasscock J(3), Armstrong J(3), Cole JB(2), Van Tassell CP(1), Sonstegard TS(1). (2014). Bovine exome sequence analysis and targeted SNP genotyping of recessive fertility defects BH1, HH2, and HH3 reveal a putative causative mutation in SMC2 for HH3. PLoS One. 9(3):e92769.
Abstract
The recent discovery of bovine haplotypes with negative effects on fertility in the Brown Swiss, Holstein, and Jersey breeds has allowed producers to identify carrier animals using commercial single nucleotide polymorphism (SNP) genotyping assays. This study was devised to identify the causative mutations underlying defective bovine embryo development contained within three of these haplotypes (Brown Swiss haplotype 1 and Holstein haplotypes 2 and 3) by combining exome capture with next generation sequencing. Of the 68,476,640 sequence variations (SV) identified, only 1,311 genome-wide SNP were concordant with the haplotype status of 21 sequenced carriers. Validation genotyping of 36 candidate SNP identified only 1 variant that was concordant to Holstein haplotype 3 (HH3), while no variants located within the refined intervals for HH2 or BH1 were concordant. The variant strictly associated with HH3 is a non-synonymous SNP (T/C) within exon 24 of the Structural Maintenance of Chromosomes 2 (SMC2) on Chromosome 8 at position 95,410,507 (UMD3.1). This polymorphism changes amino acid 1135 from phenylalanine to serine and causes a non-neutral, non-tolerated, and evolutionarily unlikely substitution within the NTPase domain of the encoded protein. Because only exome capture sequencing was used, we could not rule out the possibility that the true causative mutation for HH3 might lie in a non-exonic genomic location. Given the essential role of SMC2 in DNA repair, chromosome condensation and segregation during cell division, our findings strongly support the non-synonymous SNP (T/C) in SMC2 as the likely causative mutation. The absence of concordant variations for HH2 or BH1 suggests either the underlying causative mutations lie within a non-exomic region or in exome regions not covered by the capture array.
Null SE(1), Medellín-Azuara J(2), Escriva-Bou A(3), Lent M(2), Lund JR(2). (2014). Optimizing the dammed: water supply losses and fish habitat gains from dam removal in California. J Environ Manage. 136:121-31.
Abstract
Dams provide water supply, flood protection, and hydropower generation benefits, but also harm native species by altering the natural flow regime and degrading aquatic and riparian habitat. Restoring some rivers reaches to free-flowing conditions may restore substantial environmental benefits, but at some economic cost. This study uses a systems analysis approach to preliminarily evaluate removing rim dams in California's Central Valley to highlight promising habitat and unpromising economic use tradeoffs for water supply and hydropower. CALVIN, an economic-engineering optimization model, is used to evaluate water storage and scarcity from removing dams. A warm and dry climate model for a 30-year period centered at 2085, and a population growth scenario for year 2050 water demands represent future conditions. Tradeoffs between hydropower generation and water scarcity to urban, agricultural, and instream flow requirements were compared with additional river kilometers of habitat accessible to anadromous fish species following dam removal. Results show that existing infrastructure is most beneficial if operated as a system (ignoring many current institutional constraints). Removing all rim dams is not beneficial for California, but a subset of existing dams are potentially promising candidates for removal from an optimized water supply and free-flowing river perspective. Removing individual dams decreases statewide delivered water by 0-2282 million cubic meters and provides access to 0 to 3200 km of salmonid habitat upstream of dams. The method described here can help prioritize dam removal, although more detailed, project-specific studies also are needed. Similarly, improving environmental protection can come at substantially lower economic cost, when evaluated and operated as a system.
Pubmed 
Null DM(1), Alvord J(1), Leavitt W(1), Wint A(1), Dahl MJ(1), Presson AP(2), Lane RH(1), DiGeronimo RJ(1), Yoder BA(1), Albertine KH(3). (2013). High-frequency nasal ventilation for 21 d maintains gas exchange with lower respiratory pressures and promotes alveolarization in preterm lambs. Pediatr Res. 75(4):507-16.
Abstract
BACKGROUND: Short-term high-frequency nasal ventilation (HFNV) of preterm neonates provides acceptable gas exchange compared to endotracheal intubation and intermittent mandatory ventilation (IMV). Whether long-term HFNV will provide acceptable gas exchange is unknown. We hypothesized that HFNV for up to 21 d would lead to acceptable gas exchange at lower inspired oxygen (O2) levels and airway pressures compared to intubation and IMV. METHODS: Preterm lambs were exposed to antenatal steroids and treated with perinatal surfactant and postnatal caffeine. Lambs were intubated and resuscitated by IMV. At ~3 h of age, half of the lambs were switched to noninvasive HFNV. Support was for 3 or 21 d. By design, Pao2 and Paco2 were not different between groups. RESULTS: At 3 d (n = 5) and 21 d (n = 4) of HFNV, fractional inspired O2 (FiO2), peak inspiratory pressure (PIP), mean airway, intratracheal, and positive end-expiratory pressures, oxygenation index, and alveolar-arterial gradient were significantly lower than matched periods of intubation and IMV. Pao2/FiO2 ratio was significantly higher at 3 and 21 d of HFNV compared to matched intubation and IMV. HFNV led to better alveolarization at 3 and 21 d. CONCLUSION: Long-term HFNV provides acceptable gas exchange at lower inspired O2 levels and respiratory pressures compared to intubation and IMV.
Nyendak MR(1), Park B, Null MD, Baseke J, Swarbrick G, Mayanja-Kizza H, Nsereko M, Johnson DF, Gitta P, Okwera A, Goldberg S, Bozeman L, Johnson JL, Boom WH, Lewinsohn DA, Lewinsohn DM; Tuberculosis Research Unit and the Tuberculosis Trials Consortium. (2013). Mycobacterium tuberculosis specific CD8(+) T cells rapidly decline with antituberculosis treatment. PLoS One. 8(12):e81564.
Abstract
RATIONALE: Biomarkers associated with response to therapy in tuberculosis could have broad clinical utility. We postulated that the frequency of Mycobacterium tuberculosis (Mtb) specific CD8(+) T cells, by virtue of detecting intracellular infection, could be a surrogate marker of response to therapy and would decrease during effective antituberculosis treatment. OBJECTIVES: We sought to determine the relationship of Mtb specific CD4(+) T cells and CD8(+) T cells with duration of antituberculosis treatment. MATERIALS AND METHODS: We performed a prospective cohort study, enrolling between June 2008 and August 2010, of HIV-uninfected Ugandan adults (n?=?50) with acid-fast bacillus smear-positive, culture confirmed pulmonary TB at the onset of antituberculosis treatment and the Mtb specific CD4(+) and CD8(+) T cell responses to ESAT-6 and CFP-10 were measured by IFN-γ ELISPOT at enrollment, week 8 and 24. RESULTS: There was a significant difference in the Mtb specific CD8(+) T response, but not the CD4(+) T cell response, over 24 weeks of antituberculosis treatment (p<0.0001), with an early difference observed at 8 weeks of therapy (p?=?0.023). At 24 weeks, the estimated Mtb specific CD8(+) T cell response decreased by 58%. In contrast, there was no significant difference in the Mtb specific CD4(+) T cell during the treatment. The Mtb specific CD4(+) T cell response, but not the CD8(+) response, was negatively impacted by the body mass index. CONCLUSIONS: Our data provide evidence that the Mtb specific CD8(+) T cell response declines with antituberculosis treatment and could be a surrogate marker of response to therapy. Additional research is needed to determine if the Mtb specific CD8(+) T cell response can detect early treatment failure, relapse, or to predict disease progression.
Null DM. (2013). Early use of surfactant and nitric oxide. J Perinatol. 33(12):909.
Abstract
Comment on J Perinatol. 2013 Dec;33(12):944-9.
Mehta AI(1), Adogwa O, Karikari IO, Thompson P, Verla T, Null UT, Friedman AH, Cheng JS, Bagley CA, Isaacs RE. (2013). Anatomical location dictating major surgical complications for intradural extramedullary spinal tumors: a 10-year single-institutional experience. J Neurosurg Spine. 19(6):701-7.
Abstract
Comment in J Neurosurg Spine. 2014 Jun;20(6):768-9.
Pubmed 
Arnold BF(1), Null C, Luby SP, Unicomb L, Stewart CP, Dewey KG, Ahmed T, Ashraf S, Christensen G, Clasen T, Dentz HN, Fernald LC, Haque R, Hubbard AE, Kariger P, Leontsini E, Lin A, Njenga SM, Pickering AJ, Ram PK, Tofail F, Winch PJ, Colford JM Jr. (2013). Cluster-randomised controlled trials of individual and combined water, sanitation, hygiene and nutritional interventions in rural Bangladesh and Kenya: the WASH Benefits study design and rationale. BMJ Open. 3(8):e003476.
Abstract
INTRODUCTION: Enteric infections are common during the first years of life in low-income countries and contribute to growth faltering with long-term impairment of health and development. Water quality, sanitation, handwashing and nutritional interventions can independently reduce enteric infections and growth faltering. There is little evidence that directly compares the effects of these individual and combined interventions on diarrhoea and growth when delivered to infants and young children. The objective of the WASH Benefits study is to help fill this knowledge gap. METHODS AND ANALYSIS: WASH Benefits includes two cluster-randomised trials to assess improvements in water quality, sanitation, handwashing and child nutrition-alone and in combination-to rural households with pregnant women in Kenya and Bangladesh. Geographically matched clusters (groups of household compounds in Bangladesh and villages in Kenya) will be randomised to one of six intervention arms or control. Intervention arms include water quality, sanitation, handwashing, nutrition, combined water+sanitation+handwashing (WSH) and WSH+nutrition. The studies will enrol newborn children (N=5760 in Bangladesh and N=8000 in Kenya) and measure outcomes at 12 and 24 months after intervention delivery. Primary outcomes include child length-for-age Z-scores and caregiver-reported diarrhoea. Secondary outcomes include stunting prevalence, markers of environmental enteropathy and child development scores (verbal, motor and personal/social). We will estimate unadjusted and adjusted intention-to-treat effects using semiparametric estimators and permutation tests. ETHICS AND DISSEMINATION: Study protocols have been reviewed and approved by human subjects review boards at the University of California, Berkeley, Stanford University, the International Centre for Diarrheal Disease Research, Bangladesh, the Kenya Medical Research Institute, and Innovations for Poverty Action. Independent data safety monitoring boards in each country oversee the trials. This study is funded by a grant from the Bill & Melinda Gates Foundation to the University of California, Berkeley. REGISTRATION: Trial registration identifiers (http://www.clinicaltrials.gov): NCT01590095 (Bangladesh), NCT01704105 (Kenya).
Wyatt AJ(1), Yount KM, Null C, Ramakrishnan U, Webb Girard A. (2013). Dairy intensification, mothers and children: an exploration of infant and young child feeding practices among rural dairy farmers in Kenya. 12. Matern Child Nutr. 2013 Aug 14.
Abstract
Agricultural strategies such as dairy intensification have potential to improve human nutrition through increased household food security. Increasing dairy productivity could also adversely affect infant and young child feeding (IYCF) practices because of increased maternal stress, demands on maternal time, and beliefs about the timing and appropriate types of complementary foods. Yet, few studies have looked rigorously at how interventions can affect young children (0-60 months). The study explores, within the context of rural dairy farming in Kenya, the relationship between level of household dairy production and selected IYCF practices using a mixed-methods approach. Six focus group discussions with women involved in dairy farming investigated their attitudes towards breastfeeding, introduction of complementary foods and child diets. Ninety-two households involved in three levels of dairy production with at least one child 0-60 months participated in a household survey. Quantitative results indicated that women from higher dairy producing households were more likely to introduce cow's milk to infants before they reached 6 months than women from households not producing any dairy. Themes from the focus group discussions demonstrated that women were familiar with exclusive breastfeeding recommendations, but indicated a preference for mixed feeding of infants. Evidence from this study can inform nutrition education programmes targeted to farmers participating in dairy interventions in rural, low-income settings to minimise potential harm to the nutritional status of children.
Pubmed 
Cepeda-Plaza M(1), Null EL, Lu Y. (2013). Metal ion as both a cofactor and a probe of metal-binding sites in a uranyl-specific DNAzyme: a uranyl photocleavage study. Nucleic Acids Res. 41(20):9361-70.
Abstract
DNAzymes are known to bind metal ions specifically to carry out catalytic functions. Despite many studies since DNAzymes were discovered nearly two decades ago, the metal-binding sites in DNAzymes are not fully understood. Herein, we adopt uranyl photocleavage to probe specific uranyl-binding sites in the 39E DNAzyme with catalytically relevant concentrations of uranyl. The results indicate that uranyl binds between T23 and C25 in the bulge loop, G11 and T12 in the stem loop of the enzyme strand, as well as between T2.4 and G3 close to the cleavage site in the substrate strand. Control experiments using two 39E DNAzyme mutants revealed a different cleavage pattern of the mutated region. Another DNAzyme, the 8-17 DNAzyme, which has a similar secondary structure but shows no activity in the presence of uranyl, indicated a different uranyl-dependent photocleavage as well. In addition, a close correlation between the concentration-dependent photocleavage and enzymatic activities is also demonstrated. Together, these experiments suggest that uranyl photocleavage has been successfully used to probe catalytically relevant uranyl-binding sites in the 39E DNAzyme. As uranyl is the cofactor of the 39E DNAzyme as well as the probe, specific uranyl binding has now been identified without disruption of the structure.
Dikmen S(1), Cole JB, Null DJ, Hansen PJ. (2013). Genome-wide association mapping for identification of quantitative trait loci for rectal temperature during heat stress in Holstein cattle. PLoS One. 8(7):e69202.
Abstract
Heat stress compromises production, fertility, and health of dairy cattle. One mitigation strategy is to select individuals that are genetically resistant to heat stress. Most of the negative effects of heat stress on animal performance are a consequence of either physiological adaptations to regulate body temperature or adverse consequences of failure to regulate body temperature. Thus, selection for regulation of body temperature during heat stress could increase thermotolerance. The objective was to perform a genome-wide association study (GWAS) for rectal temperature (RT) during heat stress in lactating Holstein cows and identify SNPs associated with genes that have large effects on RT. Records on afternoon RT where the temperature-humidity index was ≥78.2 were obtained from 4,447 cows sired by 220 bulls, resulting in 1,440 useable genotypes from the Illumina BovineSNP50 BeadChip with 39,759 SNP. For GWAS, 2, 3, 4, 5, and 10 adjacent SNP were averaged to identify consensus genomic regions associated with RT. The largest proportion of SNP variance (0.07 to 0.44%) was explained by markers flanking the region between 28,877,547 and 28,907,154 bp on Bos taurus autosome (BTA) 24. That region is flanked by U1 (28,822,883 to 28,823,043) and NCAD (28,992,666 to 29,241,119). In addition, the SNP at 58,500,249 bp on BTA 16 explained 0.08% and 0.11% of the SNP variance for 2- and 3-SNP analyses, respectively. That contig includes SNORA19, RFWD2 and SCARNA3. Other SNPs associated with RT were located on BTA 16 (close to CEP170 and PLD5), BTA 5 (near SLCO1C1 and PDE3A), BTA 4 (near KBTBD2 and LSM5), and BTA 26 (located in GOT1, a gene implicated in protection from cellular stress). In conclusion, there are QTL for RT in heat-stressed dairy cattle. These SNPs could prove useful in genetic selection and for identification of genes involved in physiological responses to heat stress.
Cochran SD(1), Cole JB, Null DJ, Hansen PJ. (2013). Single nucleotide polymorphisms in candidate genes associated with fertilizing ability of sperm and subsequent embryonic development in cattle. Biol Reprod. 89(3):69.
Abstract
Fertilization and development of the preimplantation embryo is under genetic control. The present goal was to test 434 single nucleotide polymorphisms (SNPs) for association with genetic variation in fertilization and early embryonic development. The approach was to produce embryos from 93 bulls using in vitro procedures (n = 3-6 replicates per bull) and relate cleavage rate (CR) and development of cleaved embryos to the blastocyst stage (BDRC) to the genotype for each SNP. Bulls were selected to have either high or low estimates for predicted transmitted ability for daughter pregnancy rate (DPR), an estimate of female fertility. The repeatability was 0.84 for CR and 0.55 for BDRC. Semen extender affected CR, with lower results for milk extender than yolk extender. There was no significant correlation between DPR and either CR or BDRC. A total of 100 SNPs had a minor allele frequency sufficiently high (>5%) to allow association analysis. There were nine genes with SNPs associated with CR (AVP, DEPP, EPAS1, HSD17B6, NT5E, SERPINE2, SLC18A2, TBC1D24, and a noncharacterized gene) and 12 genes with SNPs associated with BDRC (C1QB, FAM5C, HSPA1A, IRF9, MON1B, PARM1, PCCB, PMM2, SLC18A2, TBC1D24, TTLL3, and WBP1). Results demonstrate that in vitro fertilization and blastocyst development are under genetic control and point out the potential importance of some previously unknown genes in these processes. Selection of cattle based on the genotype at one or more of these 19 loci may prove useful in conjunction with other genetic markers for improving genetic ability for fertility.
Schmerer MW(1), Null RW, Shankland M. (2013). Developmental transition to bilaterally symmetric cell divisions is regulated by Pax-mediated transcription in embryos of the leech Helobdella austinensis. Dev Biol. 382(1):149-59.
Abstract
The leech embryo develops by spiral cleavage, and establishes the symmetry properties of its adult body plan through the bilaterally symmetric divisions of mesodermal proteloblast DM″ and ectodermal proteloblast DNOPQ?. We here show that transcriptional inhibitors α-amanitin and actinomycin D specifically disrupt the symmetry and orientation of these two proteloblast cell divisions while having no apparent effect on the timing or geometry of other divisions. Transcriptional inhibition had a similar effect on both proteloblasts, i.e. cytokinesis was highly asymmetric and the cleavage plane roughly orthogonal to that seen during normal development. These findings suggest that zygotic gene product(s) are required, either directly or indirectly, for the correct placement of the proteloblast cleavage furrow. The same phenotypes were also observed following in vivo expression of dominant-negative Pax gene constructs. These dominant-negative phenotypes depended on protein/DNA interaction, and could be rescued by coexpression of full length Pax proteins. However, symmetric cleavage of the mesodermal proteloblast was rescued by full length constructs of either Hau-Paxβ1 or Hau-Pax2/5/8, while only Hau-Paxβ1 rescued the symmetry of ectodermal cleavage. We conclude that both proteloblasts need Pax-mediated transcription to adopt their normally symmetric cleavage patterns, but differ in terms of the specific Pax proteins required. The implication of these findings for the evolution of spiral cleavage is discussed.
Pubmed 
Khan SA(1), Adogwa O, Gan TJ, Null UT, Verla T, Gokhale S, White WD, Britz GW, Zomorodi AR, James ML, McDonagh DL. (2013). Effect of 6% hydroxyethyl starch 130/0.4 in 0.9% sodium chloride (Voluven®) on complications after subarachnoid hemorrhage: a retrospective analysis. Springerplus. 2(1):314. Print 2013 Dec.
Abstract
BACKGROUND: 6% Hydroxyethyl Starch 130/0.4 in 0.9% Sodium Chloride (Voluven®; 6% HES 130/0.4) is a colloid often used for fluid resuscitation in patients with subarachnoid hemorrhage (SAH), despite a lack of safety data for this use. The purpose of our study was to evaluate the effect of 6% HES 130/0.4 on major complications associated with SAH. METHODS: Medical records of all patients presenting between May 2010 and September 2012 with aneurysmal SAH were analyzed. Patients were divided in two groups based on the administration of 6% HES 130/0.4; HES group (n=57) and Non-HES group (n=72). The primary outcome included a composite of three major complications associated with SAH: Delayed Cerebral Ischemia (DCI), Hydrocephalus (HCP) requiring cerebrospinal fluid (CSF) shunting, and Rebleeding. RESULTS: The study groups were similar with respect to most characteristics except the incidences of hypertension, ischemic heart disease, Fisher grade and lowest hemoglobin during stay. The odds of developing the primary composite outcome was higher in the HES group [OR= 3.1(1.30-7.36), p=0.01]. The patients in the HES group had a significantly longer median duration of hospital (19 vs 14 days) and Neurointensive Care Unit stay (14 vs 10 days) compared to the Non HES group. CONCLUSION: We observed increased complications after SAH with 6% HES 130/0.4 (Voluven®) administration. An adequately powered prospective randomized controlled trial into the safety of 6% HES 130/0.4 in this patient population is warranted.
Lewinsohn DM(1), Swarbrick GM, Cansler ME, Null MD, Rajaraman V, Frieder MM, Sherman DR, McWeeney S, Lewinsohn DA. (2013). Human Mycobacterium tuberculosis CD8 T Cell Antigens/Epitopes Identified by a Proteomic Peptide Library. PLoS One. 8(6):e67016. Print 2013.
Abstract
Identification of CD8(+) T cell antigens/epitopes expressed by human pathogens with large genomes is especially challenging, yet necessary for vaccine development. Immunity to tuberculosis, a leading cause of mortality worldwide, requires CD8(+) T cell immunity, yet the repertoire of CD8 antigens/epitopes remains undefined. We used integrated computational and proteomic approaches to screen 10% of the Mycobacterium tuberculosis (Mtb) proteome for CD8 Mtb antigens. We designed a weighting schema based upon a Multiple Attribute Decision Making:framework to select 10% of the Mtb proteome with a high probability of containing CD8(+) T cell epitopes. We created a synthetic peptide library consisting of 15-mers overlapping by 11 aa. Using the interferon-γ ELISPOT assay and Mtb-infected dendritic cells as antigen presenting cells, we screened Mtb-specific CD8(+) T cell clones restricted by classical MHC class I molecules (MHC class Ia molecules), that were isolated from Mtb-infected humans, against this library. Three novel CD8 antigens were unambiguously identified: the EsxJ family (Rv1038c, Rv1197, Rv3620c, Rv2347c, Rv1792), PE9 (Rv1088), and PE_PGRS42 (Rv2487c). The epitopes are B5701-restricted EsxJ24-34, B3905-restricted PE953-67, and B3514-restricted PE_PGRS4248-56, respectively. The utility of peptide libraries in identifying unknown epitopes recognized by classically restricted CD8(+) T cells was confirmed, which can be applied to other intracellular pathogens with large size genomes. In addition, we identified three novel Mtb epitopes/antigens that may be evaluated for inclusion in vaccines and/or diagnostics for tuberculosis.
Cochran SD(1), Cole JB, Null DJ, Hansen PJ. (2013). Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle. BMC Genet. 14:49.
Abstract
BACKGROUND: Identification of single nucleotide polymorphisms (SNPs) for specific genes involved in reproduction might improve reliability of genomic estimates for these low-heritability traits. Semen from 550 Holstein bulls of high (≥ 1.7; n = 288) or low (≤ -2; n = 262) daughter pregnancy rate (DPR) was genotyped for 434 candidate SNPs using the Sequenom MassARRAY® system. Three types of SNPs were evaluated: SNPs previously reported to be associated with reproductive traits or physically close to genetic markers for reproduction, SNPs in genes that are well known to be involved in reproductive processes, and SNPs in genes that are differentially expressed between physiological conditions in a variety of tissues associated in reproductive function. Eleven reproduction and production traits were analyzed. RESULTS: A total of 40 SNPs were associated (P < 0.05) with DPR. Among these were genes involved in the endocrine system, cell signaling, immune function and inhibition of apoptosis. A total of 10 genes were regulated by estradiol. In addition, 22 SNPs were associated with heifer conception rate, 33 with cow conception rate, 36 with productive life, 34 with net merit, 23 with milk yield, 19 with fat yield, 13 with fat percent, 19 with protein yield, 22 with protein percent, and 13 with somatic cell score. The allele substitution effect for SNPs associated with heifer conception rate, cow conception rate, productive life and net merit were in the same direction as for DPR. Allele substitution effects for several SNPs associated with production traits were in the opposite direction as DPR. Nonetheless, there were 29 SNPs associated with DPR that were not negatively associated with production traits. CONCLUSION: SNPs in a total of 40 genes associated with DPR were identified as well as SNPs for other traits. It might be feasible to include these SNPs into genomic tests of reproduction and other traits. The genes associated with DPR are likely to be important for understanding the physiology of reproduction. Given the large number of SNPs associated with DPR that were not negatively associated with production traits, it should be possible to select for DPR without compromising production.
McClure M(1), Kim E, Bickhart D, Null D, Cooper T, Cole J, Wiggans G, Ajmone-Marsan P, Colli L, Santus E, Liu GE, Schroeder S, Matukumalli L, Van Tassell C, Sonstegard T. (2013). Fine mapping for Weaver syndrome in Brown Swiss cattle and the identification of 41 concordant mutations across NRCAM, PNPLA8 and CTTNBP2. 20. PLoS One. 2013;8(3):e59251.
Abstract
Bovine Progressive Degenerative Myeloencephalopathy (Weaver Syndrome) is a recessive neurological disease that has been observed in the Brown Swiss cattle breed since the 1970's in North America and Europe. Bilateral hind leg weakness and ataxia appear in afflicted animals at 6 to 18 months of age, and slowly progresses to total loss of hind limb control by 3 to 4 years of age. While Weaver has previously been mapped to Bos taurus autosome (BTA) 4?46-56 Mb and a diagnostic test based on the 6 microsatellite (MS) markers is commercially available, neither the causative gene nor mutation has been identified; therefore misdiagnosis can occur due to recombination between the diagnostic MS markers and the causative mutation. Analysis of 34,980 BTA 4 SNPs genotypes derived from the Illumina BovineHD assay for 20 Brown Swiss Weaver carriers and 49 homozygous normal bulls refined the Weaver locus to 48-53 Mb. Genotyping of 153 SNPs, identified from whole genome sequencing of 10 normal and 10 carrier animals, across a validation set of 841 animals resulted in the identification of 41 diagnostic SNPs that were concordant with the disease. Except for one intergenic SNP all are associated with genes expressed in nervous tissues: 37 distal to NRCAM, one non-synonymous (serine to asparagine) in PNPLA8, one synonymous and one non-synonymous (lysine to glutamic acid) in CTTNBP2. Haplotype and imputation analyses of 7,458 Brown Swiss animals with Illumina BovineSNP50 data and the 41 diagnostic SNPs resulted in the identification of only one haplotype concordant with the Weaver phenotype. Use of this haplotype and the diagnostic SNPs more accurately identifies Weaver carriers in both Brown Swiss purebred and influenced herds.
Cole JB(1), Null DJ. (2013). Visualization of the transmission of direct genomic values for paternal and maternal chromosomes for 15 traits in US Brown Swiss, Holstein, and Jersey cattle. J Dairy Sci. 96(4):2713-26.
Abstract
Haplotypes are available for 220,671 Brown Swiss, Holstein, and Jersey bulls and cows that received genomic evaluations in August 2012. Differences in least squares means of direct genomic values (DGV) for paternal and maternal haplotypes of Bos taurus autosomes 1, 6, 14, and 18 for lifetime net merit were significant in all but one case. Those chromosomes were chosen to represent cases with and without known quantitative trait loci, and other chromosomes may differ as well. Paternal haplotypes had higher DGV than maternal haplotypes in most cases, and differences were larger when quantitative trait loci were present. Longer chromosomes generally accounted for more variance than shorter chromosomes, and differences among breeds were consistent with known mutations of large effect. For example, Bos taurus autosome 18 accounted for 2.5, 7, and 2.6% of the variance in lifetime net merit for Brown Swiss (BS), Holsteins, and Jerseys, respectively. Distributions of the number of positive DGV inherited from sires and dams were negatively skewed in all breeds, and modes were slightly higher for paternally than maternally derived haplotypes in Holsteins and BS (22 vs. 20 and 22 vs. 21, respectively) and slightly lower in BS (17 vs. 19). Graphical representations of DGV are available to all users through a query on the Animal Improvement Programs Laboratory (ARS, USDA, Beltsville, MD) web site. Query results were also used to illustrate several quantitative genetic principles using genotype information from real animals. For example, offspring DGV can be compared with parental DGV to demonstrate that a parent transmits the average value of its 2 chromosomes to its progeny. The frequency of DGV with positive and negative values in animals of different ages can be used to show how selection affects allele frequencies. The effect of selection for alleles with large effects versus those with small effects is demonstrated using an animal with undesirable alleles for a marker with a large effect but many desirable alleles for markers with small effects. Strategies for the use of those data in selection programs are being studied, and work is underway to add data on conformation traits to the system.
Pubmed 
Mahabaleshwarkar RK(1), Yang Y, Datar MV, Bentley JP, Strum MW, Banahan BF, Null KD. (2013). Risk of adverse cardiovascular outcomes and all-cause mortality associated with concomitant use of clopidogrel and proton pump inhibitors in elderly patients. Curr Med Res Opin. 29(4):315-23.
Abstract
OBJECTIVE: To examine the effect of concomitant use of clopidogrel and PPIs in a national sample of elderly Medicare beneficiaries (age ≥65 years). METHODS: A nested case-control design was employed. A cohort of Medicare beneficiaries who initiated clopidogrel and did not have any gap of ≥30 days between clopidogrel fills between July 1, 2006 and December 31, 2008 was identified from a 5% national sample of Medicare claims data. Within this cohort, cases (beneficiaries who experienced any major cardiovascular event [MCE] [acute myocardial infarction, stroke, coronary artery bypass graft, or percutaneous coronary intervention] or all-cause mortality) and controls (beneficiaries who did not experience any MCE or all-cause mortality) were identified from inpatient and outpatient claims. Cases and controls were matched on age and the time to first clopidogrel fill. Conditional logistic regression was performed on the matched sample to evaluate the association between concomitant use of clopidogrel and PPIs and adverse health outcomes (MCEs and all-cause mortality). RESULTS: A total of 43,159 clopidogrel users were identified. Among them, 15,415 (35.7%) received clopidogrel and a PPI concomitantly at any time during the study period, 3502 (8.1%) experienced a MCE, 7306 (17.1%) died, and a total of 9908 (22.8%) experienced the primary composite outcome (any MCE or all-cause mortality) during follow-up. The odds ratio (OR) for the primary composite outcome was 1.26 (95% confidence interval [CI]: 1.18-1.35). Secondary analyses indicated that elderly patients using clopidogrel and a PPI concomitantly were more likely to experience all-cause mortality (OR: 1.40; 95% CI: 1.29-1.53) as compared to those receiving clopidogrel only, but not MCEs (OR: 1.06; 95% CI: 0.95-1.18). CONCLUSIONS: Concomitant use of clopidogrel and PPIs was associated with a slightly increased risk of all-cause mortality but not MCEs.
Sonstegard TS(1), Cole JB, VanRaden PM, Van Tassell CP, Null DJ, Schroeder SG, Bickhart D, McClure MC. (2013). Identification of a nonsense mutation in CWC15 associated with decreased reproductive efficiency in Jersey cattle. 23. PLoS One. 2013;8(1):e54872.
Abstract
With the recent advent of genomic tools for cattle, several recessive conditions affecting fertility have been identified and selected against, such as deficiency of uridine monophosphate synthase, complex vertebral malformation, and brachyspina. The current report refines the location of a recessive haplotype affecting fertility in Jersey cattle using crossover haplotypes, discovers the causative mutation using whole genome sequencing, and examines the gene's role in embryo loss. In an attempt to identify unknown recessive lethal alleles in the current dairy population, a search using deep Mendelian sampling of 5,288 Jersey cattle was conducted for high-frequency haplotypes that have a deficit of homozygotes at the population level. This search led to the discovery of a putative recessive lethal in Jersey cattle on Bos taurus autosome 15. The haplotype, denoted JH1, was associated with reduced fertility, and further investigation identified one highly-influential Jersey bull as the putative source ancestor. By combining SNP analysis of whole-genome sequences aligned to the JH1 interval and subsequent SNP validation a nonsense mutation in CWC15 was identified as the likely causative mutation underlying the fertility phenotype. No homozygous recessive individuals were found in 749 genotyped animals, whereas all known carriers and carrier haplotypes possessed one copy of the mutant allele. This newly identified lethal has been responsible for a substantial number of spontaneous abortions in Jersey dairy cattle throughout the past half-century. With the mutation identified, selection against the deleterious allele in breeding schemes will aid in reducing the incidence of this defect in the population. These results also show that carrier status can be imputed with high accuracy. Whole-genome resequencing proved to be a powerful strategy to rapidly identify a previously mapped deleterious mutation in a known carrier of a recessive lethal allele.
Moss A(1), Smith S, Null D, Long Roth S, Tragoudas U. (2013). Farm to School and Nutrition Education: Positively Affecting Elementary School-Aged Children's Nutrition Knowledge and Consumption Behavior. Child Obes. 9(1):51-6.
Abstract
BACKGROUND: Good nutrition is crucial. School-aged children battle social and health issues such as poor nutrition, childhood obesity, and minimal nutrition knowledge. This study was a quasi-experimental design analyzing the effects of the Coordinated Approach to Child Health (CATCH) nutrition curriculum with a Farm to School program to assess nutrition knowledge of 3(rd) grade students, and to increase fruit and vegetable consumption behavior. METHODS: Third grade boys and girls (n=65) participated in this study. The intervention consisted of two nutrition education classes and a farm tour. Data were collected at baseline and postintervention. Surveys assessed nutrition knowledge, fruit and vegetable consumption behavior, and awareness of farms and farmers. Chi-squared tests of independence were performed to examine the relation between the baseline and postintervention responses. RESULTS: Significant differences were found concerning knowledge of fiber (p<0.001). Knowledge of vitamins and minerals, reported vegetable consumption behavior at school, and farm exposure were also significant (p<0.05). CONCLUSIONS: These findings suggest that CATCH nutrition education and farm tours can positively affect school-aged children's nutrition knowledge and fruit and vegetable consumption behavior.
Arrington CB(1), Bleyl SB, Matsunami N, Bowles NE, Leppert TI, Demarest BL, Osborne K, Yoder BA, Byrne JL, Schiffman JD, Null DM, DiGeronimo R, Rollins M, Faix R, Comstock J, Camp NJ, Leppert MF, Yost HJ, Brunelli L. (2012). A family-based paradigm to identify candidate chromosomal regions for isolated congenital diaphragmatic hernia. Am J Med Genet A. 158A(12):3137-47.
Abstract
Congenital diaphragmatic hernia (CDH) is a developmental defect of the diaphragm that causes high newborn mortality. Isolated or non-syndromic CDH is considered a multifactorial disease, with strong evidence implicating genetic factors. As low heritability has been reported in isolated CDH, family-based genetic methods have yet to identify the genetic factors associated with the defect. Using the Utah Population Database, we identified distantly related patients from several extended families with a high incidence of isolated CDH. Using high-density genotyping, seven patients were analyzed by homozygosity exclusion rare allele mapping (HERAM) and phased haplotype sharing (HapShare), two methods we developed to map shared chromosome regions. Our patient cohort shared three regions not previously associated with CDH, that is, 2q11.2-q12.1, 4p13 and 7q11.2, and two regions previously involved in CDH, that is, 8p23.1 and 15q26.2. The latter regions contain GATA4 and NR2F2, two genes implicated in diaphragm formation in mice. Interestingly, three patients shared the 8p23.1 locus and one of them also harbored the 15q26.2 segment. No coding variants were identified in GATA4 or NR2F2, but a rare shared variant was found in intron 1 of GATA4. This work shows the role of heritability in isolated CDH. Our family-based strategy uncovers new chromosomal regions possibly associated with disease, and suggests that non-coding variants of GATA4 and NR2F2 may contribute to the development of isolated CDH. This approach could speed up the discovery of the genes and regulatory elements causing multifactorial diseases, such as isolated CDH.
Pubmed 
Null DB(1), Weiland CM, Camlibel AR. (2012). Postmenopausal bleeding-first steps in the workup. J Fam Pract. 61(10):597-604.
Abstract
Is it endometrial cancer? When a postmenopausal woman presents with unexpected vaginal bleeding, this algorithm and review can help you answer that question without delay.
VanRaden PM(1), Null DJ, Sargolzaei M, Wiggans GR, Tooker ME, Cole JB, Sonstegard TS, Connor EE, Winters M, van Kaam JB, Valentini A, Van Doormaal BJ, Faust MA, Doak GA. (2012). Genomic imputation and evaluation using high-density Holstein genotypes. J Dairy Sci. 96(1):668-78.
Abstract
Genomic evaluations for 161,341 Holsteins were computed by using 311,725 of 777,962 markers on the Illumina BovineHD Genotyping BeadChip (HD). Initial edits with 1,741 HD genotypes from 5 breeds revealed that 636,967 markers were usable but that half were redundant. Holstein genotypes were from 1,510 animals with HD markers, 82,358 animals with 45,187 (50K) markers, 1,797 animals with 8,031 (8K) markers, 20,177 animals with 6,836 (6K) markers, 52,270 animals with 2,683 (3K) markers, and 3,229 nongenotyped dams (0K) with >90% of haplotypes imputable because they had 4 or more genotyped progeny. The Holstein HD genotypes were from 1,142 US, Canadian, British, and Italian sires, 196 other sires, 138 cows in a US Department of Agriculture research herd (Beltsville, MD), and 34 other females. Percentages of correctly imputed genotypes were tested by applying the programs findhap and FImpute to a simulated chromosome for an earlier population that had only 1,112 animals with HD genotypes and none with 8K genotypes. For each chip, 1% of the genotypes were missing and 0.02% were incorrect initially. After imputation of missing markers with findhap, percentages of genotypes correct were 99.9% from HD, 99.0% from 50K, 94.6% from 6K, 90.5% from 3K, and 93.5% from 0K. With FImpute, 99.96% were correct from HD, 99.3% from 50K, 94.7% from 6K, 91.1% from 3K, and 95.1% from 0K genotypes. Accuracy for the 3K and 6K genotypes further improved by approximately 2 percentage points if imputed first to 50K and then to HD instead of imputing all genotypes directly to HD. Evaluations were tested by using imputed actual genotypes and August 2008 phenotypes to predict deregressed evaluations of US bulls proven after August 2008. For 28 traits tested, the estimated genomic reliability averaged 61.1% when using 311,725 markers vs. 60.7% when using 45,187 markers vs. 29.6% from the traditional parent average. Squared correlations with future data were slightly greater for 16 traits and slightly less for 12 with HD than with 50K evaluations. The observed 0.4 percentage point average increase in reliability was less favorable than the 0.9 expected from simulation but was similar to actual gains from other HD studies. The largest HD and 50K marker effects were often located at very similar positions. The single-breed evaluation tested here and previous single-breed or multibreed evaluations have not produced large gains. Increasing the number of HD genotypes used for imputation above 1,074 did not improve the reliability of Holstein genomic evaluations.
Pubmed 
Harshfield E(1), Lantagne D, Turbes A, Null C. (2012). Evaluating the sustained health impact of household chlorination of drinking water in rural Haiti. Am J Trop Med Hyg. 87(5):786-95.
Abstract
The Jolivert Safe Water for Families program has sold sodium hypochlorite solution (chlorine) and conducted household visits in rural Haiti since 2002. To assess the impact of the program on diarrheal disease, in 2010 we conducted a survey and water quality testing in 201 program participants and 425 control households selected at random. Fifty-six percent of participants (versus 10% of controls) had free chlorine residuals between 0.2 and 2.0 mg/L, indicating correct water treatment. Using intention-to-treat analysis, we found that significantly fewer children < 5 in participant households had an episode of diarrhea in the previous 48 hours (32% versus 52%; P < 0.001) with 59% reduced odds (odds ratio = 0.41, 95% confidence interval = 0.21-0.79). Treatment-on-treated estimates of the odds of diarrhea indicated larger program effects for participants who met more stringent verifications of participation. Diarrheal disease reduction in this long-term program was comparable with that seen in short-term randomized, controlled interventions, suggesting that household chlorination can be an effective long-term water treatment strategy.
Cole JB(1), Ehrlich JL, Null DJ. (2012). Short communication: Projecting milk yield using best prediction and the MilkBot lactation model. J Dairy Sci. 95(7):4041-4.
Abstract
The accuracy and precision of 3 lactation models was estimated by summarizing means and variability in projection error for next-test milk and actual 305-d milk yield (M305) for 50-d intervals in a large Dairy Herd Improvement Association data set. Lactations were grouped by breed (Holstein, Jersey, and crossbred) and parity (first vs. later). A smaller, single-herd data set with both Dairy Herd Improvement Association data and daily milk weights was used to compare M305 calculated from test-day data with M305 computed by summing daily milk weights. The lactation models tested were best prediction (BP), the nonlinear MilkBot (MB) model, and a null model (NM) based on a stepwise function. The accuracy of the models was ranked (best to worst) MB, BP, and NM for later-parity cows and MB, NM, and BP for first-parity cows, with MB achieving accuracy in projecting daily milk of 0.5 kg or better in most groups. The models generally showed better accuracy after 50 d in milk. Best prediction and NM had low accuracy for crossbred cows and first-parity Holstein and Jersey cows. The MB model appears to be more precise than BP, and NM had low precision, especially for M305. Regression of model-generated M305 on summed M305 showed BP and MB to be equally efficient in ranking lactations, but MB was better at quantifying differences.
Pubmed 
Dikmen S(1), Cole JB, Null DJ, Hansen PJ. (2012). Heritability of rectal temperature and genetic correlations with production and reproduction traits in dairy cattle. J Dairy Sci. 95(6):3401-5.
Abstract
Genetic selection for body temperature during heat stress might be a useful approach to reduce the magnitude of heat stress effects on production and reproduction. Objectives of the study were to estimate the genetic parameters of rectal temperature (RT) in dairy cows in freestall barns under heat stress conditions and to determine the genetic and phenotypic correlations of rectal temperature with other traits. Afternoon RT were measured in a total of 1,695 lactating Holstein cows sired by 509 bulls during the summer in North Florida. Genetic parameters were estimated with Gibbs sampling, and best linear unbiased predictions of breeding values were predicted using an animal model. The heritability of RT was estimated to be 0.17 ± 0.13. Predicted transmitting abilities for rectal temperature changed 0.0068 ± 0.0020°C/yr from (birth year) 2002 to 2008. Approximate genetic correlations between RT and 305-d milk, fat, and protein yields, productive life, and net merit were significant and positive, whereas approximate genetic correlations between RT and somatic cell count score and daughter pregnancy rate were significant and negative. Rectal temperature during heat stress has moderate heritability, but genetic correlations with economically important traits mean that selection for RT could lead to lower productivity unless methods are used to identify genes affecting RT that do not adversely affect other traits of economic importance.
Pubmed 
Miner KD(1), Mukherjee A, Gao YG, Null EL, Petrik ID, Zhao X, Yeung N, Robinson H, Lu Y. (2012). A designed functional metalloenzyme that reduces O2 to H2O with over one thousand turnovers. Angew Chem Int Ed Engl. 51(23):5589-92.
Abstract
PMCID: PMC3461324 PMID: 22539151 [PubMed - indexed for MEDLINE]
Null DM(1), Yoder BA, DiGeronimo RJ. (2012). Early neonatal research at Wilford Hall US Air Force Medical Center. Pediatrics. 129 Suppl 1:S20-6.
Abstract
PMID: 22300825 [PubMed - indexed for MEDLINE]
Rollins MD(1), Yoder BA, Moore KR, Barnhart DC, Jones C, Null DM, DiGeronimo RJ. (2012). Utility of neuroradiographic imaging in predicting outcomes after neonatal extracorporeal membrane oxygenation. J Pediatr Surg. 47(1):76-80.
Abstract
BACKGROUND: The need for routine neuroimaging after extracorporeal membrane oxygenation (ECMO) and the optimal radiographic study remains unclear. We sought to evaluate the correlation between findings on head ultrasound (HUS) and magnetic resonance imaging (MRI) and determine the association of these findings to neurodevelopmental outcome. METHODS: A retrospective review was performed (2003-2010) to identify neonates who had a MRI after ECMO. Each MRI was reviewed by a single pediatric neuroradiologist. Neurodevelopmental data was collected from the high-risk neonatal follow-up clinic. RESULTS: Fifty neonates had a MRI (venoarterial 37, venovenous 13) after ECMO. HUS was abnormal in 24%, whereas MRI was abnormal in 62%. All infants with an abnormal HUS had an abnormal MRI, but an additional 50% of patients with a normal HUS had an abnormal MRI. Venoarterial ECMO was significantly associated with an abnormal MRI. Follow-up data was available for 26 neonates. The only predictor of abnormal neurodevelopment was the need for supplemental tube feeds at discharge. CONCLUSIONS: MRI identified significantly more abnormalities compared to routine HUS after neonatal ECMO. However, neither MRI nor HUS findings correlated with early neurodevelopmental outcome. Feeding ability at discharge was the overall best predictor of neurologic impairment in survivors.
Pubmed 
Grundstein A(1), Null J, Meentemeyer V. ( 34. Geogr Rev. 2011;101(4):353-70. ). Weather, geography, and vehicle-related hyperthermia in children. 34. Geogr Rev. 2011;101(4):353-70.
Abstract
Vehicle-related hyperthermia is an unfortunate tragedy that leads to the accidental deaths of children each year. This research utilizes the most extensive dataset of child vehicle-related hyperthermia deaths in the United States, including 414 deaths between 1998 and 2008. Deaths follow a seasonal pattern, with a peak in July and no deaths in December or January. Also, deaths occurred over a wide range of temperature and radiation levels and across virtually all regions, although most of them took place across the southern United States. In particular, the Phoenix, Houston, Dallas, and Las Vegas metropolitan areas had the greatest number of deaths. We utilize our vehicle hyperthermia index (vhi) to compare expected deaths versus actual deaths in a metropolitan area, based on the number of children in the area who are under the age of five and on the frequency of hot days in the area. The vhi indicates that the Memphis, West Palm Beach-Boca Raton, and Las Vegas metropolitan areas are the most dangerous places for vehicle-related hyperthermia. We conclude by discussing several recommendations with public health policy implications.
VanRaden PM(1), Olson KM, Null DJ, Hutchison JL. (2011). Harmful recessive effects on fertility detected by absence of homozygous haplotypes. J Dairy Sci. 94(12):6153-61.
Abstract
Five new recessive defects were discovered in Holsteins, Jerseys, and Brown Swiss by examining haplotypes that had a high population frequency but were never homozygous. The method required genotypes only from apparently normal individuals and not from affected embryos. Genotypes from the BovineSNP50 BeadChip (Illumina, San Diego, CA) were examined for 58,453 Holsteins, 5,288 Jerseys, and 1,991 Brown Swiss with genotypes in the North American database. Haplotypes with a length of ≤ 75 markers were obtained. Eleven candidate haplotypes were identified, with the earliest carrier born before 1980; 7 to 90 homozygous haplotypes were expected, but none were observed in the genomic data. Expected numbers were calculated using either the actual mating pattern or assuming random mating. Probability of observing no homozygotes ranged from 0.0002 for 7 to 10??? for 90 expected homozygotes. Phenotypic effects were confirmed for 5 of the 11 candidate haplotypes using 14,911,387 Holstein, 830,391 Jersey, and 68,443 Brown Swiss records for conception rate. Estimated effect for interaction of carrier service sire with carrier maternal grandsire ranged from -3.0 to -3.7 percentage points, which was slightly smaller than the -3.9 to -4.6 percentage points expected for lethal recessives but slightly larger than estimated effects for previously known lethal alleles of -2.5 percentage points for brachyspina and -2.9 percentage points for complex vertebral malformation. Conception rate was coded as a success only if the gestation went to term or the cow was confirmed to be pregnant. Estimated effect of carrier interaction for stillbirth rate based on 10,876,597 Holstein and 25,456 Jersey records was small. Thus, lethal effects may include conception, gestation, and stillbirth losses. Carrier frequency has been >20% for many years for the confirmed defect in Jerseys and is currently 16% for the defect in Brown Swiss. The 3 defects discovered in Holsteins have carrier frequencies of 2.7 to 6.4% in the current population. For previously known defects, map locations and lack of homozygotes were consistent with the literature and lethal recessive inheritance, but numbers of expected homozygotes for some were small because of low frequency. Very large genotypic and phenotypic data sets allow efficient detection of smaller and less frequent effects. Haplotype tests can help breeders avoid carrier matings for such defects and reduce future frequencies.
Pubmed 
Lancioni C(1), Nyendak M, Kiguli S, Zalwango S, Mori T, Mayanja-Kizza H, Balyejusa S, Null M, Baseke J, Mulindwa D, Byrd L, Swarbrick G, Scott C, Johnson DF, Malone L, Mudido-Musoke P, Boom WH, Lewinsohn DM, Lewinsohn DA; Tuberculosis Research Unit. (2011). CD8+ T cells provide an immunologic signature of tuberculosis in young children. Am J Respir Crit Care Med. 185(2):206-12.
Abstract
RATIONALE: The immunologic events surrounding primary Mycobacterium tuberculosis infection and development of tuberculosis remain controversial. Young children who develop tuberculosis do so quickly after first exposure, thus permitting study of immune response to primary infection and disease. We hypothesized that M. tuberculosis-specific CD8(+) T cells are generated in response to high bacillary loads occurring during tuberculosis. OBJECTIVES: To determine if M. tuberculosis-specific T cells are generated among healthy children exposed to M. tuberculosis and children with tuberculosis. METHODS: Enzyme-linked immunosorbent spot assays were used to measure IFN-γ production in response to M. tuberculosis-specific proteins ESAT-6/CFP-10 by peripheral blood mononuclear cells and CD8(+) T cells isolated from Ugandan children hospitalized with tuberculosis (n = 96) or healthy tuberculosis contacts (n = 62). MEASUREMENTS AND MAIN RESULTS: The proportion of positive CD8(+) T-cell assays and magnitude of CD8(+) T-cell responses were significantly greater among young (<5 yr) tuberculosis cases compared with young contacts (P = 0.02, Fisher exact test, P = 0.01, Wilcoxon rank-sum, respectively). M. tuberculosis-specific T-cell responses measured in peripheral blood mononuclear cells were equivalent between groups. CONCLUSIONS: Among young children, M. tuberculosis-specific CD8(+) T cells develop in response to high bacillary loads, as occurs during tuberculosis, and are unlikely to be found after M. tuberculosis exposure. T-cell responses measured in peripheral blood mononuclear cells are generated after M. tuberculosis exposure alone, and thus cannot distinguish exposure from disease. In young children, IFN-γ-producing M. tuberculosis-specific CD8(+) T cells provide an immunologic signature of primary M. tuberculosis infection resulting in disease.
Null D(1), Walz D. (2011). Nonhealing vulvar ulcer. Am Fam Physician. 84(3):311-2.
Abstract
PMID: 21842778 [PubMed - indexed for MEDLINE]
Sinha I(1), Null K, Wolter W, Suckow MA, King T, Pinto JT, Sinha R. (2011). Methylseleninic acid downregulates hypoxia-inducible factor-1α in invasive prostate cancer. Int J Cancer. 130(6):1430-9.
Abstract
Alternative strategies are needed to control growth of advanced and hormone refractory prostate cancer. In this regard, we investigated the efficacy of methylseleninic acid (MSeA), a penultimate precursor to the highly reactive selenium metabolite, methylselenol, to inhibit growth of invasive and hormone refractory rat (PAIII) and human (PC-3 and PC-3M) prostate cancer cells. Our results demonstrate that MSeA inhibits PAIII cell growth in vitro as well as reduces weights of tumors generated by PAIII cells treated ex vivo. A significant reduction in the number of metastatic lung foci by MSeA treatment was also noted in Lobund-Wistar rats. The PAIII cells along with PC-3, DU145 and PC-3M cells undergo apoptosis after MSeA treatments in both normoxia and hypoxia. Treatment of metastatic rat and human prostate cancer cell lines with MSeA decreased hypoxia-inducible factor-1α (HIF-1α) levels in a dose-dependent manner. Additionally, HIF-1α transcription activity both in normoxic and hypoxic conditions is reduced after MSeA treatment of prostate cancer cells. Furthermore, VEGF and GLUT1, downstream targets of HIF-1α, were also reduced in prostate cancer cells after MSeA treatment. Our study illustrates the efficacy of MSeA in controlling growth of hormone refractory prostate cancer by downregulating HIF-1α, which is possibly occurring through stabilization or increase in prolyl hydroxylase activity.
Pubmed 
Cole JB(1), Null DJ, De Vries A. (2011). Short communication: best prediction of 305-day lactation yields with regional and seasonal effects. J Dairy Sci. 94(3):1601-4.
Abstract
In the United States, lactation yields are calculated using best prediction (BP), a method in which test-day (TD) data are compared with breed- and parity-specific herd lactation curves that do not account for differences among regions of the country or seasons of calving. Complete data from 538,090 lactations of 348,123 Holstein cows with lactation lengths between 250 and 500 d, records made in a single herd, at least 5 reported TD, and twice-daily milking were extracted from the national dairy database and used to construct regional and seasonal lactation curves. Herds were assigned to 1 of 7 regions of the country, individual lactations were assigned to 3-mo seasons of calving, and lactation curves for milk, fat, and protein yields were estimated by parity group for regions, seasons, and seasons within regions. Multiplicative pre-adjustment factors (MF) also were computed. The resulting lactation curves and MF were tested on a validation data set of 891,806 lactations from 400,000 Holstein cows sampled at random from the national dairy database. Mature-equivalent milk, fat, and protein yields were calculated using the standard and adjusted curves and MF, and differences between 305-d mature-equivalent yields were tested for significance. Yields calculated using 50-d intervals from 50 to 250 d in milk (DIM) and using all TD to 500 DIM allowed comparisons of predictions for records in progress (RIP). Differences in mature-equivalent milk ranged from 0 to 51 kg and were slightly larger for first-parity than for later parity cows. Milk and components yields did not differ significantly in any case. Correlations of yields for 50-d intervals with those using all TD were similar across analyses. Yields for RIP were slightly more accurate when adjusted for regional and seasonal differences.
Pubmed 
Zwane AP(1), Zinman J, Van Dusen E, Pariente W, Null C, Miguel E, Kremer M, Karlan DS, Hornbeck R, Giné X, Duflo E, Devoto F, Crepon B, Banerjee A. (2011). Being surveyed can change later behavior and related parameter estimates. Proc Natl Acad Sci U S A. 108(5):1821-6.
Abstract
Does completing a household survey change the later behavior of those surveyed? In three field studies of health and two of microlending, we randomly assigned subjects to be surveyed about health and/or household finances and then measured subsequent use of a related product with data that does not rely on subjects' self-reports. In the three health experiments, we find that being surveyed increases use of water treatment products and take-up of medical insurance. Frequent surveys on reported diarrhea also led to biased estimates of the impact of improved source water quality. In two microlending studies, we do not find an effect of being surveyed on borrowing behavior. The results suggest that limited attention could play an important but context-dependent role in consumer choice, with the implication that researchers should reconsider whether, how, and how much to survey their subjects.
Gold MC(1), Cerri S, Smyk-Pearson S, Cansler ME, Vogt TM, Delepine J, Winata E, Swarbrick GM, Chua WJ, Yu YY, Lantz O, Cook MS, Null MD, Jacoby DB, Harriff MJ, Lewinsohn DA, Hansen TH, Lewinsohn DM. (2010). Human mucosal associated invariant T cells detect bacterially infected cells. PLoS Biol. 8(6):e1000407.
Abstract
Control of infection with Mycobacterium tuberculosis (Mtb) requires Th1-type immunity, of which CD8+ T cells play a unique role. High frequency Mtb-reactive CD8+ T cells are present in both Mtb-infected and uninfected humans. We show by limiting dilution analysis that nonclassically restricted CD8+ T cells are universally present, but predominate in Mtb-uninfected individuals. Interestingly, these Mtb-reactive cells expressed the Valpha7.2 T-cell receptor (TCR), were restricted by the nonclassical MHC (HLA-Ib) molecule MR1, and were activated in a transporter associated with antigen processing and presentation (TAP) independent manner. These properties are all characteristics of mucosal associated invariant T cells (MAIT), an "innate" T-cell population of previously unknown function. These MAIT cells also detect cells infected with other bacteria. Direct ex vivo analysis demonstrates that Mtb-reactive MAIT cells are decreased in peripheral blood mononuclear cells (PBMCs) from individuals with active tuberculosis, are enriched in human lung, and respond to Mtb-infected MR1-expressing lung epithelial cells. Overall, these findings suggest a generalized role for MAIT cells in the detection of bacterially infected cells, and potentially in the control of bacterial infection.
Null SE(1), Viers JH, Mount JF. (2010). Hydrologic response and watershed sensitivity to climate warming in California's Sierra Nevada. PLoS One. 5(4):e9932.
Abstract
This study focuses on the differential hydrologic response of individual watersheds to climate warming within the Sierra Nevada mountain region of California. We describe climate warming models for 15 west-slope Sierra Nevada watersheds in California under unimpaired conditions using WEAP21, a weekly one-dimensional rainfall-runoff model. Incremental climate warming alternatives increase air temperature uniformly by 2 degrees, 4 degrees, and 6 degrees C, but leave other climatic variables unchanged from observed values. Results are analyzed for changes in mean annual flow, peak runoff timing, and duration of low flow conditions to highlight which watersheds are most resilient to climate warming within a region, and how individual watersheds may be affected by changes to runoff quantity and timing. Results are compared with current water resources development and ecosystem services in each watershed to gain insight into how regional climate warming may affect water supply, hydropower generation, and montane ecosystems. Overall, watersheds in the northern Sierra Nevada are most vulnerable to decreased mean annual flow, southern-central watersheds are most susceptible to runoff timing changes, and the central portion of the range is most affected by longer periods with low flow conditions. Modeling results suggest the American and Mokelumne Rivers are most vulnerable to all three metrics, and the Kern River is the most resilient, in part from the high elevations of the watershed. Our research seeks to bridge information gaps between climate change modeling and regional management planning, helping to incorporate climate change into the development of regional adaptation strategies for Sierra Nevada watersheds.
Peterson S(1), Duncan DP, Null DB, Roth SL, Gill L. (2010). Positive changes in perceptions and selections of healthful foods by college students after a short-term point-of-selection intervention at a dining hall. J Am Coll Health. 58(5):425-31.
Abstract
OBJECTIVE: Determine the effects of a short-term, multi-faceted, point-of-selection intervention on college students' perceptions and selection of 10 targeted healthful foods in a university dining hall and changes in their self-reported overall eating behaviors. PARTICIPANTS: 104 college students, (age 18-23) completed pre-I and post-I surveys. METHODS: Pre-survey collected at dining hall in April 2007, followed by 3-week intervention then post-survey collected via email. Healthy choice indicators, large signs, table tents, flyers and colorful photographs with "benefit-based messages" promoted targeted foods. Response rate to both surveys was 38%. RESULTS: Significantly more participants reported that healthful choices were clearly identified in the dining hall after the intervention. Over 20% of participants reported becoming more aware of healthful food choices in the dining hall after the intervention. Significant increases in self-reported intake were reported for cottage cheese and low-fat salad dressing, with a trend toward increased consumption of fresh fruit. Seven of the 14 assessed eating behaviors had significant changes in the desired direction. Increased awareness of healthful foods was the top reason for self-reported changes in overall eating behaviors. CONCLUSION: Short-term, multi-faceted, point-of-selection marketing of healthful foods in university dining halls may be beneficial for improving college students' perceptions and selections of targeted healthful foods in the dining hall and may improve overall eating behaviors of college students.
Null EL(1), Lu Y. (2009). Rapid determination of enantiomeric ratio using fluorescent DNA or RNA aptamers. Analyst. 135(2):419-22.
Abstract
The natural chirality of DNA and RNA aptamers has been used to develop fluorescent agents to determine the enantiomeric ratio of adenosine and arginine, respectively. The quantification is based on structure-switching DNA or RNA aptamers labeled with fluorophore and quencher, allowing chiral detection down to 0.1 : 99.9 (L : D) for arginine after calibration. Such a method provides a general platform for simple, low-cost and high throughput detection and quantification of chirality of a broad range of molecules.
Desai D(1), Sinha I, Null K, Wolter W, Suckow MA, King T, Amin S, Sinha R. (2010). Synthesis and antitumor properties of selenocoxib-1 against rat prostate adenocarcinoma cells. Int J Cancer. 127(1):230-8.
Abstract
Hormone refractory prostate cancer poses a huge problem and standard of care chemotherapy has not been very successful. We used a novel strategy to combine properties of 2 well-studied class of compounds (selenium and COX-2 inhibitor) and examined the resulting effectiveness against prostate cancer. Bearing in mind that sulfonamide moiety and pyrazole ring is important for the proapoptotic activity of Celecoxib, we synthesized a selenium derivative, Selenocoxib-1, by modifying Celecoxib at position 3 of the pyrazole ring. The PAIII cells derived from a metastatic prostate tumor that arose spontaneously in a Lobund-Wistar (LW) rat were used to examine the efficacy of Selenocoxib-1 in vitro. In addition, human metastatic prostate cancer cells, PC-3M, were tested for antitumor effect of Selenocoxib-1 in vitro. The IC(50) in PAIII and PC-3M cells for Selenocoxib-1 was about 5 microM, while for Celecoxib it was more than 20 microM. Selenocoxib-1 induced apoptosis in a dose-dependent manner in the PAIII cells. COX-2 expression in PAIII cells was downregulated by Celecoxib and Selenocoxib-1 at 20 and 5 microM, respectively; the COX-2 activity was, however, not affected by Selenocoxib-1. Following treatment with Selenocoxib-1, PAIII cells resulted in dose-dependent decrease in HIF-1alpha, p-AKT and Bcl-2 levels. A reduction in weights was observed in subcutaneous tumors produced by PAIII cells pretreated with Selenocoxib-1 as compared to Celecoxib in LW rats. Further, following 1 week Selenocoxib-1 treatment of PAIII tumors resulted in significant reduction of tumor weights. This study demonstrates that Selenocoxib-1 is more effective against prostate cancer than Celecoxib.
Kochunov P(1), Coyle T, Lancaster J, Robin DA, Hardies J, Kochunov V, Bartzokis G, Stanley J, Royall D, Schlosser AE, Null M, Fox PT. (2009). Processing speed is correlated with cerebral health markers in the frontal lobes as quantified by neuroimaging. Neuroimage. 49(2):1190-9.
Abstract
We explored relationships between decline in cognitive processing speed (CPS) and change in frontal lobe MRI/MRS-based indices of cerebral integrity in 38 healthy adults (age 57-90 years). CPS was assessed using a battery of four timed neuropsychological tests: Grooved Pegboard, Coding, Symbol Digit Modalities Test and Category Fluency (Fruits and Furniture). The neuropsychological tests were factor analyzed to extract two components of CPS: psychomotor (PM) and psychophysical (PP). MRI-based indices of cerebral integrity included three cortical measurements per hemisphere (GM thickness, intergyral and sulcal spans) and two subcortical indices (fractional anisotropy (FA), measured using track-based spatial statistics (TBSS), and the volume of hyperintense WM (HWM)). MRS indices included levels of choline-containing compounds (GPC+PC), phosphocreatine plus creatine (PCr+Cr), and N-acetylaspartate (NAA), measured bilaterally in the frontal WM bundles. A substantial fraction of the variance in the PM-CPS (58%) was attributed to atrophic changes in frontal WM, observed as increases in sulcal span, declines in FA values and reductions in concentrations of NAA and choline-containing compounds. A smaller proportion (20%) of variance in the PP-CPS could be explained by bilateral increases in frontal sulcal span and increases in HWM volumes.
Burch PT(1), Cowley CG, Holubkov R, Null D, Lambert LM, Kouretas PC, Hawkins JA. (2009). Coarctation repair in neonates and young infants: is small size or low weight still a risk factor? J Thorac Cardiovasc Surg. 138(3):547-52.
Abstract
OBJECTIVE: Previous reports of neonatal coarctation repair demonstrate a high rate of recurrent arch obstruction in small neonates. This study assesses the effect of patient size on reintervention and survival in neonates and infants undergoing repair of simple aortic coarctation. METHODS: From 1996 to 2006, 167 neonates and infants younger than 90 days with simple coarctation underwent repair. Median patient age was 16 days (range, 1-85 days). Median patient weight was 3.4 kg (range, 0.8-6.0 kg), with 29 patients weighing less than 2.5 kg. All 167 patients included in the study underwent repair through a left thoracotomy. RESULTS: There was 1 early death (1/167, 0.6%). Median follow-up of 4.8 years (range, 0-11.8 years) demonstrated 2 late deaths unrelated to recurrent coarctation. Eighteen patients underwent intervention for recurrent arch obstruction a median of 0.48 years postoperatively (range, 0.14-9.8 years). All were treated with balloon angioplasty and have required no additional intervention. Actuarial freedom from reintervention was 90% at 1 year and 89% at 5 years for infants weighing more than 2.5 kg and 89% at 1 year and 86% at 5 years (P = .31) for infants weighing less than 2.5 kg. There was no difference between survival or reintervention for neonates 30 days of age or younger compared with infants 31 to 90 days of age. Use of polypropylene sutures and female sex did correlate with increased reintervention. CONCLUSIONS: Low weight does not affect survival or reintervention rates after coarctation repair in neonates and infants less than 3 months of age. Balloon angioplasty is an effective treatment for recurrent obstruction after coarctation repair in infancy. In the current era, timing of the operation should be based on clinical status.
Null R(1), Wei J. ( 48. Int J Electron Healthc. 2009;5(1):48-67. ). Value increasing business model for e-hospital. 48. Int J Electron Healthc. 2009;5(1):48-67.
Abstract
This paper developed a business value increasing model for electronic hospital (e-hospital) based on electronic value chain analysis. From this model, 58 hospital electronic business (e-business) solutions were developed. Additionally, this paper investigated the adoption patterns of these 58 e-business solutions within six US leading hospitals. The findings show that only 36 of 58 or 62% of the e-business solutions are fully or partially implemented within the six hospitals. Ultimately, the research results will be beneficial to managers and executives for accelerating e-business adoptions for e-hospital.
Cole JB(1), Null DJ. (2009). Genetic evaluation of lactation persistency for five breeds of dairy cattle. J Dairy Sci. 92(5):2248-58.
Abstract
Cows with high lactation persistency tend to produce less milk than expected at the beginning of lactation and more than expected at the end. Best prediction of lactation persistency is calculated as a function of trait-specific standard lactation curves and linear regressions of test-day deviations on days in milk. Because regression coefficients are deviations from a tipping point selected to make yield and lactation persistency phenotypically uncorrelated it should be possible to use 305-d actual yield and lactation persistency to predict yield for lactations with later endpoints. The objectives of this study were to calculate (co)variance components and breeding values for best predictions of lactation persistency of milk (PM), fat (PF), protein (PP), and somatic cell score (PSCS) in breeds other than Holstein, and to demonstrate the calculation of prediction equations for 400-d actual milk yield. Data included lactations from Ayrshire, Brown Swiss, Guernsey (GU), Jersey (JE), and Milking Shorthorn (MS) cows calving since 1997. The number of sires evaluated ranged from 86 (MS) to 3,192 (JE), and mean sire estimated breeding value for PM ranged from 0.001 (Ayrshire) to 0.10 (Brown Swiss); mean estimated breeding value for PSCS ranged from -0.01 (MS) to -0.043 (JE). Heritabilities were generally highest for PM (0.09 to 0.15) and lowest for PSCS (0.03 to 0.06), with PF and PP having intermediate values (0.07 to 0.13). Repeatabilities varied considerably between breeds, ranging from 0.08 (PSCS in GU, JE, and MS) to 0.28 (PM in GU). Genetic correlations of PM, PF, and PP with PSCS were moderate and favorable (negative), indicating that increasing lactation persistency of yield traits is associated with decreases in lactation persistency of SCS, as expected. Genetic correlations among yield and lactation persistency were low to moderate and ranged from -0.55 (PP in GU) to 0.40 (PP in MS). Prediction equations for 400-d milk yield were calculated for each breed by regression of both 305-d yield and 305-d yield and lactation persistency on 400-d yield. Goodness-of-fit was very good for both models, but the addition of lactation persistency to the model significantly improved fit in all cases. Routine genetic evaluations for lactation persistency, as well as the development of prediction equations for several lactation end-points, may provide producers with tools to better manage their herds.
Cole JB(1), Null DJ, Vanraden PM. (2009). Best prediction of yields for long lactations. J Dairy Sci. 92(4):1796-810.
Abstract
Lactation records of any reasonable length now can be processed with the selection index method known as best prediction (BP). Previous prediction programs were limited to the 305-d standard used since 1935. Best prediction was implemented in 1998 to calculate lactation records in USDA genetic evaluations, replacing the test interval method used since 1969 to calculate lactation records. Best prediction is more complex but also more accurate, particularly when testing is less frequent. Programs were reorganized to output better graphics, give users simpler access to options, and provide additional output, such as BP of daily yields. Test-day data for 6 breeds were extracted from the national dairy database, and lactation lengths were required to be > or =500 d (Ayrshire, Milking Shorthorn) or > or =800 d (all others). Average yield and SD at any day in milk (DIM) were estimated by fitting 3-parameter Wood's curves (milk, fat, protein) and 4-parameter exponential functions (somatic cell score) to means and SD of 15- (< or =300 DIM) and 30-d (>300 DIM) intervals. Correlations among TD yields were estimated using an autoregressive matrix to account for biological changes and an identity matrix to model daily measurement error. Autoregressive parameters (r) were estimated separately for first (r = 0.998) and later parities (r = 0.995). These r values were slightly larger than previous estimates due to the inclusion of the identity matrix. Correlations between traits were modified so that correlations between somatic cell score and other traits may be nonzero. The new lactation curves and correlation functions were validated by extracting TD data from the national database, estimating 305-d yields using the original and new programs, and correlating those results. Daily BP of yield were validated using daily milk weights from on-farm meters in university research herds. Correlations ranged from 0.900 to 0.988 for 305-d milk yield. High correlations ranged from 0.844 to 0.988 for daily yields, although correlations were as low as 0.015 on d 1 of lactation, which may be due to calving-related disorders that are not accounted for by BP. Correlations between 305-d yield calculated using 50-d intervals from 50 to 250 DIM and 305-yield calculated using all TD to 500 DIM increased as TD data accumulated. Many cows can profitably produce for >305 DIM, and the revised program provides a flexible tool to model these records.
Dohner ML(1), Wiedmeier SE, Stoddard RA, Null D Jr, Lambert DK, Burnett J, Baer VL, Hunt JC, Henry E, Christensen RD. (2009). Very high users of platelet transfusions in the neonatal intensive care unit. Transfusion. 49(5):869-72.
Abstract
BACKGROUND: In neonatal intensive care unit (NICU) practice, a small percentage of the patients receive a large proportion of the platelet (PLT) transfusions administered. This study sought to better define this very-high-user group. To accomplish this, records of all NICU patients in a multihospital health care system who, during a recent 5(1/2)-year period, received 20 or more PLT transfusions were examined. STUDY DESIGN AND METHODS: Electronic medical record repositories of Intermountain Healthcare neonates with dates of birth from January 1, 2002, through June 30, 2007, who received 20 or more PLT transfusions were identified. The causes of the thrombocytopenia were sought, whether each transfusion given was a treatment for bleeding versus prophylaxis was determined, whether each transfusion was compliant with our transfusion guidelines was judged, and the outcomes were tabulated. RESULTS: During this period, 45 patients received 20 or more PLT transfusions (median, 29; range, 20-79). Medical conditions could be categorized into six diagnoses: 1) extracorporeal membrane oxygenation (ECMO) for congenital diaphragmatic hernia (CDH; n = 13), 2) fungal sepsis (n = 8), 3) ECMO for reasons other than CDH (n = 8), 4) necrotizing enterocolitis (n = 7), 5) bacterial sepsis (n = 7), and 6) congenital hyporegenerative thrombocytopenia (n = 2). Nineteen percent of the transfusions were ordered for oozing, bruising, or bleeding and 81 percent for prophylaxis. Thirty-six percent of transfusions were given in violation of our transfusion guidelines. Forty-nine percent of the high users died, but no deaths were due to hemorrhage. All survivors developed chronic lung disease, and all survivors weighing less than 1250 g at birth developed retinopathy of prematurity. CONCLUSIONS: Almost all patients that received 20 or more PLT transfusions had an acquired, consumptive thrombocytopenia. All could have received fewer transfusions had the guidelines already in place been observed. Eighty-one percent fewer PLT transfusions would have been administered had the paradigm been transfusing only if oozing, bruising, or bleeding was present.
Whitty LA(1), Murray JD, Null WE, Elwood ET, Jones GE. (2009). An arteriovenous malformation of the external ear in the pediatric population: A case report and review of the literature. 52. Can J Plast Surg. 2009 Winter;17(4):e45-7.
Abstract
The literature regarding arteriovenous malformations of the external ear is sparse. A case of a patient clinically diagnosed with an arteriovenous malformation of the external ear that was managed empirically with surgical excision, without recurrence, is presented. The pathogenesis, clinical presentation, radiological work up and management options regarding arteriovenous malformations are reviewed.
Singh U(1), Null K, Sinha R. (2008). In vitro growth inhibition of mouse mammary epithelial tumor cells by methylseleninic acid: involvement of protein kinases. Mol Nutr Food Res. 52(11):1281-8.
Abstract
Methylseleninic acid (MSeA) is a synthetic organoselenium form known to be effective against mammary carcinogenesis in vivo. Using the synchronized mouse mammary epithelial tumor cell (TM6) model, we have previously shown that 5 microM MSeA significantly inhibits cell growth and induces a reversible growth arrest in the G1 phase. In the present study, we examined the effects of MSeA on Rb, cyclin dependent kinase 2 (cdk2), cdk4, cyclin E and cyclin D1. Growth arrest of cells was accompanied by a reduction in total cdk2 kinase and cyclin E-associated cdk2 kinase activities. The p27 levels associated with cdk2 were elevated during the cell cycle. In addition, growth inhibition correlated with a relative increase in the hypophosphorylated form of Rb in MSeA-treated cells and Egr1 was elevated in MSeA-treated cells. The Kinetworks Protein Kinase Screen (KPKS 1.0) was used to examine 75 protein kinases. MSeA treatment resulted in differential expression of several protein-serine/threonine kinases, protein-tyrosine kinases and protein-threonine/tyrosine kinases. Some of these kinases are being reported for the first time as being altered by MSeA. The outcome of these experiments will be of significance since these kinases are known to be involved in survival and/or apoptotic pathways of tumor cells.
Null B(1), Liu CW, Hedehus M, Conolly S, Davis RW. (2008). High-resolution, in vivo magnetic resonance imaging of Drosophila at 18.8 Tesla. PLoS One. 3(7):e2817.
Abstract
High resolution MRI of live Drosophila was performed at 18.8 Tesla, with a field of view less than 5 mm, and administration of manganese or gadolinium-based contrast agents. This study demonstrates the feasibility of MR methods for imaging the fruit fly Drosophila with an NMR spectrometer, at a resolution relevant for undertaking future studies of the Drosophila brain and other organs. The fruit fly has long been a principal model organism for elucidating biology and disease, but without capabilities like those of MRI. This feasibility marks progress toward the development of new in vivo research approaches in Drosophila without the requirement for light transparency or destructive assays.
Reyburn B(1), Li M, Metcalfe DB, Kroll NJ, Alvord J, Wint A, Dahl MJ, Sun J, Dong L, Wang ZM, Callaway C, McKnight RA, Moyer-Mileur L, Yoder BA, Null DM, Lane RH, Albertine KH. (2008). Nasal ventilation alters mesenchymal cell turnover and improves alveolarization in preterm lambs. Am J Respir Crit Care Med. 178(4):407-18.
Abstract
RATIONALE: Bronchopulmonary dysplasia (BPD) is a frequent cause of morbidity in preterm infants that is characterized by prolonged need for ventilatory support in an intensive care environment. BPD is characterized histopathologically by persistently thick, cellular distal airspace walls. In normally developing lungs, by comparison, remodeling of the immature parenchymal architecture is characterized by thinning of the future alveolar walls, a process predicated on cell loss through apoptosis. OBJECTIVES: We hypothesized that minimizing lung injury, using high-frequency nasal ventilation to provide positive distending pressure with minimal assisted tidal volume displacement, would increase apoptosis and decrease proliferation among mesenchymal cells in the distal airspace walls compared with a conventional mode of support (intermittent mandatory ventilation). METHODS: Accordingly, we compared two groups of preterm lambs: one group managed by high-frequency nasal ventilation and a second group managed by intermittent mandatory ventilation. Each group was maintained for 3 days. MEASUREMENTS AND MAIN RESULTS: Oxygenation and ventilation targets were sustained with lower airway pressures and less supplemental oxygen in the high-frequency nasal ventilation group, in which alveolarization progressed. Thinning of the distal airspace walls was accompanied by more apoptosis, and less proliferation, among mesenchymal cells of the high-frequency nasal ventilation group, based on morphometric, protein abundance, and mRNA expression indices of apoptosis and proliferation. CONCLUSIONS: Our study shows that high-frequency nasal ventilation preserves the balance between mesenchymal cell apoptosis and proliferation in the distal airspace walls, such that alveolarization progresses.
Lien WH(1), Klezovitch O, Null M, Vasioukhin V. (2008). alphaE-catenin is not a significant regulator of beta-catenin signaling in the developing mammalian brain. J Cell Sci. 121(Pt 9):1357-62.
Abstract
beta-Catenin is a crucial mediator of the canonical Wnt-signaling pathway. alpha-catenin is a major beta-catenin-binding protein, and overexpressed alpha-catenin can negatively regulate beta-catenin activity. Thus, alpha-catenin may be an important modulator of the Wnt pathway. We show here that endogenous alpha-catenin has little impact on the transcriptional activity of beta-catenin in developing mammalian organisms. We analyzed beta-catenin signaling in mice with conditional deletion of alphaE-catenin (Ctnna1) in the developing central nervous system. This mutation results in brain hyperplasia and we investigated whether activation of beta-catenin signaling may be at least partially responsible for this phenotype. To reveal potential quantitative or spatial changes in beta-catenin signaling, we used mice carrying a beta-catenin-signaling reporter transgene. In addition, we analyzed the expression of known endogenous targets of the beta-catenin pathway and the amount and localization of beta-catenin in mutant progenitor cells. We found that although loss of alphaE-catenin resulted in disruption of intercellular adhesion and hyperplasia in the developing brain, beta-catenin signaling was not altered. We conclude that endogenous alphaE-catenin has no significant impact on beta-catenin transcriptional activities in the developing mammalian brain.
Klezovitch O(1), Risk M, Coleman I, Lucas JM, Null M, True LD, Nelson PS, Vasioukhin V. (2008). A causal role for ERG in neoplastic transformation of prostate epithelium. Proc Natl Acad Sci U S A. 105(6):2105-10.
Abstract
A significant proportion of human prostate cancers carry a chromosomal rearrangement resulting in the overexpression of the ETS transcription factor, ERG; however, the functional significance of this event is poorly understood. We report here that up-regulation of ERG transcript is sufficient for the initiation of prostate neoplasia. In agreement with measurements of ERG transcripts, we found that ERG protein is expressed in neoplastic human prostate epithelium. Overexpression of ERG in prostate cell lines increased cell invasion. Moreover, targeted expression of this transcript in vivo in luminal prostate epithelial cells of transgenic mice results in initiation of prostate neoplasia observed as the development of focal precancerous prostatic intraepithelial neoplasia (PIN). Similar to human cancers, luminal epithelial cells in these PIN lesions displace diminishing in numbers basal epithelial cells and establish direct contact with the stromal cell compartment. Loss of basal cells is considered to be one of the critical hallmarks of human prostate cancer; however, the mechanisms responsible for this event were unknown. We propose that up-regulation of ERG in human prostate cancer activates cell invasion programs that subsequently displace basal cells by neoplastic epithelium. Our data demonstrate that ERG plays an important causal role in the transformation of prostate epithelium and should be considered as a target for prevention or early therapeutic intervention.
Lewinsohn DA(1), Winata E, Swarbrick GM, Tanner KE, Cook MS, Null MD, Cansler ME, Sette A, Sidney J, Lewinsohn DM. (2007). Immunodominant tuberculosis CD8 antigens preferentially restricted by HLA-B. PLoS Pathog. 3(9):1240-9.
Abstract
CD8(+) T cells are essential for host defense to intracellular bacterial pathogens such as Mycobacterium tuberculosis (Mtb), Salmonella species, and Listeria monocytogenes, yet the repertoire and dominance pattern of human CD8 antigens for these pathogens remains poorly characterized. Tuberculosis (TB), the disease caused by Mtb infection, remains one of the leading causes of infectious morbidity and mortality worldwide and is the most frequent opportunistic infection in individuals with HIV/AIDS. Therefore, we undertook this study to define immunodominant CD8 Mtb antigens. First, using IFN-gamma ELISPOT and synthetic peptide arrays as a source of antigen, we measured ex vivo frequencies of CD8(+) T cells recognizing known immunodominant CD4(+) T cell antigens in persons with latent tuberculosis infection. In addition, limiting dilution was used to generate panels of Mtb-specific T cell clones. Using the peptide arrays, we identified the antigenic specificity of the majority of T cell clones, defining several new epitopes. In all cases, peptide representing the minimal epitope bound to the major histocompatibility complex (MHC)-restricting allele with high affinity, and in all but one case the restricting allele was an HLA-B allele. Furthermore, individuals from whom the T cell clone was isolated harbored high ex vivo frequency CD8(+) T cell responses specific for the epitope, and in individuals tested, the epitope represented the single immunodominant response within the CD8 antigen. We conclude that Mtb-specific CD8(+) T cells are found in high frequency in infected individuals and are restricted predominantly by HLA-B alleles, and that synthetic peptide arrays can be used to define epitope specificities without prior bias as to MHC binding affinity. These findings provide an improved understanding of immunodominance in humans and may contribute to a development of an effective TB vaccine and improved immunodiagnostics.
Honey G(1), Bleak T, Karp T, MacRitchie A, Null D Jr. (2007). Use of the Duotron transporter high frequency ventilator during neonatal transport. Neonatal Netw. 26(3):167-74.
Abstract
In the past, transport of neonates with severe respiratory failure was hampered by the lack of an appropriate transport ventilator capable of providing high frequency ventilation (HFV). This article reports on the experiences of the Intermountain Health Care Life Flight Program in selecting a high frequency ventilator and preparing the transport team members for its use. Once the use of the Duotron ventilator was initiated, pre- and posttransport data were collected for the first 134 neonates requiring HFV on transport. Analysis of the data determined that 96 percent of the infants were successftslly transported using the Duotron ventilator. Inspired oxygen requirements staved the same or improved in the majority of intubated patients for whom comparison data xvere available. Ventilation and acid-base balance improved. Although HFV has been a common therapy in neonatal care for some time, its adoption for use during transport required modification and considerable education for transport team members.
Daniels NA(1), Gouveia S, Null D, Gildengorin GL, Winston CA. (2006). Acceptance of pneumococcal vaccine under standing orders by race and ethnicity. J Natl Med Assoc. 98(7):1089-94.
Abstract
PURPOSE: To assess whether and how pneumococcal vaccine acceptance occurs after nurse recommendation varies by race/ethnicity. METHODS: We prospectively evaluated nurses' standing orders to assess and vaccinate high-risk patients in a general medicine practice. RESULTS: Of 370 adult patients surveyed (60% nonwhite), 78 (21%) declined vaccination following nurse recommendation, and 43 (12%) persisted in declining after physician consultation. Three-hundred-twenty-seven (88%) patients accepted vaccination: 292 (79%) accepted following nurse recommendation and 35 (9%) following physician consultation. African Americans (19%) were significantly more likely to decline compared with whites (8%) and Asians (5%) (P= 0.01). Reasons for refusal included believing vaccination was unnecessary (32%), fearing shots in general (21%), fearing vaccine-induced illness (26%) and wanting more informotion regarding the vaccine (9%). CONCLUSION: Standing orders, physicians' firm recommendations and addressing patients' vaccine-related concerns may reduce racial/ethnic disparities in vaccination.
Ballard RA(1), Truog WE, Cnaan A, Martin RJ, Ballard PL, Merrill JD, Walsh MC, Durand DJ, Mayock DE, Eichenwald EC, Null DR, Hudak ML, Puri AR, Golombek SG, Courtney SE, Stewart DL, Welty SE, Phibbs RH, Hibbs AM, Luan X, Wadlinger SR, Asselin JM, Coburn CE; NO CLD Study Group. (2006). Inhaled nitric oxide in preterm infants undergoing mechanical ventilation. N Engl J Med. 355(4):343-53.
Abstract
Erratum in N Engl J Med. 2007 Oct 4;357(14):1444-5.
BACKGROUND: Bronchopulmonary dysplasia in premature infants is associated with prolonged hospitalization, as well as abnormal pulmonary and neurodevelopmental outcome. In animal models, inhaled nitric oxide improves both gas exchange and lung structural development, but the use of this therapy in infants at risk for bronchopulmonary dysplasia is controversial. METHODS: We conducted a randomized, stratified, double-blind, placebo-controlled trial of inhaled nitric oxide at 21 centers involving infants with a birth weight of 1250 g or less who required ventilatory support between 7 and 21 days of age. Treated infants received decreasing concentrations of nitric oxide, beginning at 20 ppm, for a minimum of 24 days. The primary outcome was survival without bronchopulmonary dysplasia at 36 weeks of postmenstrual age. RESULTS: Among 294 infants receiving nitric oxide and 288 receiving placebo birth weight (766 g and 759 g, respectively), gestational age (26 weeks in both groups), and other characteristics were similar. The rate of survival without bronchopulmonary dysplasia at 36 weeks of postmenstrual age was 43.9 percent in the group receiving nitric oxide and 36.8 percent in the placebo group (P=0.042). The infants who received inhaled nitric oxide were discharged sooner (P=0.04) and received supplemental oxygen therapy for a shorter time (P=0.006). There were no short-term safety concerns. CONCLUSIONS: Inhaled nitric oxide therapy improves the pulmonary outcome for premature infants who are at risk for bronchopulmonary dysplasia when it is started between 7 and 21 days of age and has no apparent short-term adverse effects. (ClinicalTrials.gov number, NCT00000548 [ClinicalTrials.gov] .).
Tay SH(1), Nordli DR Jr, Bonilla E, Null E, Monaco S, Hirano M, DiMauro S. (2006). Aortic rupture in mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes. Arch Neurol. 63(2):281-3.
Abstract
BACKGROUND: Microangiopathy has been well described in the brain and muscle of patients with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). OBJECTIVE: To describe a patient with the common A3243G/MELAS point mutation who had aortic rupture and whose mother also died of large vessel rupture. DESIGN: Case report. SETTING: Collaboration between a primary care hospital and 2 academic tertiary care hospitals. RESULTS: Histologically, there was marked disarray of the smooth muscle architecture of the aorta, and immunohistochemical staining with antibodies against the mitochondrial DNA-encoded cytochrome-C oxidase I subunit showed uniformly decreased immunostaining of the endothelial and smooth muscle cells of the aorta and vasa vasorum. Polymerase chain reaction and restriction fragment length polymorphism analysis showed that the mutation load was 40.5% in blood but 85.3% in the blood vessels. CONCLUSIONS: The severe vasculopathy in this patient is probably directly related to the high mutation load in the blood vessels. Although aortic rupture is an unusual manifestation of MELAS, it is an important potential complication in patients undergoing minor surgical procedures.
Null D Jr(1), Pollara B, Dennehy PH, Steichen J, Sánchez PJ, Givner LB, Carlin D, Landry B, Top FH Jr, Connor E. (2005). Safety and immunogenicity of palivizumab (Synagis) administered for two seasons. Pediatr Infect Dis J. 24(11):1021-3.
Abstract
To evaluate the safety and immunogenicity of palivizumab, 55 children who received palivizumab in the IMpact-RSV trial received 5 monthly doses of 15 mg/kg palivizumab (Synagis) during the subsequent year. The single child with an antipalivizumab titer of >1/40 had no associated serious adverse events and had expected serum palivizumab trough concentrations. Second year palivizumab prophylaxis was safe and well-tolerated.
McLaren C(1), Null J, Quinn J. (2005). Heat stress from enclosed vehicles: moderate ambient temperatures cause significant temperature rise in enclosed vehicles. Pediatrics. 116(1):e109-12.
Abstract
OBJECTIVE: Each year, children die from heat stroke after being left unattended in motor vehicles. In 2003, the total was 42, up from a national average of 29 for the past 5 years. Previous studies found that on days when ambient temperatures exceeded 86 degrees F, the internal temperatures of the vehicle quickly reached 134 to 154 degrees F. We were interested to know whether similarly high temperatures occurred on clear sunny days with more moderate temperatures. The objective of this study was to evaluate the degree of temperature rise and rate of rise in similar and lower ambient temperatures. In addition, we evaluated the effect of having windows "cracked" open. METHODS: In this observational study, temperature rise was measured continuously over a 60-minute period in a dark sedan on 16 different clear sunny days with ambient temperatures ranging from 72 to 96 degrees F. On 2 of these days, additional measurements were made with the windows opened 1.5 inches. Analysis of variance was used to compare how quickly the internal vehicle temperature rose and to compare temperature rise when windows were cracked open 1.5 inches. RESULTS: Regardless of the outside ambient temperature, the rate of temperature rise inside the vehicle was not significantly different. The average mean increase was 3.2 degrees F per 5-minute interval, with 80% of the temperature rise occurring during the first 30 minutes. The final temperature of the vehicle depended on the starting ambient temperature, but even at the coolest ambient temperature, internal temperatures reached 117 degrees F. On average, there was an approximately 40 degrees F increase in internal temperature for ambient temperatures spanning 72 to 96 degrees F. Cracking windows open did not decrease the rate of temperature rise in the vehicle (closed: 3.4 degrees F per 5 minutes; opened: 3.1 degrees F per 5 minutes or the final maximum internal temperature. CONCLUSIONS: Even at relatively cool ambient temperatures, the temperature rise in vehicles is significant on clear, sunny days and puts infants at risk for hyperthermia. Vehicles heat up rapidly, with the majority of the temperature rise occurring within the first 15 to 30 minutes. Leaving the windows opened slightly does not significantly slow the heating process or decrease the maximum temperature attained. Increased public awareness and parental education of heat rise in motor vehicles may reduce the incidence of hyperthermia death and improve child passenger safety.
Hougland KT(1), Hanna AM, Meyers R, Null D. (2005). Increasing prevalence of gastroschisis in Utah. J Pediatr Surg. 40(3):535-40.
Abstract
BACKGROUND: Recent studies provide conflicting information about gastroschisis prevalence trends. The authors proposed that prevalence of gastroschisis in live births has increased in Utah and that characteristics of these infants would provide clinically useful information about treatment and outcomes. METHODS: Primary Children's Medical Center (PCMC) is the sole pediatric surgical referral hospital for Utah. The authors used both pediatric surgical and neonatal databases to identify gastroschisis cases at PCMC from 1971 through 2002. Only infants whose mothers had a primary residence in Utah were included. Individual charts were reviewed for infant characteristics for cases from 1998 through 2002. Utah Vital Statistics Reports were used to determine live birth rates and general infant and maternal characteristics. RESULTS: Gastroschisis prevalence increased from 0.36 to 3.92 cases per 10,000 live births over 31 years (P < .001). Young maternal age, primigravida status, and tobacco use were associated risk factors. Using the time required to achieve full enteric feedings at targeted volume and caloric density as a measurement of outcome, we found no association between delivery mode or surgical closure type (primary or secondary) and time to full feedings. Higher birth weight was associated with decreased time to full feedings (P = .03). CONCLUSIONS: Gastroschisis prevalence has increased 10-fold over the past 3 decades in Utah.
de Felice C(1), Latini G, Parrini S, Bianciardi G, Toti P, Kopotic RJ, Null DM. (2004). Oral mucosal microvascular abnormalities: an early marker of bronchopulmonary dysplasia. Pediatr Res. 56(6):927-31.
Abstract
An abnormal pulmonary vasculature has been reported as an important component of bronchopulmonary dysplasia (BPD). We tested the hypothesis of an early abnormal vascular network pattern in infants with BPD. Fifteen infants with BPD (nine boys and six girls; gestational age 27.5 +/- 2.0 wk; birth weight 850 +/- 125 g) and 15 sex- and gestational age-matched infants (nine boys and six girls; gestational age 27.6 +/- 2.6 wk; birth weight 865 +/- 135 g) were examined on postnatal days 1 and 28. BPD infants showed a significantly higher prevalence of histologic chorioamnionitis (p = 0.009), as well as higher intubation duration (p = 0.0004), oxygen supplementation (p < 0.0001), and initial illness severity (p = 0.0002) than the BPD-negative population. The lower gingival and vestibular oral mucosa was chosen as the study area. The blood vessel area was determined, and the oral vascular networks were characterized by analyzing their complexity (D, at two scales: D 1-46, D 1-15), tortuosity (Dmin), and randomness (L-Z) of the vascular loops. Infants with BPD showed a significantly lower blood vessel area as well as a higher vascular network complexity (D 1-46, D 1-15, and L-Z) than control subjects (p < 0.0001). Our findings provide a new early clinical sign in BPD and stress the importance of an early disorder in the oral mucosal vascularization process in the disease pathogenesis.
Arbeitman MN(1), Fleming AA, Siegal ML, Null BH, Baker BS. (2004). A genomic analysis of Drosophila somatic sexual differentiation and its regulation. Development. 131(9):2007-21.
Abstract
In virtually all animals, males and females are morphologically, physiologically and behaviorally distinct. Using cDNA microarrays representing one-third of Drosophila genes to identify genes expressed sex-differentially in somatic tissues, we performed an expression analysis on adult males and females that: (1) were wild type; (2) lacked a germline; or (3) were mutant for sex-determination regulatory genes. Statistical analysis identified 63 genes sex-differentially expressed in the soma, 20 of which have been confirmed by RNA blots thus far. In situ hybridization experiments with 11 of these genes showed they were sex-differentially expressed only in internal genital organs. The nature of the products these genes encode provides insight into the molecular physiology of these reproductive tissues. Analysis of the regulation of these genes revealed that their adult expression patterns are specified by the sex hierarchy during development, and that doublesex probably functions in diverse ways to set their activities.
Null AP(1), Benson LM, Muddiman DC. ( 68. Rapid Commun Mass Spectrom. 2003;17(24):2699-706. ). Enzymatic strategies for the characterization of nucleic acids by electrospray ionization mass spectrometry. 68. Rapid Commun Mass Spectrom. 2003;17(24):2699-706.
Abstract
Electrospray ionization mass spectrometry (ESI-MS) is a powerful technique used for the identification and characterization of DNA polymorphisms. Continual improvement in instrument design assures high mass measurement accuracy, sensitivity, and resolving power. This work describes an eclectic array of enzymatic strategies we have invoked in order to detect single-nucleotide polymorphisms by ESI-MS, although other applications may be envisioned. One strategy combines the use of two enzymes, exonuclease III and lambda exonuclease, to provide a ladder of single-stranded DNA fragments for straightforward sequence identification by mass spectrometry. A second strategy combines restriction enzymes to screen for polymorphisms present within specific amplicons. Finally, we describe the use of stable-isotope-labeled nucleotides for the determination of length and base composition of a PCR product.
Pubmed 
Gerbert B(1), Berg-Smith S, Mancuso M, Caspers N, McPhee S, Null D, Wofsy J. (2003). Using innovative video doctor technology in primary care to deliver brief smoking and alcohol intervention. Health Promot Pract. 4(3):249-61.
Abstract
Given physicians' increased responsibilities and time constraints, it is increasingly difficult for primary care physicians to assume a major role in delivering smoking and alcohol assessment and intervention. The authors developed an innovative use of computer technology in the form of a "video doctor" to support physicians with this. In this article, two brief interventions, delivered by an interactive, multimedia video doctor, that reduce primary care patients' smoking and alcohol use are detailed: (a) a patient-centered advice message and (b) a brief motivational intervention. The authors are testing the use of the video doctor to deliver these interventions in a randomized, controlled study, Project Choice. A pilot study testing the feasibility and acceptability of the video doctor suggests it was well received and accepted by patients (n = 52) and potentially provides an innovative, cost-effective, and practical way to support providers' efforts to reduce smoking and alcohol use in primary care populations.
Munley PH(1), Null U. (2003). An approach for practice administration and interpretation of the MMPI-2. Psychol Rep. 93(1):69-72.
Abstract
This paper discusses one teaching approach for practice administration and interpretation of the MMPI-2 in a graduate course on personality assessment. After graduate students practice scoring and interpreting the MMPI-2 inventory completed with specific response sets and practice interpreting MMPI-2 profiles of cases prepared for teaching and practice in interpretation, students practice administering and interpreting the MMPI-2 for volunteer test-takers who complete the inventory with a simulated stress response set. The simulated stress response set approach may be helpful in providing skill-building practice in the early phases of learning the MMPI-2 with volunteer test-takers, while minimizing some of the ethical concerns raised regarding actual administrations with nonclient volunteers in the early phases of training in assessment.
Null AP(1), Muddiman DC. ( 71. Rapid Commun Mass Spectrom. 2003;17(15):1714-22. ). Determination of a correction to improve mass measurement accuracy of isotopically unresolved polymerase chain reaction amplicons by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. 71. Rapid Commun Mass Spectrom. 2003;17(15):1714-22.
Abstract
The experimental determination of average mass by mass spectrometry is limited for large molecules due to the negative bias introduced by the natural distribution of isotopic abundances. This results in the measurement of the top-of-centroid (ToC) as opposed to the true centroid. We have developed a practical correction factor that is applied to the ToC measurement to largely remove the systematic bias introduced by nature. The correction factor is calculated easily using the average molecular mass (<100 kDa) of the analyte molecule and the full-width half maximum resolving power (<3,500) of the measurement. In addition, an approach to calculating resolving power is described that accurately predicts resolving power achievable for Fourier transform ion cyclotron resonance (FT-ICR) mass analysis of large molecules. A combination of internal calibration with a dual-electrospray source and application of the correction factor to average mass measurements improved the mass error from 192.5 to -35.0 ppm for a 44 kDa PCR amplicon.
Pubmed 
Bhat BG(1), Rapp SR, Beaudry JA, Napawan N, Butteiger DN, Hall KA, Null CL, Luo Y, Keller BT. (2003). Inhibition of ileal bile acid transport and reduced atherosclerosis in apoE-/- mice by SC-435. J Lipid Res. 44(9):1614-21.
Abstract
Blocking intestinal bile acid absorption by inhibiting the apical sodium codependent bile acid transporter (ASBT) is a target for increasing hepatic bile acid synthesis and reducing plasma LDL cholesterol. SC-435 was identified as a potent inhibitor of ASBT (IC50 = 1.5 nM) in cells transfected with the human ASBT gene. Dietary administration of 3 mg/kg to 30 mg/kg SC-435 to apolipoprotein E-/- (apoE-/-) mice increased fecal bile acid excretion by >2.5-fold. In vivo inhibition of ASBT also resulted in significant increases of hepatic mRNA levels for cholesterol 7alpha-hydroxylase and HMG-CoA reductase. Administration of 10 mg/kg SC-435 for 12 weeks to apoE-/- mice lowered serum total cholesterol by 35% and reduced aortic root lesion area by 65%. Treatment of apoE-/- mice also resulted in decreased expression of ileal bile acid binding protein and hepatic nuclear hormone receptor small heterodimer partner, direct target genes of the farnesoid X receptor (FXR), suggesting a possible role of FXR in SC-435 modulation of cholesterol homeostasis. In dogs, SC-435 treatment reduced serum total cholesterol levels by </=12% and, in combination with atorvastatin treatment, caused an additional reduction of 25%. These results suggest that specific inhibition of ASBT is a novel therapeutic approach for treatment of hypercholesterolemia resulting in a decreased risk for atherosclerosis.
Benson LM(1), Null AP, Muddiman DC. (2003). Advantages of Thermococcus kodakaraenis (KOD) DNA Polymerase for PCR-mass spectrometry based analyses. J Am Soc Mass Spectrom. 14(6):601-4.
Abstract
The advantages of the thermostable DNA polymerase from Thermococcus kodakaraensis (KOD) are demonstrated for PCR amplification with subsequent detection by mass spectrometry. Commonly used DNA polymerases for PCR amplification include those from Thermus aquaticus (Taq) and Pyrococcus furiosus (Pfu). A 116 base-pair PCR product derived from a vWA locus was amplified by Taq, Pfu, or KOD DNA polymerase and compared by agarose gel electrophoresis and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). KOD DNA polymerase demonstrated a 2- to 3-fold increase in PCR product formation compared to Pfu or Taq, respectively, and generated blunt-ended PCR product which allows facile interpretation of the mass spectrum. Additionally, we demonstrate the advantage of using high magnetic fields to obtain unit resolution of the same 116 base pair (approximately 72 kDa) PCR product at high m/z.
Null AP(1), Nepomuceno AI, Muddiman DC. (2003). Implications of hydrophobicity and free energy of solvation for characterization of nucleic acids by electrospray ionization mass spectrometry. Anal Chem. 75(6):1331-9.
Abstract
Electrospray ionization (ESI) is a dynamic process that, when coupled with mass spectrometry (MS), serves as an invaluable tool for analysis of biomolecules. Our group, as well as others, has observed that there is a bias in signal intensity for one strand of a PCR amplicon over the complementary strand in an ESI mass spectrum. In this report, we have investigated the contributions of hydrophobicity and free energy of solvation to relative signal intensities in ESI-MS spectra of nucleic acids. We developed approaches for predicting which strand of the PCR amplicon will be the most intense: one based on a rate equation for calculating ion flux using values from the literature for hydrophobicity and free energy of solvation and the other based on the percentage of the relatively hydrophilic guanines present in the strand. A trend in signal intensity for deoxyribonucleotide triphosphates, oligonucleotides, and PCR amplicons was observed that was consistent with our model. On the basis of the observation that increased hydrophobicity correlates with greater signal intensity, we selectively enhanced the signal intensity of a 20-mer with the addition of an alkyl chain to the 5' terminus, which subsequently improved the limit of detection to 1 nM, an improvement by 1 order of magnitude. This was extended to a 53-bp PCR amplicon by modifying one primer with the hydrophobic moiety, which resulted in a 16% increase in signal intensity. We capitalized on this result to determine allele frequencies from pooled DNA for single-nucleotide polymorphisms down to 1%.
Arbeitman MN(1), Furlong EE, Imam F, Johnson E, Null BH, Baker BS, Krasnow MA, Scott MP, Davis RW, White KP. (2002). Gene expression during the life cycle of Drosophila melanogaster. Science. 297(5590):2270-5.
Abstract
Erratum in Science 2002 Nov 8;298(5596):1172.
Pubmed 
Flora JW(1), Null AP, Muddiman DC. (2002). Dual-micro-ESI source for precise mass determination on a quadrupole time-of-flight mass spectrometer for genomic and proteomic applications. Anal Bioanal Chem. 373(7):538-46.
Abstract
A universal dual-electrospray (ESI) source is demonstrated on a quadrupole orthogonal-accelerated time-of-flight mass spectrometer (Q-ToF-MS) for both genomic and proteomic applications. This facile source modification enables internal calibration for consistent mass measurements by a mainstream MS platform and requires no mixing of analyte and calibrant prior to ion formation. In this report, the dual-sprayer is demonstrated in the negative-ion mode for internal calibration of polymerase chain reaction (PCR) amplicons generated from synthetic and genomic templates as well as a proteolytic digest of a naturally phosphorylated protein. For all PCR amplicons, experimentally determined average mass measurements are well within the instrument specifications of better than 0.01%. For the proteolytic fragments of the phosphoprotein, average mass errors of the isotopically resolved peptides are better than 10 ppm.
Null AP(1), Hudson J, Gorbsky GJ. (2002). Both alpha and beta isoforms of mammalian DNA topoisomerase II associate with chromosomes in mitosis. Cell Growth Differ. 13(7):325-33.
Abstract
Two isoforms of DNA topoisomerase II, alpha and beta, coded by separate genes, are expressed in actively cycling vertebrate cells. Some previous studies have suggested that only topoisomerase II alpha remains associated with chromosomes at mitosis. Here, the distributions of topoisomerase II alpha and beta in mitosis were studied by subcellular fractionation and by immunolocalization. Both isoforms of topoisomerase II were found to remain associated with mitotic chromatin. Topoisomerase II alpha was distributed along chromosome arms throughout mitosis and was highly concentrated at centromeres until mid-anaphase, particularly in some cell types. Topoisomerase II beta showed weak concentration at centromeres in early mitosis in some cell types and was distributed along chromosome arms at every stage of mitosis through telophase. These studies suggest that in most cells both the major topoisomerase II isoforms may play roles in chromatin remodeling during M phase.
Null AP(1), George LT, Muddiman DC. (2002). Evaluation of sample preparation techniques for mass measurements of PCR products using ESI-FT-ICR mass spectrometry. J Am Soc Mass Spectrom. 13(4):338-44.
Abstract
Elimination of PCR buffer components and alkali metal cations (i.e., Na+, K+) is of critical importance to allow for accurate mass measurements of PCR products for genotyping and sequencing applications. Ethanol precipitation followed by microdialysis has been repeatedly shown to efficiently desalt PCR products for analysis by mass spectrometry and is considered the gold standard. Alternative cleanup techniques that are compatible with automation are explored here with the intent of expanding the bottleneck that exists between the production of PCR products and analysis by electrospray ionization mass spectrometry (ESI-MS). Numerous combinations of approaches were evaluated that included PCR purification kits and alcohol precipitations. The data shown here support alternative approaches to an ethanol precipitation followed by microdialysis that have comparable desalting efficiency and can be utilized for cleanup of PCR products generated from single reactions.
Null AP(1), Muddima DC. (2002). CEPH family 1362 STR database: an online resource for characterization of PCR products using electrospray ionization mass spectrometry. J Am Soc Mass Spectrom. 13(1):89-90.
Abstract
An online database has been established in order to validate electrospray ionization mass spectrometry (ESI-MS) for genotyping and to publicize the procedures developed in our laboratory for the characterization of PCR products by ESI-MS. Genotypes derived from short tandem repeat (STR) loci that were obtained using ESI Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) have been posted for fifteen members of the CEPH family 1362 pedigree. The website provides specific information such as PCR parameters, PCR product cleanup approaches, and ESI solution compositions to enable other laboratories to reproduce our data. Links are provided to related websites in an effort to integrate information regarding the CEPH family, STR genotyping, and mass spectrometry. The database, currently available at http://www.people.vcu.edu/ -dcmuddim/genotype/ will be routinely updated with genotypes from additional STR loci including PCR parameters as well as PCR cleanup strategies as further developments are completed.
Null AP(1), Hannis JC, Muddiman DC. (2001). Genotyping of simple and compound short tandem repeat loci using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Anal Chem. 73(18):4514-21.
Abstract
The utility of electrospray ionization Fourier transform ion cyclotron resonance (ESI-FIICR) mass spectrometry as a new approach for genotyping short tandem repeats (STRs) is demonstrated. STRs are currently valued as a powerful source of genetic information with repeats that range in structure from simple to hypervariable. Two tetranucleotide STR loci were chosen to evaluate ESI-FTICR mass spectrometry as a tool for genotyping: HUM-TH01, a simple STR with nonconsensus alleles, and vWA, a compound STR with nonconsensus alleles. For HUM-TH01, the genotype (i.e., repeat number of each allele) was determined for each of 30 individuals using mass measurements of double-stranded amplicons. Low-intensity peaks observed in the spectra of amplicons derived from heterozygous individuals were identified by mass as heteroduplexes that had formed between nonhomologous strands. Mass measurement of the double-stranded vWA amplicon was not sufficient for determining whether the individual was homozygous for allele subtype 18 or 18' since the amplicons differ by only 0.99 Da. Therefore, single-stranded amplicons were generated by incorporating a phosphorylated primer, prepared using T4 polynucleotide kinase, into the PCR phase and subsequently digesting the bottom strand using lambda-exonuclease. Accurate mass measurements were obtained for the single-stranded amplicons using internal calibration and the addition of a correction factor to adjust for the natural variation of isotopic abundances, confirming that the individual is homozygous for allele 18. Our results clearly demonstrate that ESI-FTICR mass spectrometry is a powerful approach to characterize both simple and compound STRs beyond the capabilities of electrophoretic technologies.
Furlong EE(1), Andersen EC, Null B, White KP, Scott MP. (2001). Patterns of gene expression during Drosophila mesoderm development. Science. 293(5535):1629-33.
Abstract
The transcription factor Twist initiates Drosophila mesoderm development, resulting in the formation of heart, somatic muscle, and other cell types. Using a Drosophila embryo sorter, we isolated enough homozygous twist mutant embryos to perform DNA microarray experiments. Transcription profiles of twist loss-of-function embryos, embryos with ubiquitous twist expression, and wild-type embryos were compared at different developmental stages. The results implicate hundreds of genes, many with vertebrate homologs, in stage-specific processes in mesoderm development. One such gene, gleeful, related to the vertebrate Gli genes, is essential for somatic muscle development and sufficient to cause neural cells to express a muscle marker.
Null AP(1), Muddiman DC. (2001). Perspectives on the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for short tandem repeat genotyping in the post-genome era. J Mass Spectrom. 36(6):589-606.
Abstract
The recent completion of the first rough draft of the human genome has provided fundamental information regarding our genetic make-up; however, the post-genome era will certainly require a host of new technologies to address complex biological questions. In particular, a rapid and accurate approach to characterize genetic markers, including short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) is demanded. STRs are the most informative of the two polymorphisms owing to their remarkable variability and even dispersity throughout eukaryotic genomes. Mass spectrometry is rapidly becoming a significant method in DNA analysis and has high probability of revolutionizing the way in which scientists probe the human genome. It is our responsibility as biomolecular mass spectrometrists to understand the issues in genetic analysis and the capabilities of mass spectrometry so that we may fulfill our role in developing a rapid, reliable technology to answer specific biological questions. This perspective is intended to familiarize the mass spectrometry community with modern genomics and to report on the current state of mass spectrometry, specifically electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry, for characterization of STRs.
Sheikh S(1), Null D, Gentile D, Bimle C, Skoner D, McCoy K, Guthrie R. (2001). Urinary leukotriene E(4) excretion during the first month of life and subsequent bronchopulmonary dysplasia in premature infants. Chest. 119(6):1749-54.
Abstract
BACKGROUND: Inflammation plays an important role in the pathogenesis of bronchopulmonary dysplasia (BPD), but the exact nature of this inflammatory process is incompletely understood. Older infants with established BPD have higher levels of urinary leukotriene E(4) (LTE(4)) compared to healthy infants of the same age. This suggests that cysteinyl leukotrienes may play a role in the abnormalities seen in BPD. OBJECTIVES: To measure urinary LTE(4) levels during the first month of life in premature infants, and to determine whether there are significant differences in premature infants who develop BPD, as compared to those who do not develop BPD. DESIGN: Prospective, blinded, controlled study. SETTING: Neonatal ICUs of a tertiary-care university hospital. METHODS: Thirty-seven premature infants (< 33 weeks of gestational age) were enrolled prospectively at birth. Urinary LTE(4) levels were measured blinded, using a standard radioimmunoassay technique at 2 days, 7 days, and 28 days of life. At 1 month of age, infants were classified as with or without BPD, based on need for supplemental oxygen, and characteristic chest radiographs. Clinical features and urinary LTE(4) were compared between the two groups. RESULTS: Mean +/- SD gestational age was 29 +/- 2.6 weeks. None of the infants had a family history of asthma. Thirteen of 37 infants were classified as having BPD at 28 days after birth. Mean gestational age in infants who developed BPD was 27 +/- 2.4 weeks, compared to 30 +/- 2 weeks in infants who did not develop BPD (p < 0.05). In infants with BPD, mean urinary LTE(4) levels of urinary creatinine were 1,762 +/- 2,003 pg/mg, 1,236 +/- 992 pg/mg, and 5,541 +/- 5,146 pg/mg at days 2, 7, and 28, respectively, compared to 1,304 +/- 1,195 pg/mg, 1,158 +/- 1,133 pg/mg, and 2,800 +/- 2,080 pg/mg in infants without BPD. LTE(4) levels at 2 days, 7 days, and 28 days did not correlate with the subsequent development of BPD. LTE(4) levels at day 28 were significantly higher than LTE(4) levels at day 2 and day 7 in both groups, even after correcting for gestational age or birth weight (p < 0.05). There was significant inverse correlation between LTE(4) levels at day 2 with gestational age and birth weight (p < 0.05). All 13 infants with BPD received steroid pulses, compared to 3 of 26 infants without BPD. Gestational age and use of postnatal steroid pulses, diuretics, and theophylline (for apnea of prematurity) were significantly associated with each other and with the subsequent development of BPD. CONCLUSION: Urinary LTE(4) levels measured on the second day of life in very-low-birth-weight infants inversely correlate with gestational age and birth weight. Urinary LTE(4) levels may reflect lung injury and/or inflammation in premature infants, not necessarily related to BPD as it is presently defined.
Anderson SA(1), Rader RK, Westlin WF, Null C, Jackson D, Lanza GM, Wickline SA, Kotyk JJ. (2000). Magnetic resonance contrast enhancement of neovasculature with alpha(v)beta(3)-targeted nanoparticles. Magn Reson Med. 44(3):433-9.
Abstract
Site-directed contrast enhancement of angiogenic vessels in vivo was demonstrated using antibody targeting of an MRI contrast agent to the alpha(v)beta(3) integrin, a molecular marker characteristic of angiogenic endothelium. The agent was tested in a rabbit corneal micropocket model, in which neovasculature is induced in the cornea using basic fibroblast growth factor. The targeted contrast agent consists of Gd-perfluorocarbon nanoparticles linked to alpha(v)beta(3) integrin antibody DM101. The animal group receiving the targeted contrast agent displayed a 25% increase in the average MR signal intensity after 90 min. Control groups in which the nanoparticles are either used alone, linked to an isotype-matched antibody, or linked to DM101 and administered following receptor blocking did not display MR contrast enhancement at similar dose levels. These findings indicate that the antibody-targeted agent enhances MR signal intensity in the capillary bed in a corneal micropocket model of angiogenesis, and is selectively retained within the angiogenic region via specific interaction with the alpha(v)beta(3) epitope.
Wan FC(1), Dettloff ML, Null MJ, White JE. (2000). Qualitative and quantitative analysis of a thermoset polymer, poly(benzoxazine), by pyrolysis-gas chromatography. J Chromatogr A. 886(1-2):217-24.
Abstract
The chemical composition of a poly(benzoxazine) thermoset polymer (a copolymer of bisphenol-A benzoxazine and tert.-butylphenol benzoxazine) has been studied by pyrolysis-gas chromatography (Py-GC). Major pyrolysates have been identified and the possible degradation pathways have been investigated. A specific pyrolysate was identified for quantitative analysis after carefully proving the linear relationship between the pyrolysate signal intensity and monomer concentration over a wide range of compositions. A method to determine the concentration of the monomer that potentially acts as a cross-linking unit has been developed. In this study, Py-GC was shown to be an excellent analytical technique for the qualitative and quantitative analysis of thermoset polymers.
Null AP(1), Hannis JC, Muddiman DC. (2000). Preparation of single-stranded PCR products for electrospray ionization mass spectrometry using the DNA repair enzyme lambda exonuclease. Analyst. 125(4):619-26.
Abstract
Electrospray ionization mass spectrometry (ESI-MS) has been utilized to obtain accurate mass measurements of intact PCR products; however, single-stranded PCR products are necessary to detect sequence modifications such as base substitutions, additions or deletions. The locations of these modifications can subsequently be determined using additional stages of mass spectrometry. The recombinant enzyme lambda exonuclease selectively digests one strand of a DNA duplex from a 5' phosphorylated end leaving the complementary strand intact. Using this rapid enzymatic step, we were able to produce single-stranded PCR products by digestion of an intact PCR product derived from the Human Tyrosine Hydroxylase (HUMTHO1) gene, which contains a tetrameric repeating motif. The non-template directed 3' adenylation common when using Taq polymerase resulted in three distinct species (blunt-ended, mono-adenylated and di-adenylated), which added complexity to the spectrum of the double-stranded product. The data from the single-stranded products shows that one strand is preferentially adenylated over the other, which cannot be determined from the mass spectrum of the double-stranded PCR product alone. The ESI-FTICR (Fourier transform ion cyclotron resonance) mass spectra of the lambda exonuclease treated PCR products exhibited less than expected signal-to-noise (S/N) ratios. This is attributed to inaccurate concentration calculations due to remaining double-stranded PCR product amplified with unphosphorylated primers, and to matrix effects contributed by the lambda exonuclease reaction buffer. To further test this hypothesis, we investigated and determined the limit of detection to be 0.27 microM using standard curve statistics for single acquisitions of a synthetic 75-mer. The concentrations of the noncoding and coding strands produced by lambda exonuclease digestion were calculated to be 0.29 and 0.37 microM, respectively, taking into account the presence of double-stranded product. The products were electrosprayed from concentrations at the limit of detection requiring the averaging of 5-10 acquisitions to produce a sufficient S/N ratio, indicating that product concentration, base composition and matrix effects play a combined, significant role in detection of lambda exonuclease treated PCR products. Although additional work will be required to further exploit this strategy, lambda exonuclease clearly provides mass spectrometrists with a method to generate single-stranded PCR products.
Rosowsky A(1), Forsch RA, Null A, Moran RG. (1999). 5-deazafolate analogues with a rotationally restricted glutamate or ornithine side chain: synthesis and binding interaction with folylpolyglutamate synthetase. J Med Chem. 42(18):3510-9.
Abstract
Rotationally restricted analogues of 5-deazapteroyl-L-glutamate and (6R,6S)-5-deaza-5,6,7,8-tetrahydropteroyl-L-glutamate with a one-carbon bridge between the amide nitrogen and the 6'-position of the p-aminobenzoyl moiety were synthesized and tested as substrates for folylpolyglutamate synthetase (FPGS), a key enzyme in folate metabolism and an important determinant of the therapeutic potency and selectivity of classical antifolates. The corresponding bridged analogues of 5-deazapteroyl-L-ornithine and (6R,6S)-5-deaza-5,6,7, 8-tetrahydropteroyl-L-ornithine were also synthesized as potential inhibitors. Condensation of diethyl L-glutamate with methyl 2-bromomethyl-4-nitrobenzoate followed by catalytic reduction of the nitro group, reductive coupling with 2-acetamido-6-formylpyrido[2, 3-d]pyrimidin-4(3H)-one in the presence of dimethylaminoborane, and acidolysis with HBr/AcOH yielded 2-L-[5-[N-(2-acetamido-4(3H)-oxopyrido[2, 3-d]pyrimidin-6-yl)methylamino]-2, 3-dihydro-1-oxo-2(1H)-isoindolyl]glutaric acid (1). When acidolysis was preceded by catalytic hydrogenation, the final product was the corresponding (6R,6S)-tetrahydro derivative 2. A similar sequence starting from methyl N(delta)-benzyloxycarbonyl-L-ornithine led to 2-L-[5-[N-(2-amino-4(3H)-oxopyrido[2, 3-d]pyrimidin-6-yl)methylamino]-2, 3-dihydro-1-oxo-2(1H)-isoindolyl]-5-aminopentanoic acid (3) and the (6R,6S)-tetrahydro derivative 4. Compounds 3 and 4 were powerful inhibitors of recombinant human FPGS, whereas 1 and 2 were exceptionally efficient FPGS substrates, with the reduced compound 2 giving a K(m) (0.018 microM) lower than that of any other substrate identified to date. (6R,6S)-5-Deazatetrahydrofolate, in which the side chain is free to rotate, was rapidly converted to long-chain polyglutamates. In contrast, the reaction of 1 and 2 was limited to the addition of a single molecule of glutamic acid. Hence rotational restriction of the side chain did not interfere with the initial FPGS-catalyzed reaction and indeed seemed to facilitate it, but the ensuing gamma-glutamyl adduct was no longer an efficient substrate for the enzyme.
Muddiman DC(1), Null AP, Hannis JC. (1999). Rapid Commun. Mass Spectrom. 13, 740 ?lpar;1999?rpar; by J. W. Hager, ?ldquo;Performance Optimization and Fringing Field Modifications of a 24?hyphen;mm Long RF?hyphen;only Quadrupole Mass Spectrometer?rdquo; Rapid Commun Mass Spectrom. 13(12):1205.
Abstract
PMID: 10407298 [PubMed - as supplied by publisher]
Muddiman DC(1), Null AP, Hannis JC. (1999). Precise mass measurement of a double-stranded 500 base-pair (309 kDa) polymerase chain reaction product by negative ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Rapid Commun Mass Spectrom. 13(12):1201-1204.
Abstract
Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) has been used to determine the mass of a double-stranded 500 base-pair (bp) polymerase chain reaction (PCR) product with an average theoretical mass of the blunt-ended (i.e. unadenylated) species of 308 859.35 Da. The PCR product was generated from the linearized bacteriophage Lambda genome which is a double-stranded template. Utilization of ethanol precipitation in tandem with a rapid microdialysis step to purify and desalt the PCR product was crucial to obtain a precise mass measurement. The PCR product (0.8 pmol/µL) was electrosprayed from a solution containing 75% acetonitrile, 25 mM piperidine, and 25 mM imidazole and was infused at a rate of 200 nL/min. The average molecular mass and the corresponding precision were determined using the charge-states ranging from 172 to 235 net negative charges. The experimental mass and corresponding precision (reported as the 95% confidence interval of the mean) was 309 406 +/- 27 Da (87 ppm). The mass accuracy was compromised due to the fact that the PCR generates multiple products when using Taq polymerase due to the non-template directed 3'-adenylation. This results in a mixture of three PCR products with nearly identical mass (i.e. blunt-ended, mono-adenylated and di-adenylated) with unknown relative abundances that were not resolved in the spectrum. Thus, the experimental mass will be a weighted average of the three species which, under our experimental conditions, reflects a nearly equal concentration of the mono- and di-adenylated species. This report demonstrates that precise mass measurements of PCR products up to 309 kDa (500 bp) can be routinely obtained by ESI-FTICR requiring low femtomole amounts. Copyright 1999 John Wiley & Sons, Ltd.
Sáez-Llorens X(1), Castaño E, Null D, Steichen J, Sánchez PJ, Ramilo O, Top FH Jr, Connor E. (1998). Safety and pharmacokinetics of an intramuscular humanized monoclonal antibody to respiratory syncytial virus in premature infants and infants with bronchopulmonary dysplasia. The MEDI-493 Study Group. Pediatr Infect Dis J. 17(9):787-91.
Abstract
BACKGROUND: Respiratory syncytial virus (RSV) is the leading cause of lower respiratory disease in infants and children. MEDI-493 (palivizumab, Synagis) is a humanized monoclonal IgG1 antibody to the fusion protein of RSV, and it is highly active in vitro against RSV A and B strains. OBJECTIVE: To describe the safety, tolerance, immunogenicity and pharmacokinetics of monthly intramuscular injections of MEDI-493 among premature infants and children with bronchopulmonary dysplasia and to compare these data with information previously obtained with intravenous dosing. DESIGN: A Phase I/II multicenter, open label, escalating dose clinical trial. PATIENT POPULATION AND DOSING REGIMEN: Children (n=65) born prematurely at < or =35 weeks of gestation who were < or =6 months of age (n=41) and children with bronchopulmonary dysplasia who were < or =24 months of age (n=24) were enrolled. From 1 to 5 monthly injections were given at doses of 5 mg/kg (n=11), 10 mg/kg (n=6) and 15 mg/kg (n=48). Serum was collected before administration of each dose, 30 days after the last dose, and 2, 7 and 14 days after the first and second doses for measurement of MEDI-493 concentrations by enzyme-linked immunosorbent assay. RESULTS: The pharmacokinetics of MEDI-493 were similar to those of other human IgG1 antibodies. Mean serum MEDI-493 concentrations were 91.1 microg/ml (range, 52.3 to 174.0) 2 days after the initial dose of 15 mg/kg and 49.2 microg/ml (range, 13.5 to 132.0) at 30 days. Monthly dosing of 15 mg/kg maintained mean trough concentrations of approximately 70 microg/ml. These concentrations were similar to previously published trough concentrations after i.v. administration. MEDI-493 injections were well-tolerated. Only three children had adverse events judged to be possibly related to MEDI-493. Ten children had transient, low titer anti-MEDI-493 binding titers (1:10 to 1:40) which were not associated with a pattern of specific adverse events or alterations of MEDI-493 concentrations. Two patients in the 5-mg/kg dose group were hospitalized for RSV; no RSV hospitalizations were found in the higher dose groups. CONCLUSIONS: MEDI-493 was safe and well-tolerated. Monthly intramuscular doses of 15 mg/kg maintained mean trough serum concentrations that were above 40 microg/ml (the value associated with 99% reduction of pulmonary RSV in the cotton rat model). These concentrations were similar to those previously reported with i.v. administration of MEDI-493.
Null SL(1), Bonser K. (1997). Multiple changes, multiple opportunities in the ambulatory surgery department. J Perianesth Nurs. 12(5):321-8.
Abstract
As one facility that was restructured and purchased, the Ambulatory Surgery Department moved through changes at a very rapid pace. The waves of change began with preoperative testing requirements and expansion of the nurse's role in accurate and appropriate testing decisions. Cross-training for radiology, pain management, infusion services, and oncology services were also incorporated. As the department became a multi-faceted area of patient care, nursing staff met the challenge of change and grew both educationally and professionally. This article identifies areas of departmental and professional opportunity and outlines the development of protocols to meet each challenge.
Paranka MS(1), Clark RH, Yoder BA, Null DM Jr. (1995). Predictors of failure of high-frequency oscillatory ventilation in term infants with severe respiratory failure. Pediatrics. 95(3):400-4.
Abstract
Comment in Pediatrics. 1996 Mar;97(3):437-8.
Pubmed 
Null S(1), Richter-Abt D, Kovac J. (1995). Development of a perioperative nursing diagnoses flow sheet. AORN J. 61(3):547-54, 557.
Abstract
Traditionally, health care professionals in the preoperative, intraoperative, and postoperative phases of care have used perioperative records that focus on the technical aspects of the care provided (eg, blood pressure, pulse measurements; equipment used) and leave room for only short narratives to document nursing care. Such formats often do not document the multitude of activities or interventions perioperative nurses provide. The question raised at the DePaul Health Center, St Louis, was: "Where do we document our nursing diagnoses and plan of care?" The response was to create a nursing diagnosis task force that investigated the feasibility of a form professional nurses could use in all phases of patient care. This investigation led to the development, implementation, and universal use of a perioperative nursing diagnoses flow sheet within the surgical services department.
Gagliardi JA(1), Chung EM, Chandnani VP, Kesling KL, Christensen KP, Null RN, Radvany MG, Hansen MF. (1994). Detection and staging of chondromalacia patellae: relative efficacies of conventional MR imaging, MR arthrography, and CT arthrography. AJR Am J Roentgenol. 163(3):629-36.
Abstract
OBJECTIVE: Chondromalacia patellae is a condition characterized by softening, fraying, and ulceration of patellar articular cartilage. We compare the sensitivity, specificity, and accuracy of conventional MR imaging, MR arthrography, and CT arthrography in detecting and staging this abnormality. SUBJECTS AND METHODS: Twenty-seven patients with pain in the anterior part of the knee were prospectively examined with MR imaging, including T1-weighted (650/16), proton density-weighted (2000/20), T2-weighted (2000/80), and spoiled two-dimensional gradient-recalled acquisition in the steady state (SPGR/)/35 degrees (51/10) with fat saturation pulse sequences. All were also examined with T1-weighted MR imaging after intraarticular injection of dilute gadopentetate dimeglumine and with double-contrast CT arthrography. Each imaging technique was evaluated independently by two observers, who reached a consensus interpretation. The signal characteristics of cartilage on MR images and contour abnormalities noted with all imaging techniques were evaluated and graded according to a modification of the classification of Shahriaree. Twenty-six of the 54 facets examined had chondromalacia shown by arthroscopy, which was used as the standard of reference. The sensitivity, specificity, and accuracy of each imaging technique in the diagnosis of each stage of chondromalacia patellae were determined and compared by using the McNemar two-tailed analysis. RESULTS: Arthroscopy showed that 28 facets were normal. Grade 1 chondromalacia patellae was diagnosed only with MR and CT arthrography in two (29%) of seven facets. Intermediate (grade 2 or 3) chondromalacia patellae was detected in two (13%) of 15 facets with T1-weighted and SPGR MR imaging, in three (20%) of 15 facets with proton density-weighted MR imaging, in seven (47%) of 15 facets with T2-weighted MR imaging, in 11 (73%) of 15 facets with CT arthrography, and in 12 (80%) of 15 facets with MR arthrography. Grade 4 was detected in three (75%) of four facets with T1-, proton density-, and T2-weighted MR imaging, two (50%) of four facets with SPGR MR imaging, and four (100%) of four facets with MR and CT arthrography. Thus, all imaging techniques were insensitive to grade 1 lesions and highly sensitive to grade 4 lesions, so that no significant difference among the techniques could be shown. CONCLUSION: All imaging techniques studied had high specificity and accuracy in the detection and grading of chondromalacia patella; however, both MR arthrography and CT arthrography were more sensitive than T1-weighted, proton density-weighted, and SPGR with fat saturation MR imaging for showing intermediate grades of chondromalacia patellae. Although the arthrographic techniques were not significantly better than T2-weighted imaging, the number of false-positive diagnoses was greatest with T2-weighted MR imaging.
Messer SC(1), Kempton T, Van Hasselt VB, Null JA, Bukstein OG. (1994). Cognitive distortions and adolescent affective disorder. Validity of the CNCEQ in an inpatient sample. Children's Negative Cognitive Error Questionnaire. Behav Modif. 18(3):339-51.
Abstract
Despite a proliferation of recent research examining childhood and adolescent depression, the area still lags behind the adult depression field, particularly in the investigation of cognitive correlates of affective psychopathology. To advance cognitive research with youth, the Children's Negative Cognitive Error Questionnaire (CNCEQ) was developed to provide a measure of cognitive distortions or errors in children and adolescents. Yet, few studies have employed the CNCEQ and no evidence exists supporting the validity of its four component cognitive error scales. The purpose of the present study was to examine the construct validity of the CNCEQ and its constituent scales through the use of factor analysis and criterion-group comparisons. Groups of adolescent psychiatric inpatients, diagnosed as affective or disruptive disordered, completed the CNCEQ following admission. Results failed to support the implicit four-factor structure of the CNCEQ, instead suggesting the appropriateness of a single-factor solution labeled "negative thinking." Despite no diagnostic group differences on the CNCEQ total or other scale scores, affective disordered patients evinced more cognitive errors on the Overgeneralizing scale. Findings suggest that the CNCEQ in its current stage of development holds promise, yet requires refinement to produce a valid measure of cognitive functioning in youth.
Cornish JD(1), Dreyer GL, Snyder GE, Kuehl TJ, Gerstmann DR, Null DM Jr, Coalson JJ, deLemos RA. (1994). Failure of acute perinatal asphyxia or meconium aspiration to produce persistent pulmonary hypertension in a neonatal baboon model. Am J Obstet Gynecol. 171(1):43-9.
Abstract
OBJECTIVE: Our purpose was to determine whether perinatal asphyxia or meconium aspiration, or both, can produce the physiologic and histologic pulmonary vascular changes associated with the meconium aspiration syndrome. STUDY DESIGN: Twenty neonatal baboons were studied in four groups: 1, control; 2, meconium aspiration; 3, asphyxia (intermittent cord compression); and 4, asphyxia with meconium aspiration. Animals were ventilated for 24 hours under ketamine, diazepam, and pancuronium. Data were analyzed by means of mixed model analysis of measures. RESULTS: Meconium significantly impaired oxygenation (p < 0.001), whereas concurrent asphyxia moderated this effect (p < 0.034). Meconium also increased the need for ventilatory support (p < 0.002). No animal had persistent pulmonary hypertension; neither systemic nor pulmonary systolic pressures differed statistically between the groups. No animal showed evidence of abnormal pulmonary arteriolar muscularization. CONCLUSION: Sublethal perinatal asphyxia or meconium aspiration were insufficient to produce either the physiologic or histologic changes of severe meconium aspiration syndrome. It is unlikely that intrapartum fetal distress alone can produce this syndrome in human neonates.
Null SL. (1994). Preadmission testing. A coordinator can be the answer. AORN J. 59(5):1051-6; 1059-60.
Abstract
PMID: 8037425 [PubMed - indexed for MEDLINE]
Kempton T(1), van Hasselt VB, Bukstein OG, Null JA. (1994). Cognitive distortions and psychiatric diagnosis in dually diagnosed adolescents. J Am Acad Child Adolesc Psychiatry. 33(2):217-22.
Abstract
OBJECTIVE: The purpose of this study was to examine the characteristics and patterns of cognitive distortions among psychiatrically hospitalized adolescents. METHOD: Measures of cognitive distortions, depression, and hopelessness were administered to 135 adolescents on two psychiatric inpatient units. Subjects were grouped according to their Axis I diagnoses: depression only, conduct disorder only, depression and substance abuse, conduct disorder and substance abuse, all three diagnoses, and none of the three diagnoses. RESULTS: Multivariate analyses of covariance indicated that differently diagnosed adolescents exhibited varying levels of cognitive distorting as measured by the Children's Negative Cognitive Errors Questionnaire (CNCEQ). In particular, adolescents with multiple Axis I diagnoses tended to score highest. On all but one of four CNCEQ subscales, the depression only group evidenced as much cognitive distortion as did the group with multiple diagnoses. However, each diagnostic grouping demonstrated its own somewhat distinct distortions based on CNCEQ subscales. CONCLUSIONS: Findings are discussed in terms of the utility of differentiating cognitive styles for subsequent treatment. It is suggested that disparate cognitive interventions could be matched with adolescents displaying particular problems.
Sekins KM(1), Coalson JJ, deLemos RA, Fields TK, Flaim SF, Guerra JM, Hopkins RM, Jackson JC, Null DM, Winter DC, et al. ( 99. Artif Cells Blood Substit Immobil Biotechnol. 1994;22(4):1381-7. ). Long-term partial liquid ventilation (PLV) with perflubron in the near-term baboon neonate. 99. Artif Cells Blood Substit Immobil Biotechnol. 1994;22(4):1381-7.
Abstract
PURPOSE: The feasibility and safety of continuous long-term (4-5 day) partial liquid ventilation (PLV) using perflubron was demonstrated in newborn baboons. PLV, a potential therapy for adult and neonatal respiratory distress syndrome (RDS), is conventional mechanical ventilation (CMV) with the lung filled to about functional residual capacity with perfluorochemical liquid. PROTOCOL: As a pilot trial for a larger preclinical study focused on the safety of extended duration PLV, three near term baboons were studied. The animals were delivered by cesarean section, anesthetized, intubated and placed on CMV. The animals were given intratracheal perflubron (30 ml/kg) and maintained on PLV for 96 hours. The transition back to gas ventilation occurred, after draining, over the fifth day (hrs 96-120). RESULTS: Two of the animals were born with normal pulmonary function, while the third developed respiratory distress prior to PLV. All the animals were adequately supported with PLV using moderate ventilator settings and low concentrations of oxygen. Perflubron distribution was enhanced by periodic rotation of the animals. Preliminary histology show vacuolated alveolar macrophages and no evidence of edema or other significant changes in the lungs. Pulmonary function in the RDS animal, after PLV treatment, showed normal gas exchange and lung mechanics. CONCLUSIONS: Three near term baboons, one with clinical RDS, tolerated 4 days of PLV followed by 1 day of CMV without complications using practical clinical management methods.
Dickey LA(1), Butler TJ, Bergmann TM, Bates ME, Null DM. ( 100. Perfusion. 1994;9(5):327-33. ). Selective heparinization of the extracorporeal membrane oxygenation circuit using continuous infusions of protamine and heparin in a short-term pig model. 100. Perfusion. 1994;9(5):327-33.
Abstract
Systemic heparinization is required for both neonatal and paediatric extracorporeal membrane oxygenation (ECMO). However, it places the patient at risk of serious haemorrhage. We report an alternative: 'selective' heparinization of the ECMO circuit using a continuous infusion of heparin near the venous catheter as blood enters the circuit, and a simultaneous protamine infusion near the arterial catheter as blood enters the patient. Theoretically, the circuit remains heparinized while the patient maintains near-normal clotting activity. Three healthy piglets were placed on venoarterial ECMO in standard fashion. When the animal and its extracorporeal circuit flow were stable, a protamine infusion was begun: 1 mg of protamine to neutralize each 100 units of infused heparin. No haemodynamic instability was noted during the five hours of each study. Mean activated clotting times (ACT) were significantly lower in all three piglets than in their respective circuits (p < 0.001). We conclude that 'selective' heparinization of the ECMO circuit is possible using continuous infusions of protamine and heparin in a short-term piglet model.
Vaddepalli P(1), Herrmann A(1), Fulton L(1), Oelschner M(1), Hillmer S(2), Stratil TF(3), Fastner A(4), Hammes UZ(4), Ott T(3), Robinson DG(2), Schneitz K(5). (2014). The C2-domain protein QUIRKY and the receptor-like kinase STRUBBELIG localize to plasmodesmata and mediate tissue morphogenesis in Arabidopsis thaliana. Development. 141(21):4139-48.
Abstract
Tissue morphogenesis in plants requires communication between cells, a process involving the trafficking of molecules through plasmodesmata (PD). PD conductivity is regulated by endogenous and exogenous signals. However, the underlying signaling mechanisms remain enigmatic. In Arabidopsis, signal transduction mediated by the receptor-like kinase STRUBBELIG (SUB) contributes to inter-cell layer signaling during tissue morphogenesis. Previous analysis has revealed that SUB acts non-cell-autonomously suggesting that SUB controls tissue morphogenesis by participating in the formation or propagation of a downstream mobile signal. A genetic screen identified QUIRKY (QKY), encoding a predicted membrane-anchored C2-domain protein, as a component of SUB signaling. Here, we provide further insight into the role of QKY in this process. We show that like SUB, QKY exhibits non-cell-autonomy when expressed in a tissue-specific manner and that non-autonomy of QKY extends across several cells. In addition, we report on localization studies indicating that QKY and SUB localize to PD but independently of each other. FRET-FLIM analysis suggests that SUB and QKY are in close contact at PD in vivo. We propose a model where SUB and QKY interact at PD to promote tissue morphogenesis, thereby linking RLK-dependent signal transduction and intercellular communication mediated by PD.
Pubmed 
Eckert C(1), Offenborn JN, Heinz T, Armarego-Marriott T, Schültke S, Zhang C, Hillmer S, Heilmann M, Schumacher K, Bock R, Heilmann I, Kudla J. (2014). The vacuolar calcium sensors CBL2 and CBL3 affect seed size and embryonic development in Arabidopsis thaliana. Plant J. 78(1):146-56.
Abstract
Stimulus-specific calcium (Ca(2+) ) signals have crucial functions in developmental processes in many organisms, and are deciphered by various Ca(2+) -binding proteins. In Arabidopsis thaliana, a signaling network consisting of calcineurin B-like (CBL) protein calcium sensors and CBL-interacting protein kinases (CIPKs) has been shown to fulfil pivotal functions at the plasma membrane in regulating ion fluxes and abiotic stress responses. However, the role of tonoplast-localized CBL proteins and especially their function in regulating developmental programs remains largely unknown. In this study, we analyzed single and double mutants of the closely related tonoplast-localized calcium sensors CBL2 and CBL3, which show either reduction of function (rf) or complete loss of function (lf). While single cbl2 or cbl3 mutants did not display discernable phenotypes, cbl2/cbl3 mutants exhibited defects in vegetative growth and were severely impaired in seed development and morphology. Seeds of the cbl2/3rf mutant were smaller in size and exhibited reduced weight and fatty acid content compared to wild-type, but accumulation of sucrose was not altered. Moreover, accumulation of inositol hexakisphosphate (InsP6 ), the major storage form of phosphorus in seeds, was significantly reduced in mutant seeds. In addition, complete loss of CBL2 and CBL3 function in cbl2/3lf resulted in a high frequency of severe defects in embryonic development. Together, our findings reveal a crucial function of Ca(2+) -controlled processes at the vacuolar membrane as determinants of seed yield and size, and demonstrate the importance of vacuolar CBL calcium sensors for plant embryogenesis.
Pubmed 
Viotti C(1), Krüger F, Krebs M, Neubert C, Fink F, Lupanga U, Scheuring D, Boutté Y, Frescatada-Rosa M, Wolfenstetter S, Sauer N, Hillmer S, Grebe M, Schumacher K. (2013). The endoplasmic reticulum is the main membrane source for biogenesis of the lytic vacuole in Arabidopsis. Plant Cell. 25(9):3434-49.
Abstract
Vacuoles are multifunctional organelles essential for the sessile lifestyle of plants. Despite their central functions in cell growth, storage, and detoxification, knowledge about mechanisms underlying their biogenesis and associated protein trafficking pathways remains limited. Here, we show that in meristematic cells of the Arabidopsis thaliana root, biogenesis of vacuoles as well as the trafficking of sterols and of two major tonoplast proteins, the vacuolar H(+)-pyrophosphatase and the vacuolar H(+)-adenosinetriphosphatase, occurs independently of endoplasmic reticulum (ER)-Golgi and post-Golgi trafficking. Instead, both pumps are found in provacuoles that structurally resemble autophagosomes but are not formed by the core autophagy machinery. Taken together, our results suggest that vacuole biogenesis and trafficking of tonoplast proteins and lipids can occur directly from the ER independent of Golgi function.
Montesinos JC(1), Langhans M, Sturm S, Hillmer S, Aniento F, Robinson DG, Marcote MJ. (2013). Putative p24 complexes in Arabidopsis contain members of the delta and beta subfamilies and cycle in the early secretory pathway. J Exp Bot. 64(11):3147-67.
Abstract
p24 proteins are a family of type I membrane proteins localized to compartments of the early secretory pathway and to coat protein I (COPI)- and COPII-coated vesicles. They can be classified, by sequence homology, into four subfamilies, named p24α, p24β, p24γ, and p24δ. In contrast to animals and fungi, plants contain only members of the p24β and p24δ subfamilies, the latter probably including two different subclasses. It has previously been shown that transiently expressed red fluorescent protein (RFP)-p24δ5 (p24δ1 subclass) localizes to the endoplasmic reticulum (ER) at steady state as a consequence of highly efficient COPI-based recycling from the Golgi apparatus. It is now shown that transiently expressed RFP-p24δ9 (p24δ2 subclass) also localizes to the ER. In contrast, transiently expressed green fluorescent protein (GFP)-p24β3 mainly localizes to the Golgi apparatus (as p24β2) and exits the ER in a COPII-dependent manner. Immunogold electron microscopy in Arabidopsis root tip cells using specific antibodies shows that endogenous p24δ9 localizes mainly to the ER but also partially to the cis-Golgi. In contrast, endogenous p24β3 mainly localizes to the Golgi apparatus. By a combination of experiments using transient expression, knock-out mutants, and co-immunoprecipitation, it is proposed that Arabidopsis p24 proteins form different heteromeric complexes (including members of the β and δ subfamilies) which are important for their stability and their coupled trafficking at the ER-Golgi interface. Evidence is also provided for a role for p24δ5 in retrograde Golgi-ER transport of the KDEL-receptor ERD2.
Romani G(1), Piotrowski A, Hillmer S, Gurnon J, Van Etten JL, Moroni A, Thiel G, Hertel B. (2013). A virus-encoded potassium ion channel is a structural protein in the chlorovirus Paramecium bursaria chlorella virus 1 virion. J Gen Virol. 94(Pt 11):2549-56.
Abstract
Most chloroviruses encode small K(+) channels, which are functional in electrophysiological assays. The experimental finding that initial steps in viral infection exhibit the same sensitivity to channel inhibitors as the viral K(+) channels has led to the hypothesis that the channels are structural proteins located in the internal membrane of the virus particles. This hypothesis was questioned recently because proteomic studies failed to detect the channel protein in virions of the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1). Here, we used a mAb raised against the functional K(+) channel from chlorovirus MA-1D to search for the viral K(+) channel in the virus particle. The results showed that the antibody was specific and bound to the tetrameric channel on the extracellular side. The antibody reacted in a virus-specific manner with protein extracts from chloroviruses that encoded channels similar to that from MA-1D. There was no cross-reactivity with chloroviruses that encoded more diverse channels or with a chlorovirus that lacked a K(+) channel gene. Together with electron microscopic imaging, which revealed labelling of individual virus particles with the channel antibody, these results establish that the viral particles contain an active K(+) channel, presumably located in the lipid membrane that surrounds the DNA in the mature virions.
Lerich A(1), Hillmer S, Langhans M, Scheuring D, van Bentum P, Robinson DG. (2012). ER Import Sites and Their Relationship to ER Exit Sites: A New Model for Bidirectional ER-Golgi Transport in Higher Plants. Front Plant Sci. 3:143.
Abstract
Per definition, ER exit sites are COPII vesiculation events at the surface of the ER and in higher plants are only visualizable in the electron microscope through cryofixation techniques. Fluorescent COPII labeling moves with Golgi stacks and locates to the interface between the ER and the Golgi. In contrast, the domain of the ER where retrograde COPI vesicles fuse, i.e., ER import sites (ERIS), has remained unclear. To identify ERIS we have employed ER-located SNAREs and tethering factors. We screened several SNAREs (SYP81, the SYP7 family, and USE1) to find a SNARE whose overexpression did not disrupt ER-Golgi traffic and which gave rise to discrete fluorescent punctae when expressed with an XFP tag. Only the Qc-SNARE SYP72 fulfilled these criteria. When coexpressed with SYP72-YFP, both the type I-membrane protein RFP-p24δ5 and the luminal marker CFP-HDEL whose ER localization are due to an efficient COPI-mediated recycling, form nodules along the tubular ER network. SYP72-YFP colocalizes with these nodules which are not seen when RFP-p24δ5 or CFP-HDEL is expressed alone or when SYP72-YFP is coexpressed with a mutant form of RFP-p24δ5 that cannot exit the ER. SYP72-YFP does not colocalize with Golgi markers, except when the Golgi stacks are immobilized through actin depolymerization. Endogenous SYP7 SNAREs, also colocalize with immobilized COPII/Golgi. In contrast, XFP-tagged versions of plant homologs to TIP20 of the Dsl1 COPI-tethering factor complex, and the COPII-tethering factor p115 colocalize perfectly with Golgi stacks irrespective of the motile status. These data suggest that COPI vesicle fusion with the ER is restricted to periods when Golgi stacks are stationary, but that when moving both COPII and COPI vesicles are tethered and collect in the ER-Golgi interface. Thus, the Golgi stack and an associated domain of the ER thereby constitute a mobile secretory and recycling unit: a unique feature in eukaryotic cells.
Montesinos JC(1), Sturm S, Langhans M, Hillmer S, Marcote MJ, Robinson DG, Aniento F. (2012). Coupled transport of Arabidopsis p24 proteins at the ER-Golgi interface. J Exp Bot. 63(11):4243-61.
Abstract
p24 proteins are a family of type I membrane proteins localized to compartments of the early secretory pathway and to coat protein I (COPI)- and COPII-coated vesicles. They can be classified, by sequence homology, into four subfamilies, named p24α, p24β, p24γ, and p24δ. In contrast to animals and fungi, plants contain only members of the p24β and p24δ subfamilies. It has previously been shown that transiently expressed red fluorescent protein (RFP)-p24δ5 localizes to the endoplasmic reticulum (ER) as a consequence of highly efficient COPI-based recycling from the Golgi apparatus. Using specific antibodies, endogenous p24δ5 has now been localized to the ER and p24β2 to the Golgi apparatus in Arabidopsis root tip cells by immunogold electron microscopy. The relative contributions of the cytosolic tail and the luminal domains to p24δ5 trafficking have also been characterized. It is demonstrated that whereas the dilysine motif in the cytoplasmic tail determines the location of p24δ5 in the early secretory pathway, the luminal domain may contribute to its distribution downstream of the Golgi apparatus. By using knock-out mutants and co-immunoprecipitation experiments, it is shown that p24δ5 and p24β2 interact with each other. Finally, it is shown that p24δ5 and p24β2 exhibit coupled trafficking at the ER-Golgi interface. It is proposed that p24δ5 and p24β2 interact with each other at ER export sites for ER exit and coupled transport to the Golgi apparatus. Once in the Golgi, p24δ5 interacts very efficiently with the COPI machinery for retrograde transport back to the ER.
Haydon MJ(1), Kawachi M, Wirtz M, Hillmer S, Hell R, Krämer U. (2012). Vacuolar nicotianamine has critical and distinct roles under iron deficiency and for zinc sequestration in Arabidopsis. Plant Cell. 24(2):724-37.
Abstract
Comment in Plant Cell. 2012 Feb;24(2):373.
Pubmed 
Hillmer S(1), Viotti C, Robinson DG. (2012). An improved procedure for low-temperature embedding of high-pressure frozen and freeze-substituted plant tissues resulting in excellent structural preservation and contrast. J Microsc. 247(1):43-7.
Abstract
Here we describe refinements in the processing of high-pressure frozen samples of delicate plant tissues for immuno-electron microscopy. These involve: shortened freeze-substitution schedules, lower temperatures during processing and polymerisation, the avoidance of temperature fluctuations and the optimisation of heat transfer from the specimens using small disposable aluminium containers. The application of these modifications leads to very good structural preservation and selective membrane contrast. As a result, the versatility of the method is increased since not only immuno-electron microscopical studies can be performed but often the quality is also quite suitable for structural investigations.
Pubmed 
Takatsuka C(1), Inoue Y, Higuchi T, Hillmer S, Robinson DG, Moriyasu Y. (2011). Autophagy in tobacco BY-2 cells cultured under sucrose starvation conditions: isolation of the autolysosome and its characterization. Plant Cell Physiol. 52(12):2074-87.
Abstract
Tobacco culture cells carry out a large-scale degradation of intracellular proteins in order to survive under sucrose starvation conditions. We have previously suggested that this bulk degradation of cellular proteins is performed by autophagy, where autolysosomes formed de novo act as the major lytic compartments. The digestion process in autolysosomes can be retarded by addition of the cysteine protease inhibitor E-64c to the culture medium, resulting in the accumulation of autolysosomes. In the present study, we have investigated several properties of autolysosomes in tobacco cells. Electron microscopy showed that the autolysosomes contain osmiophilic particles, some of which resemble partially degraded mitochondria. It also revealed the presence of two kinds of autolysosome precursor structures; one resembled the isolation membrane and the other the autophagosome of mammalian cells. Immunofluorescence microscopy showed that autolysosomes contain acid phosphatase, in accordance with cytochemical enzyme analyses by light and electron microscopy in a previous study. Autolysosomes isolated by cell fractionation on Percoll gradients showed the localization of acid phosphatase, vacuolar H(+)-ATPase and cysteine protease. These results show that starvation-induced autophagy in tobacco cells follows a macroautophagic-type response similar to that described for other eukaryotes. However, our results indicate that, although the plant vacuole is often described as being equivalent to the lysosome of the animal cell, a new low pH lytic compartment-the autolysosome-also contributes to proteolytic degradation when tobacco cells are subjected to sucrose deprivation.
Scheuring D(1), Viotti C, Krüger F, Künzl F, Sturm S, Bubeck J, Hillmer S, Frigerio L, Robinson DG, Pimpl P, Schumacher K. (2011). Multivesicular bodies mature from the trans-Golgi network/early endosome in Arabidopsis. Plant Cell. 23(9):3463-81.
Abstract
The plant trans-Golgi network/early endosome (TGN/EE) is a major hub for secretory and endocytic trafficking with complex molecular mechanisms controlling sorting and transport of cargo. Vacuolar transport from the TGN/EE to multivesicular bodies/late endosomes (MVBs/LEs) is assumed to occur via clathrin-coated vesicles, although direct proof for their participation is missing. Here, we present evidence that post-TGN transport toward lytic vacuoles occurs independently of clathrin and that MVBs/LEs are derived from the TGN/EE through maturation. We show that the V-ATPase inhibitor concanamycin A significantly reduces the number of MVBs and causes TGN and MVB markers to colocalize in Arabidopsis thaliana roots. Ultrastructural analysis reveals the formation of MVBs from the TGN/EE and their fusion with the vacuole. The localization of the ESCRT components VPS28, VPS22, and VPS2 at the TGN/EE and MVBs/LEs indicates that the formation of intraluminal vesicles starts already at the TGN/EE. Accordingly, a dominant-negative mutant of VPS2 causes TGN and MVB markers to colocalize and blocks vacuolar transport. RNA interference-mediated knockdown of the annexin ANNAT3 also yields the same phenotype. Together, these data indicate that MVBs originate from the TGN/EE in a process that requires the action of ESCRT for the formation of intraluminal vesicles and annexins for the final step of releasing MVBs as a transport carrier to the vacuole.
Sorieul M(1), Langhans M, Guetzoyan L, Hillmer S, Clarkson G, Lord JM, Roberts LM, Robinson DG, Spooner RA, Frigerio L. (2011). An Exo2 derivative affects ER and Golgi morphology and vacuolar sorting in a tissue-specific manner in arabidopsis. Traffic. 12(11):1552-62.
Abstract
We screened a panel of compounds derived from Exo2 - a drug that perturbs post-Golgi compartments and trafficking in mammalian cells - for their effect on the secretory pathway in Arabidopsis root epidermal cells. While Exo2 and most related compounds had no significant effect, one Exo2 derivative, named LG8, induced severe morphological alterations in both the Golgi (at high concentrations) and the endoplasmic reticulum (ER). LG8 causes the ER to form foci of interconnecting tubules, which at the ultrastructural level appear similar to those previously reported in Arabidopsis roots after treatment with the herbicide oryzalin. In cotyledonary leaves, LG8 causes redistribution of a trans Golgi network (TGN) marker to the vacuole. LG8 affects the anterograde secretory pathway by inducing secretion of vacuolar cargo and preventing the brassinosteroid receptor BRI1 from reaching the plasma membrane. Uptake and arrival at the TGN of the endocytic marker FM4-64 is not affected. Unlike the ADP ribosylation factor-GTP exchange factor (ARF-GEF) inhibitor brefeldin A (BFA), LG8 affects these post-Golgi events without causing the formation of BFA bodies. Up to concentrations of 50 µm, the effects of LG8 are reversible.
Pubmed 
Wang H(1), Zhuang XH, Hillmer S, Robinson DG, Jiang LW. (2011). Vacuolar sorting receptor (VSR) proteins reach the plasma membrane in germinating pollen tubes. Mol Plant. 4(5):845-53.
Abstract
Vacuolar sorting receptors (VSRs) are type I integral membrane proteins that mediate the vacuolar transport of soluble cargo proteins via prevacuolar compartments (PVCs) in plants. Confocal immunofluorescent and immunogold Electron Microscope (EM) studies have localized VSRs to PVCs or multivesicular bodies (MVBs) and trans-Golgi network (TGN) in various plant cell types, including suspension culture cells, root cells, developing and germinating seeds. Here, we provide evidence that VSRs reach plasma membrane (PM) in growing pollen tubes. Both immunofluorescent and immunogold EM studies with specific VSR antibodies show that, in addition to the previously demonstrated PVC/MVB localization, VSRs also localize to PM in lily and tobacco pollen tubes prepared from chemical fixation or high-pressure freezing/frozen substitution. Such a PM localization suggests an additional role of VSR proteins in mediating protein transport to PM and endocytosis in growing pollen tubes. Using a high-speed Spinning Disc Confocal Microscope, the possible fusion between VSR-positive PVC organelles and the PM was also observed in living tobacco pollen tubes transiently expressing the PVC reporter GFP-VSR. In contrast, the lack of a prominent PM localization of GFP-VSR in living pollen tubes may be due to the highly dynamic situation of vesicular transport in this fast-growing cell type.
Loos A(1), Van Droogenbroeck B, Hillmer S, Grass J, Pabst M, Castilho A, Kunert R, Liang M, Arcalis E, Robinson DG, Depicker A, Steinkellner H. (2011). Expression of antibody fragments with a controlled N-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of Arabidopsis. Plant Physiol. 155(4):2036-48.
Abstract
Intracellular trafficking and subcellular deposition are critical factors influencing the accumulation and posttranslational modifications of proteins. In seeds, these processes are not yet fully understood. In this study, we set out to investigate the intracellular transport, final destination, N-glycosylation status, and stability of the fusion of recombinant single-chain variable fragments to the crystallizing fragment of an antibody (scFv-Fc) of two antiviral monoclonal antibodies (2G12 and HA78). The scFv-Fcs were expressed in Arabidopsis (Arabidopsis thaliana) seeds and leaves both as secretory molecules and tagged with an endoplasmic reticulum (ER) retention signal. We demonstrate differential proteolytic degradation of scFv-Fcs in leaves versus seeds, with higher degradation in the latter organ. In seeds, we show that secretory versions of HA78 scFv-Fcs are targeted to the extracellular space but are deposited in newly formed ER-derived vesicles upon KDEL tagging. These results are in accordance with the obtained N-glycosylation profiles: complex-type and ER-typical oligomannosidic N-glycans, respectively. HA78 scFv-Fcs, expressed in seeds of an Arabidopsis glycosylation mutant lacking plant-specific N-glycans, exhibit custom-made human-type N-glycosylation. In contrast, 2G12 scFv-Fcs carry exclusively ER-typical oligomannosidic N-glycans and were deposited in newly formed ER-derived vesicles irrespective of the targeting signals. HA78 scFv-Fcs exhibited efficient virus neutralization activity, while 2G12 scFv-Fcs were inactive. We demonstrate the efficient generation of scFv-Fcs with a controlled N-glycosylation pattern. However, our results also reveal aberrant subcellular deposition and, as a consequence, unexpected N-glycosylation profiles. Our attempts to elucidate intracellular protein transport in seeds contributes to a better understanding of this basic cell biological mechanism and is a step toward the versatile use of Arabidopsis seeds as an alternative expression platform for pharmaceutically relevant proteins.
Wang J(1), Ding Y, Wang J, Hillmer S, Miao Y, Lo SW, Wang X, Robinson DG, Jiang L. (2010). EXPO, an exocyst-positive organelle distinct from multivesicular endosomes and autophagosomes, mediates cytosol to cell wall exocytosis in Arabidopsis and tobacco cells. Plant Cell. 22(12):4009-30.
Abstract
The exocyst protein complex mediates vesicle fusion with the plasma membrane. By expressing an (X)FP-tagged Arabidopsis thaliana homolog of the exocyst protein Exo70 in suspension-cultured Arabidopsis and tobacco (Nicotiana tabacum) BY-2 cells, and using antibodies specific for Exo70, we detected a compartment, which we term EXPO (for exocyst positive organelles). Standard markers for the Golgi apparatus, the trans-Golgi network/early endosome, and the multivesicular body/late endosome in plants do not colocalize with EXPO. Inhibitors of the secretory and endocytic pathways also do not affect EXPO. Exo70E2-(X)FP also locates to the plasma membrane (PM) as discrete punctae and is secreted outside of the cells. Immunogold labeling of sections cut from high-pressure frozen samples reveal EXPO to be spherical double membrane structures resembling autophagosomes. However, unlike autophagosomes, EXPOs are not induced by starvation and do not fuse with the lytic compartment or with endosomes. Instead, they fuse with the PM, releasing a single membrane vesicle into the cell wall. EXPOs are also found in other cell types, including root tips, root hair cells, and pollen grains. EXPOs therefore represent a form of unconventional secretion unique to plants.
Loos A(1), Van Droogenbroeck B, Hillmer S, Grass J, Kunert R, Cao J, Robinson DG, Depicker A, Steinkellner H. (2011). Production of monoclonal antibodies with a controlled N-glycosylation pattern in seeds of Arabidopsis thaliana. Plant Biotechnol J. 9(2):179-92.
Abstract
Seed-specific expression is an appealing alternative technology for the production of recombinant proteins in transgenic plants. Whereas attractive yields of recombinant proteins have been achieved by this method, little attention has been paid to the intracellular deposition and the quality of such products. Here, we demonstrate a comparative study of two antiviral monoclonal antibodies (mAbs) (HA78 against Hepatitis A virus; 2G12 against HIV) expressed in seeds of Arabidopsis wild-type (wt) plants and glycosylation mutants lacking plant specific N-glycan residues. We demonstrate that 2G12 is produced with complex N-glycans at great uniformity in the wt as well as in the glycosylation mutant, carrying a single dominant glycosylation species, GnGnXF and GnGn, respectively. HA78 in contrast, contains additionally to complex N-glycans significant amounts of oligo-mannosidic structures, which are typical for endoplasmic reticulum (ER)-retained proteins. A detailed subcellular localization study demonstrated the deposition of both antibodies virtually exclusively in the extracellular space, illustrating their efficient secretion. In addition, although a KDEL-tagged version of 2G12 exhibited an ER-typical N-glycosylation pattern, it was surprisingly detected in protein storage vacuoles. The different antibody variants showed different levels of degradation with hardly any degradation products detectable for HA78 carrying GnGnXF glycans. Finally, we demonstrate functional integrity of the HA78 and 2G12 glycoforms using viral inhibition assays. Our data therefore demonstrate the usability of transgenic seeds for the generation of mAbs with a controlled N-glycosylation pattern, thus expanding the possibilities for the production of optimally glycosylated proteins with enhanced biological activities for the use as human therapeutics.
Pubmed 
Schott A(1), Ravaud S, Keller S, Radzimanowski J, Viotti C, Hillmer S, Sinning I, Strahl S. (2010). Arabidopsis stromal-derived Factor2 (SDF2) is a crucial target of the unfolded protein response in the endoplasmic reticulum. J Biol Chem. 285(23):18113-21.
Abstract
Stresses increasing the load of unfolded proteins that enter the endoplasmic reticulum (ER) trigger a protective response termed the unfolded protein response (UPR). Stromal cell-derived factor2 (SDF2)-type proteins are highly conserved throughout the plant and animal kingdoms. In this study we have characterized AtSDF2 as crucial component of the UPR in Arabidopsis thaliana. Using a combination of biochemical and cell biological methods, we demonstrate that SDF2 is induced in response to ER stress conditions causing the accumulation of unfolded proteins. Transgenic reporter plants confirmed induction of SDF2 during ER stress. Under normal growth conditions SDF2 is highly expressed in fast growing, differentiating cells and meristematic tissues. The increased production of SDF2 due to ER stress and in tissues that require enhanced protein biosynthesis and secretion, and its association with the ER membrane qualifies SDF2 as a downstream target of the UPR. Determination of the SDF2 three-dimensional crystal structure at 1.95 A resolution revealed the typical beta-trefoil fold with potential carbohydrate binding sites. Hence, SDF2 might be involved in the quality control of glycoproteins. Arabidopsis sdf2 mutants display strong defects and morphological phenotypes during seedling development specifically under ER stress conditions, thus establishing that SDF2-type proteins play a key role in the UPR.
Wang H(1), Tse YC, Law AH, Sun SS, Sun YB, Xu ZF, Hillmer S, Robinson DG, Jiang L. (2009). Vacuolar sorting receptors (VSRs) and secretory carrier membrane proteins (SCAMPs) are essential for pollen tube growth. Plant J. 61(5):826-38.
Abstract
Vacuolar sorting receptors (VSRs) are type-I integral membrane proteins that mediate biosynthetic protein traffic in the secretory pathway to the vacuole, whereas secretory carrier membrane proteins (SCAMPs) are type-IV membrane proteins localizing to the plasma membrane and early endosome (EE) or trans-Golgi network (TGN) in the plant endocytic pathway. As pollen tube growth is an extremely polarized and highly dynamic process, with intense anterograde and retrograde membrane trafficking, we have studied the dynamics and functional roles of VSR and SCAMP in pollen tube growth using lily (Lilium longiflorum) pollen as a model. Using newly cloned lily VSR and SCAMP cDNA (termed LIVSR and LISCAMP, respectively), as well as specific antibodies against VSR and SCAMP1 as tools, we have demonstrated that in growing lily pollen tubes: (i) transiently expressed GFP-VSR/GFP-LIVSR is located throughout the pollen tubes, excepting the apical clear-zone region, whereas GFP-LISCAMP is mainly concentrated in the tip region; (ii) VSRs are localized to the multivesicular body (MVB) and vacuole, whereas SCAMPs are localized to apical endocytic vesicles, TGN and vacuole; and (iii) microinjection of VSR or SCAMP antibodies and LlVSR small interfering RNAs (siRNAs) significantly reduced the growth rate of the lily pollen tubes. Taken together, both VSR and SCAMP are required for pollen tube growth, probably working together in regulating protein trafficking in the secretory and endocytic pathways, which need to be coordinated in order to support pollen tube elongation.
Benz C(1), Engstler M, Hillmer S, Clayton C. (2009). Depletion of 14-3-3 proteins in bloodstream-form Trypanosoma brucei inhibits variant surface glycoprotein recycling. Int J Parasitol. 40(5):629-34.
Abstract
Bloodstream-form Trypanosoma brucei have two 14-3-3 proteins, which are required for parasite multiplication. We here describe the effects of 14-3-3 depletion on vesicular transport of variant surface glycoprotein (VSG). 14-3-3 depletion had no detectable effect on de novo synthesis and trafficking of VSG to the cell surface, or on VSG endocytosis. Despite strong inhibition of cell division, the flagellar pocket was not enlarged and the ultrastructure of internal organelles appeared normal. The Rab11-positive recycling endosome compartment was, however, fivefold smaller than normal, and the rate of return of recycling VSG to the surface was correspondingly reduced. Down-regulating 14-3-3 also prevented enlargement of the flagellar pocket by clathrin depletion. These results suggest that there is a remarkably specific requirement for 14-3-3 in normal functioning of the Rab11-positive recycling endosome compartment.
Pubmed 
Niemes S(1), Langhans M, Viotti C, Scheuring D, San Wan Yan M, Jiang L, Hillmer S, Robinson DG, Pimpl P. (2009). Retromer recycles vacuolar sorting receptors from the trans-Golgi network. Plant J. 61(1):107-21.
Abstract
Receptor-mediated sorting processes in the secretory pathway of eukaryotic cells rely on mechanisms to recycle the receptors after completion of transport. Based on this principle, plant vacuolar sorting receptors (VSRs) are thought to recycle after dissociating of receptor-ligand complexes in a pre-vacuolar compartment. This recycling is mediated by retromer, a cytosolic coat complex that comprises sorting nexins and a large heterotrimeric subunit. To analyse retromer-mediated VSR recycling, we have used a combination of immunoelectron and fluorescence microscopy to localize the retromer components sorting nexin 1 (SNX1) and sorting nexin 2a (SNX2a) and the vacuolar sorting protein VPS29p. All retromer components localize to the trans-Golgi network (TGN), which is considered to represent the early endosome of plants. In addition, we show that inhibition of retromer function in vivo by expression of SNX1 or SNX2a mutants as well as transient RNAi knockdown of all sorting nexins led to accumulation of the VSR BP80 at the TGN. Quantitative protein transport studies and live-cell imaging using fluorescent vacuolar cargo molecules revealed that arrival of these VSR ligands at the vacuole is not affected under these conditions. Based on these findings, we propose that the TGN is the location of retromer-mediated recycling of VSRs, and that transport towards the lytic vacuole downstream of the TGN is receptor-independent and occurs via maturation, similar to transition of the early endosome into the late endosome in mammalian cells.
Van Son L(1), Tiedemann J, Rutten T, Hillmer S, Hinz G, Zank T, Manteuffel R, Bäumlein H. (2009). The BURP domain protein AtUSPL1 of Arabidopsis thaliana is destined to the protein storage vacuoles and overexpression of the cognate gene distorts seed development. Plant Mol Biol. 71(4-5):319-29.
Abstract
BURP domain proteins comprise a broadly distributed, plant-specific family of functionally poorly understood proteins. VfUSP (Vicia faba Unknown Seed Protein) is the founding member of this family. The BURP proteins are characterized by a highly conserved C-terminal protein domain with a characteristic cysteine-histidine pattern. The Arabidopsis genome contains five BURP-domain encoding genes. Three of them are similar to the non-catalytic beta-subunit of the polygalacturonase of tomato and form a distinct subgroup. The remaining two genes are AtRD22 and AtUSPL1. The deduced product of AtUSPL1 is similar in size and sequence to VfUSP and that of the Brassica napus BNM2 gene which is expressed during microspore-derived embryogenesis. The protein products of BURP genes have not been found, especially that of VfUSP despite a great deal of interest arising from copious transcription of the gene in seeds. Here, we demonstrate that VfUSP and AtUSPL1 occur in cellular compartments essential for seed protein synthesis and storage, like the Golgi cisternae, dense vesicles, prevaculoar vesicles and the protein storage vacuoles in the parenchyma cells of cotyledons. Ectopic expression of AtUSPL1 leads to a shrunken seed phenotype; these seeds show structural alterations in their protein storage vacuoles and lipid vesicles. Furthermore, there is a reduction in the storage protein content and a perturbation in the seed fatty acid composition. However, loss of AtUSP1 gene function due to T-DNA insertions does not lead to a phenotypic change under laboratory conditions even though the seeds have less storage proteins. Thus, USP is pertinent to seed development but its role is likely shared by other proteins that function well enough under the laboratory growth conditions.
Lam SK(1), Cai Y, Hillmer S, Robinson DG, Jiang L. (2008). SCAMPs highlight the developing cell plate during cytokinesis in tobacco BY-2 cells. Plant Physiol. 147(4):1637-45.
Abstract
We previously demonstrated that rice (Oryza sativa) SECRETORY CARRIER MEMBRANE PROTEIN1 (OsSCAMP1)-yellow fluorescent protein in transgenic tobacco (Nicotiana tabacum) Bright Yellow-2 cells locates to the plasma membrane and to motile punctate structures, which represent the trans-Golgi network/early endosome and are tubular-vesicular in nature. Here, we now show that SCAMPs are diverted to the cell plate during cytokinesis dividing Bright Yellow-2 cells. As cells progress from metaphase to cytokinesis, punctate OsSCAMP1-labeled structures begin to collect in the future division plane. Together with the internalized endosomal marker FM4-64, they then become incorporated into the cell plate as it forms and expands. This was confirmed by immunogold electron microscopy. We also monitored for the Golgi apparatus and the prevacuolar compartment (PVC)/multivesicular body. Golgi stacks tend to accumulate in the vicinity of the division plane, but the signals are clearly separate to the cell plate. The situation with the PVC (labeled by green fluorescent protein-BP-80) is not so clear. Punctate BP-80 signals are seen at the advancing periphery of the cell plate, which was confirmed by immunogold electron microscopy. Specific but weak labeling was observed in the cell plate, but no evidence for a fusion of the PVC/multivesicular body with the cell plate could be obtained. Our data, therefore, support the notion that cell plate formation is mainly a secretory process involving mass incorporation of domains of the trans-Golgi network/early endosome membrane. We regard the involvement of multivesicular late endosomes in this process to be equivocal.
Olbrich A(1), Hillmer S, Hinz G, Oliviusson P, Robinson DG. (2007). Newly formed vacuoles in root meristems of barley and pea seedlings have characteristics of both protein storage and lytic vacuoles. Plant Physiol. 145(4):1383-94.
Abstract
Comment in Plant Physiol. 2008 Mar;146(3):1024-5; author reply 1026-7.
Pubmed 
Langhans M(1), Hawes C, Hillmer S, Hummel E, Robinson DG. (2007). Golgi regeneration after brefeldin A treatment in BY-2 cells entails stack enlargement and cisternal growth followed by division. Plant Physiol. 145(2):527-38.
Abstract
Brefeldin A (BFA) treatment stops secretion and leads to the resorption of much of the Golgi apparatus into the endoplasmic reticulum. This effect is reversible upon washing out the drug, providing a situation for studying Golgi biogenesis. In this investigation Golgi regeneration in synchronized tobacco BY-2 cells was followed by electron microscopy and by the immunofluorescence detection of ARF1, which localizes to the rims of Golgi cisternae and serves as an indicator of COPI vesiculation. Beginning as clusters of vesicles that are COPI positive, mini-Golgi stacks first become recognizable 60 min after BFA washout. They continue to increase in terms of numbers and length of cisternae for a further 90 min before overshooting the size of control Golgi stacks. As a result, increasing numbers of dividing Golgi stacks were observed 120 min after BFA washout. BFA-regeneration experiments performed on cells treated with BFA (10 microg mL(-1)) for only short periods (30-45 min) showed that the formation of ER-Golgi hybrid structures, once initiated by BFA treatment, is an irreversible process, the further incorporation of Golgi membranes into the ER continuing during a subsequent drug washout. Application of the protein kinase A inhibitor H-89, which effectively blocks the reassembly of the Golgi apparatus in mammalian cells, also prevented stack regeneration in BY-2 cells, but only at very high, almost toxic concentrations (>200 microm). Our data suggest that under normal conditions mitosis-related Golgi stack duplication may likely occur via cisternal growth followed by fission.
Hinz G(1), Colanesi S, Hillmer S, Rogers JC, Robinson DG. (2007). Localization of vacuolar transport receptors and cargo proteins in the Golgi apparatus of developing Arabidopsis embryos. Traffic. 8(10):1452-64.
Abstract
Using immunogold electron microscopy, we have investigated the relative distribution of two types of vacuolar sorting receptors (VSR) and two different types of lumenal cargo proteins, which are potential ligands for these receptors in the secretory pathway of developing Arabidopsis embryos. Interestingly, both cargo proteins are deposited in the protein storage vacuole, which is the only vacuole present during the bent-cotyledon stage of embryo development. Cruciferin and aleurain do not share the same pattern of distribution in the Golgi apparatus. Cruciferin is mainly detected in the cis and medial cisternae, especially at the rims where storage proteins aggregate into dense vesicles (DVs). Aleurain is found throughout the Golgi stack, particularly in the trans cisternae and trans Golgi network where clathrin-coated vesicles (CCVs) are formed. Nevertheless, aleurain was detected in both DV and CCV. VSR-At1, a VSR that recognizes N-terminal vacuolar sorting determinants (VSDs) of the NPIR type, localizes mainly to the trans Golgi and is hardly detectable in DV. Receptor homology-transmembrane-RING H2 domain (RMR), a VSR that recognizes C-terminal VSDs, has a distribution that is very similar to that of cruciferin and is found in DV. Our results do not support a role for VSR-At1 in storage protein sorting, instead RMR proteins because of their distribution similar to that of cruciferin in the Golgi apparatus and their presence in DV are more likely candidates. Aleurain, which has an NPIR motif and seems to be primarily sorted via VSR-At1 into CCV, also possesses putative hydrophobic sorting determinants at its C-terminus that could allow the additional incorporation of this protein into DV.
Wang J(1), Li Y, Lo SW, Hillmer S, Sun SS, Robinson DG, Jiang L. (2007). Protein mobilization in germinating mung bean seeds involves vacuolar sorting receptors and multivesicular bodies. Plant Physiol. 143(4):1628-39.
Abstract
Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination. Here we test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating seeds. We demonstrate that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean (Vigna radiata) seed germination. Immunogold electron microscopy with VSR antibodies demonstrate that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in day 1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at day 3 germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating seeds. Further confocal immunofluorescence and immunogold electron microscopy studies demonstrate that VSR and aleurain colocalize to MVBs as well as PSVs in germinating seeds. Thus, MVBs in germinating seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination.
Hummel E(1), Schmickl R, Hinz G, Hillmer S, Robinson DG. (2007). Brefeldin A action and recovery in Chlamydomonas are rapid and involve fusion and fission of Golgi cisternae. 27. Plant Biol (Stuttg). 2007 Jul;9(4):489-501.
Abstract
CHLAMYDOMONAS NOCTIGAMA has a non-motile Golgi apparatus consisting of several Golgi stacks adjacent to transitional ER. These domains are characterized by vesicle-budding profiles and the lack of ribosomes on the side of the ER proximal to the Golgi stacks. Immunogold labelling confirms the presence of COPI-proteins at the periphery of the Golgi stacks, and COPII-proteins at the ER-Golgi interface. After addition of BFA (10 microg/ml) a marked increase in the number of vesicular profiles lying between the ER and the Golgi stacks is seen. Serial sections of cells do not provide any evidence for the existence of tubular connections between the ER and the Golgi stacks, supporting the notion that COPI- but not COPII-vesicle production is affected by BFA. The fusion of COPII-vesicles at the CIS-Golgi apparatus apparently requires the presence of retrograde COPI-vesicles. After 15 min the cisternae of neighbouring Golgi stacks begin to fuse forming "mega-Golgis", which gradually curl before fragmenting into clusters of vesicles and tubules. These are surrounded by the transitional ER on which vesicle-budding profiles are still occasionally visible. Golgi remnants continue to survive for several hours and do not completely disappear. Washing out BFA leads to a very rapid reassembly of Golgi cisternae. At first, clusters of vesicles are seen adjacent to transitional ER, then "mini Golgis" are seen whose cisternae grow in length and number to produce "mega Golgis". These structures then divide by vertical fission to produce Golgi stacks of normal size and morphology roughly 60 min after drug wash-out.
Van Droogenbroeck B(1), Cao J, Stadlmann J, Altmann F, Colanesi S, Hillmer S, Robinson DG, Van Lerberge E, Terryn N, Van Montagu M, Liang M, Depicker A, De Jaeger G. (2007). Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds. Proc Natl Acad Sci U S A. 104(4):1430-5.
Abstract
Production of high-value recombinant proteins in transgenic seeds is an attractive and economically feasible alternative to conventional systems based on mammalian cells and bacteria. In contrast to leaves, seeds allow high-level accumulation of recombinant proteins in a relatively small volume and a stable environment. We demonstrate that single-chain variable fragment (scFv)-Fc antibodies, with N-terminal signal sequence and C-terminal KDEL tag, can accumulate to very high levels as bivalent IgG-like antibodies in Arabidopsis thaliana seeds and illustrate that a plant-produced anti-hepatitis A virus scFv-Fc has similar antigen-binding and in vitro neutralizing activities as the corresponding full-length IgG. As expected, most scFv-Fc produced in seeds contained only oligomannose-type N-glycans, but, unexpectedly, 35-40% was never glycosylated. A portion of the scFv-Fc was found in endoplasmic reticulum (ER)-derived compartments delimited by ribosome-associated membranes. Additionally, consistent with the glycosylation data, large amounts of the recombinant protein were deposited in the periplasmic space, implying a direct transport from the ER to the periplasmic space between the plasma membrane and the cell wall. Aberrant localization of the ER chaperones calreticulin and binding protein (BiP) and the endogenous seed storage protein cruciferin in the periplasmic space suggests that overproduction of recombinant scFv-Fc disturbs normal ER retention and protein-sorting mechanisms in the secretory pathway.
Lam SK(1), Siu CL, Hillmer S, Jang S, An G, Robinson DG, Jiang L. (2007). Rice SCAMP1 defines clathrin-coated, trans-golgi-located tubular-vesicular structures as an early endosome in tobacco BY-2 cells. Plant Cell. 19(1):296-319.
Abstract
We recently identified multivesicular bodies (MVBs) as prevacuolar compartments (PVCs) in the secretory and endocytic pathways to the lytic vacuole in tobacco (Nicotiana tabacum) BY-2 cells. Secretory carrier membrane proteins (SCAMPs) are post-Golgi, integral membrane proteins mediating endocytosis in animal cells. To define the endocytic pathway in plants, we cloned the rice (Oryza sativa) homolog of animal SCAMP1 and generated transgenic tobacco BY-2 cells expressing yellow fluorescent protein (YFP)-SCAMP1 or SCAMP1-YFP fusions. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that YFP-SCAMP1 fusions and native SCAMP1 localize to the plasma membrane and mobile structures in the cytoplasm of transgenic BY-2 cells. Drug treatments and confocal immunofluorescence studies demonstrated that the punctate cytosolic organelles labeled by YFP-SCAMP1 or SCAMP1 were distinct from the Golgi apparatus and PVCs. SCAMP1-labeled organelles may represent an early endosome because the internalized endocytic markers FM4-64 and AM4-64 reached these organelles before PVCs. In addition, wortmannin caused the redistribution of SCAMP1 from the early endosomes to PVCs, probably as a result of fusions between the two compartments. Immunogold electron microscopy with high-pressure frozen/freeze-substituted samples identified the SCAMP1-positive organelles as tubular-vesicular structures at the trans-Golgi with clathrin coats. These early endosomal compartments resemble the previously described partially coated reticulum and trans-Golgi network in plant cells.
Tse YC(1), Lo SW, Hillmer S, Dupree P, Jiang L. (2006). Dynamic response of prevacuolar compartments to brefeldin a in plant cells. Plant Physiol. 142(4):1442-59.
Abstract
Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs) in the secretory pathway. Using transgenic tobacco (Nicotiana tabacum) Bright-Yellow-2 (BY-2) cells expressing membrane-anchored yellow fluorescent protein (YFP) reporters marking Golgi or PVCs, we have recently demonstrated that PVCs are mobile multivesicular bodies defined by vacuolar sorting receptor proteins. Here, we demonstrate that Golgi and PVCs have different sensitivity in response to brefeldin A (BFA) treatment in living tobacco BY-2 cells. BFA at low concentrations (5-10 microg mL(-1)) induced YFP-marked Golgi stacks to form both endoplasmic reticulum-Golgi hybrid structures and BFA-induced aggregates, but had little effect on YFP-marked PVCs in transgenic BY-2 cells at both confocal and immunogold electron microscopy levels. However, BFA at high concentrations (50-100 microg mL(-1)) caused both YFP-marked Golgi stacks and PVCs to form aggregates in a dose- and time-dependent manner. Normal Golgi or PVC signals can be recovered upon removal of BFA from the culture media. Confocal immunofluorescence and immunogold electron microscopy studies with specific organelle markers further demonstrate that the PVC aggregates are distinct, but physically associated, with Golgi aggregates in BFA-treated cells and that PVCs might lose their internal vesicle structures at high BFA concentration. In addition, vacuolar sorting receptor-marked PVCs in root-tip cells of tobacco, pea (Pisum sativum), mung bean (Vigna radiata), and Arabidopsis (Arabidopsis thaliana) upon BFA treatment are also induced to form similar aggregates. Thus, we have demonstrated that the effects of BFA are not limited to endoplasmic reticulum and Golgi, but extend to PVC in the endomembrane system, which might provide a quick tool for distinguishing Golgi from PVC for its identification and characterization, as well as a possible new tool in studying PVC-mediated protein traffic in plant cells.
Oliviusson P(1), Heinzerling O, Hillmer S, Hinz G, Tse YC, Jiang L, Robinson DG. (2006). Plant retromer, localized to the prevacuolar compartment and microvesicles in Arabidopsis, may interact with vacuolar sorting receptors. Plant Cell. 18(5):1239-52.
Abstract
Receptors for acid hydrolases destined for the lytic compartment in yeast and mammalian cells are retrieved from intermediate, endosomal organelles with the help of a pentameric protein complex called the retromer. We cloned the Arabidopsis thaliana homologs of the three yeast proteins (Vps35, Vps29, and Vps26) constituting the larger subunit of retromer and prepared antisera against them. With these antibodies, we demonstrated the presence of a retromer-like protein complex in salt extracts prepared from Arabidopsis microsomes. This complex is associated with membranes that coequilibrate with prevacuolar compartment markers and with high-density sedimenting membranes. Immunogold negative staining identified these membranes as 90-nm-diameter coated microvesicles. Confocal laser scanning immunofluorescence studies performed on tobacco (Nicotiana tabacum) BY-2 cells revealed high degrees of colabeling between all three retromer antisera and the prevacuolar compartment (PVC) markers PEP12 and vacuolar sorting receptor VSR(At-1). The presence of plant retromer at the surface of multivesicular bodies was also demonstrated by immunogold labeling of sections obtained from high-pressure frozen/freeze-substituted specimens. Treatment of BY-2 cells with wortmannin led to swelling of the PVC and a separation of the VPS35 and VSR signals. Preliminary data suggesting that retromer interacts with the cytosolic domain of a VSR were obtained by immunoprecipitation experiments performed on detergent-solubilized microsomes with Vps35 antibodies.
Pimpl P(1), Taylor JP, Snowden C, Hillmer S, Robinson DG, Denecke J. (2005). Golgi-mediated vacuolar sorting of the endoplasmic reticulum chaperone BiP may play an active role in quality control within the secretory pathway. Plant Cell. 18(1):198-211.
Abstract
Quality control in the endoplasmic reticulum (ER) prevents the arrival of incorrectly or incompletely folded proteins at their final destinations and targets permanently misfolded proteins for degradation. Such proteins have a high affinity for the ER chaperone BiP and are finally degraded via retrograde translocation from the ER lumen back to the cytosol. This ER-associated protein degradation (ERAD) is currently thought to constitute the main disposal route, but there is growing evidence for a vacuolar role in quality control. We show that BiP is transported to the vacuole in a wortmannin-sensitive manner in tobacco (Nicotiana tabacum) and that it could play an active role in this second disposal route. ER export of BiP occurs via COPII-dependent transport to the Golgi apparatus, where it competes with other HDEL receptor ligands. When HDEL-mediated retrieval from the Golgi fails, BiP is transported to the lytic vacuole via multivesicular bodies, which represent the plant prevacuolar compartment. We also demonstrate that a subset of BiP-ligand complexes is destined to the vacuole and differs from those likely to be disposed of via the ERAD pathway. Vacuolar disposal could act in addition to ERAD to maximize the efficiency of quality control in the secretory pathway.
Tse YC(1), Mo B, Hillmer S, Zhao M, Lo SW, Robinson DG, Jiang L. (2004). Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells. Plant Cell. 16(3):672-93.
Abstract
Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs). We have demonstrated recently that vacuolar sorting receptor (VSR) proteins are concentrated on PVCs. In this study, we generated transgenic Nicotiana tabacum (tobacco) BY-2 cell lines expressing two yellow fluorescent protein (YFP)-fusion reporters that mark PVC and Golgi organelles. Both transgenic cell lines exhibited typical punctate YFP signals corresponding to distinct PVC and Golgi organelles because the PVC reporter colocalized with VSR proteins, whereas the Golgi marker colocalized with mannosidase I in confocal immunofluorescence. Brefeldin A induced the YFP-labeled Golgi stacks but not the YFP-marked PVCs to form typical enlarged structures. By contrast, wortmannin caused YFP-labeled PVCs but not YFP-labeled Golgi stacks to vacuolate. VSR antibodies labeled multivesicular bodies (MVBs) on thin sections prepared from high-pressure frozen/freeze substituted samples, and the enlarged PVCs also were indentified as MVBs. MVBs were further purified from BY-2 cells and found to contain VSR proteins via immunogold negative staining. Similar to YFP-labeled Golgi stacks, YFP-labeled PVCs are mobile organelles in BY-2 cells. Thus, we have unequivocally identified MVBs as PVCs in N. tabacum BY-2 cells. Uptake studies with the styryl dye FM4-64 strongly indicate that PVCs also lie on the endocytic pathway of BY-2 cells.
Laporte C(1), Vetter G, Loudes AM, Robinson DG, Hillmer S, Stussi-Garaud C, Ritzenthaler C. (2003). Involvement of the secretory pathway and the cytoskeleton in intracellular targeting and tubule assembly of Grapevine fanleaf virus movement protein in tobacco BY-2 cells. Plant Cell. 15(9):2058-75.
Abstract
Grapevine fanleaf virus (GFLV) is one of a large class of plant viruses whose cell-to-cell transport involves the passage of virions through tubules composed of virus-encoded movement protein (MP). The tubules are embedded within modified plasmodesmata, but the mechanism of targeting of MP to these sites is unknown. To study intracellular GFLV MP trafficking, a green fluorescent protein-MP fusion (GFP:MP) was expressed in transgenic tobacco BY-2 suspension cells under the control of an inducible promoter. We show that GFP:MP is targeted preferentially to calreticulin-labeled foci within the youngest cross walls, where it assembles into tubules. During cell division, GFP:MP colocalizes in the cell plate with KNOLLE, a cytokinesis-specific syntaxin, and both proteins are linked physically, as shown by coimmunoprecipitation of the two proteins from the same microsomal fraction. In addition, treatment with various drugs has revealed that a functional secretory pathway, but not the cytoskeleton, is required for tubule formation. However, correct GFP:MP targeting to calreticulin-labeled foci seems to be cytoskeleton dependent. Finally, biochemical analyses have revealed that at least a fraction of the MP behaves as an intrinsic membrane protein. These findings support a model in which GFP:MP would be transported to specific sites via Golgi-derived vesicles along two different pathways: a microtubule-dependent pathway in normal cells and a microfilament-dependent default pathway when microtubules are depolymerized.
Voncken F(1), van Hellemond JJ, Pfisterer I, Maier A, Hillmer S, Clayton C. (2003). Depletion of GIM5 causes cellular fragility, a decreased glycosome number, and reduced levels of ether-linked phospholipids in trypanosomes. J Biol Chem. 278(37):35299-310.
Abstract
Microbody division in mammalian cells, trypanosomes, and yeast depends on the PEX11 microbody membrane proteins. The function of PEX11 is not understood, and the suggestion that it affects microbody (peroxisome) numbers in mammals and yeast, because it plays a role in beta-oxidation of fatty acids, is controversial. PEX11 and two PEX11-related proteins, GIM5A and GIM5B, are the predominant membrane proteins of the microbodies (glycosomes) of Trypanosoma brucei. The compartmentation of glycosomal enzymes is essential in trypanosomes. Deletion of the GIM5A gene from the form of the parasite that lives in the mammalian blood has no effect on trypanosome growth, but depletion of GIM5B on a gim5a null background causes death. We show here that procyclic trypanosomes, adapted for life in the Tsetse fly vector, survive without GIM5A and with very low levels of GIM5B. The depleted cells have fewer glycosomes than usual and are osmotically fragile, which is a novel observation for a microbody defect. Thus trypanosomes require both GIM5B and PEX11 for the maintenance of normal glycosome numbers. Procyclic cells lacking GIM5A, like mouse cells partially defective in PEX11, have fewer ether-linked phospholipids, even when GIM5B levels are not reduced. Metabolite measurements on GIM5A/B-depleted bloodstream form trypanosomes suggested a change in the flux through the glycolytic pathway. We conclude that PEX11 family proteins play important roles in determining microbody membrane structure, with secondary effects on a subset of microbody metabolic pathways.
Sohn EJ(1), Kim ES, Zhao M, Kim SJ, Kim H, Kim YW, Lee YJ, Hillmer S, Sohn U, Jiang L, Hwang I. (2003). Rha1, an Arabidopsis Rab5 homolog, plays a critical role in the vacuolar trafficking of soluble cargo proteins. Plant Cell. 15(5):1057-70.
Abstract
Rab proteins are members of the Ras superfamily of small GTP binding proteins and play important roles in various intracellular trafficking steps. We investigated the role of Rha1, an Arabidopsis Rab5 homolog, in intracellular trafficking in Arabidopsis protoplasts. In the presence of a dominant-negative mutant of Rha1, soluble vacuolar cargo proteins such as sporamin:green fluorescent protein (Spo:GFP) and Arabidopsis aleurain like protein:GFP are not delivered to the central vacuole; instead, they accumulate as a diffuse or punctate staining pattern within the cell. Spo:GFP at the punctate stains observed in the presence of hemagglutinin:Rha1[S24N] is colocalized with endogenous vacuolar sorting receptor (VSR(At-1)), which is known to localize primarily to the prevacuolar compartment, whereas Spo:GFP in the diffuse pattern is associated with tonoplasts. Furthermore, expression of Rha1[S24N] causes the secretion of a portion of the vacuolar proteins into medium. However, the inhibitory effect of Rha1[S24N] on vacuolar trafficking is relieved partially by coexpressed wild-type Rha1. Based on these results, we propose that Rha1 plays a critical role in the trafficking of soluble cargoes from the prevacuolar compartment to the central vacuole.
Pich A(1), Manteuffel R, Hillmer S, Scholz G, Schmidt W. (2001). Fe homeostasis in plant cells: does nicotianamine play multiple roles in the regulation of cytoplasmic Fe concentration? Planta. 213(6):967-76.
Abstract
The cellular and intracellular localization of the non-proteogenic amino acid nicotianamine (NA) in leaves and root elongation zones was immunochemically investigated in pea (Pisum sativum L.) and tomato (Lycopersicon esculentum Mill.) plants grown under various iron regimes and in three mutants defective in the regulation of iron uptake. Strongest immunostaining was observed in the over-accumulating pea mutants brz and dgl, and in iron-loaded wild-type plants. Fe concentration and NA level paralleled staining intensity, indicating that NA synthesis is induced by high iron availability. While label was mainly present in the cytoplasm under normal (10 microM) Fe supply and under Fe deprivation, most of the labeling was present in the vacuole in iron-loaded plants. This pattern resembled the distribution of NA in Fe over-accumulating mutants, indicating the possible importance of vacuolar sequestration in the detoxification of excess Fe. Based on the dependence of the cellular distribution of NA on the iron nutritional status of the plant, a possible role of NA in buffering free Fe in root and leaf cells was inferred. We show here for the first time that the NA concentration is increased in response to iron overload, indicating that, besides other classes of intracellular metal-binding ligands, NA may play an essential role in iron tolerance.
Törmäkangas K(1), Hadlington JL, Pimpl P, Hillmer S, Brandizzi F, Teeri TH, Denecke J. (2001). A vacuolar sorting domain may also influence the way in which proteins leave the endoplasmic reticulum. Plant Cell. 13(9):2021-32.
Abstract
Protein sorting to plant vacuoles is known to be dependent on a considerable variety of protein motifs recognized by a family of sorting receptors. This can involve either traffic from the endoplasmic reticulum (ER) through the Golgi apparatus or direct ER-to-vacuole transport. Barley aspartic protease (Phytepsin) was shown previously to reach the vacuole via trafficking through the Golgi apparatus. Here we show that Phytepsin normally exits the ER in a COPII-mediated manner, because the Phytepsin precursor accumulates in the ER upon specific inhibition of the formation of COPII vesicles in vivo. Phytepsin differs from its yeast and mammalian counterparts by the presence of a saposin-like plant-specific insert (PSI). Deletion of this domain comprising 104 amino acids causes efficient secretion of the truncated molecule (Phytepsin Delta PSI) without affecting the enzymatic activity of the enzyme. Interestingly, deletion of the PSI also changes the way in which Phytepsin exits the ER. Inhibition of COPII vesicle formation causes accumulation of the Phytepsin precursor in the ER but has no effect on the secretion of Phytepsin Delta PSI. This suggests either that vacuolar sorting commences at the ER export step and involves recruitment into COPII vesicles or that the PSI domain carries two signals, one for COPII-dependent export from the ER and one for vacuolar delivery from the Golgi. The relevance of these observations with respect to the bulk flow model of secretory protein synthesis is discussed.
Hillmer S(1), Movafeghi A, Robinson DG, Hinz G. (2001). Vacuolar storage proteins are sorted in the cis-cisternae of the pea cotyledon Golgi apparatus. J Cell Biol. 152(1):41-50.
Abstract
Developing pea cotyledons contain functionally different vacuoles, a protein storage vacuole and a lytic vacuole. Lumenal as well as membrane proteins of the protein storage vacuole exit the Golgi apparatus in dense vesicles rather than in clathrin-coated vesicles (CCVs). Although the sorting receptor for vacuolar hydrolases BP-80 is present in CCVs, it is not detectable in dense vesicles. To localize these different vacuolar sorting events in the Golgi, we have compared the distribution of vacuolar storage proteins and of alpha-TIP, a membrane protein of the protein storage vacuole, with the distribution of the vacuolar sorting receptor BP-80 across the Golgi stack. Analysis of immunogold labeling from cryosections and from high pressure frozen samples has revealed a steep gradient in the distribution of the storage proteins within the Golgi stack. Intense labeling for storage proteins was registered for the cis-cisternae, contrasting with very low labeling for these antigens in the trans-cisternae. The distribution of BP-80 was the reverse, showing a peak in the trans-Golgi network with very low labeling of the cis-cisternae. These results indicate a spatial separation of different vacuolar sorting events in the Golgi apparatus of developing pea cotyledons.
Pimpl P(1), Movafeghi A, Coughlan S, Denecke J, Hillmer S, Robinson DG. (2000). In situ localization and in vitro induction of plant COPI-coated vesicles. Plant Cell. 12(11):2219-36.
Abstract
Comment in Plant Cell. 2000 Nov;12(11):2009-11.
Pubmed 
Bauly JM(1), Sealy IM, Macdonald H, Brearley J, Dröge S, Hillmer S, Robinson DG, Venis MA, Blatt MR, Lazarus CM, Napier RM. (2000). Overexpression of auxin-binding protein enhances the sensitivity of guard cells to auxin. Plant Physiol. 124(3):1229-38.
Abstract
To explore the role of auxin-binding protein (ABP1) in planta, a number of transgenic tobacco (Nicotiana tabacum) lines were generated. The wild-type KDEL endoplasmic reticulum targeting signal was mutated to HDEL, another common retention sequence in plants, and to KEQL or KDELGL to compromise its activity. The auxin-binding kinetics of these forms of ABP1 were found to be similar to those of ABP1 purified from maize (Zea mays). To test for a physiological response mediated by auxin, intact guard cells of the transgenic plants were impaled with double-barreled microelectrodes, and auxin-dependent changes in K(+) currents were recorded under voltage clamp. Exogenous auxin affected inwardly and outwardly rectifying K(+) currents in a dose-dependent manner. Auxin sensitivity was markedly enhanced in all plants overexpressing ABP1, irrespective of the form present. Immunogold electron microscopy was used to investigate the localization of ABP1 in the transgenic plants. All forms were detected in the endoplasmic reticulum and the KEQL and KDELGL forms passed further across the Golgi stacks than KDEL and HDEL forms. However, neither electron microscopy nor silver-enhanced immunogold epipolarization microscopy revealed differences in cell surface ABP1 abundance for any of the plants, including control plants, which indicated that overexpression of ABP1 alone was sufficient to confer increased sensitivity to added auxin. Jones et al. ([1998] Science 282: 1114-1117) found increased cell expansion in transgenic plants overexpressing wild-type ABP1. Single cell recordings extend this observation, with the demonstration that the auxin sensitivity of guard cell K(+) currents is mediated, at least in part, by ABP1.
Fischer J(1), Becker C, Hillmer S, Horstmann C, Neubohn B, Schlereth A, Senyuk V, Shutov A, Müntz K. (2000). The families of papain- and legumain-like cysteine proteinases from embryonic axes and cotyledons of Vicia seeds: developmental patterns, intracellular localization and functions in globulin proteolysis. Plant Mol Biol. 43(1):83-101.
Abstract
Families of papain- and legumain-like cysteine proteinases (CPR) were found in Vicia seeds. cDNAs and antibodies were used to follow organ specificity and the developmental course of CPR-specific mRNAs and polypeptides. Four papain-like cysteine proteinases (CPR1, CPR2, proteinase A and CPR4) from vetch seeds (Vicia sativa L.) were analysed. CPR2 and its mRNA were already found in dry embryonic axes. CPR1 was only detected there during early germination. Both CPR1 and CPR2 strongly increased later during germination. In cotyledons, both CPR1 and CPR2 were only observed one to two days later than in the axis. Proteinase A was not found in axes. In cotyledons it could only be detected several days after seeds had germinated. CPR4 mRNA and polypeptide were already present in embryonic axes and cotyledons during seed maturation and decreased in both organs during germination. Purified CPR1, CPR2 and proteinase A exhibited partially different patterns of globulin degradation products in vitro. Although the cDNA-deduced amino acid sequence of the precursor of proteinase A has an N-terminal signal peptide, the enzyme was not found in vacuoles whereas the other papain-like CPRs showed vacuolar localization. Four different legumain-like cysteine proteinases (VsPB2, proteinase B, VnPB1 and VnPB2) of Vicia species were analysed. Proteinase B and VnPB1 mRNAs were detected in cotyledons and seedling organs after seeds had germinated. Proteinase B degraded globulins isolated from mature vetch seeds in vitro. VsPB2 and proteinase B are localized to protein bodies of maturing seeds and seedlings, respectively, of V. sativa. Like VsPB2 from V sativa, also VnPB2 of V. narbonensis corresponds to vacuolar processing enzymes (betaVPE). Based on these results different functions in molecular maturation and mobilization of storage proteins could be attributed to the various members of the CPR families.
Piedras P(1), Rivas S, Dröge S, Hillmer S, Jones JD. (2000). Functional, c-myc-tagged Cf-9 resistance gene products are plasma-membrane localized and glycosylated. Plant J. 21(6):529-36.
Abstract
The Cf-9 resistance gene from tomato confers resistance to races of the fungal pathogen Cladosporium fulvum that express the corresponding avirulence gene, Avr9. Avr9 encodes a secreted peptide. To investigate Cf-9 function, we tagged the Cf-9 protein with a triple myc epitope at either the amino- or carboxy-terminus of the mature protein. Tobacco plants carrying these constructs activate a defence response to Avr9 peptide. The Cf-9 sequence predicts a protein of 94 kDa, with 22 glycosylation sites. Using c-myc antibodies, c-myc : Cf-9 protein was detected as a unique band with a molecular size of 160 kDa. The band shifted to approximately 105 kDa after glucosidase treatment, indicating that Cf-9 protein is highly glycosylated. Plasma membranes were isolated using two-phase partitioning, and c-myc : Cf-9 was enriched in these fractions, indicating that Cf-9 is a plasma membrane protein. This was confirmed by silver-enhanced immunogold labelling of tobacco protoplasts carrying the amino-terminal c-myc tag; a higher labelling density was observed on the surface of protoplasts derived from c-myc : Cf-9 tobacco compared to untransformed control. The presence of Cf-9 in the plasma membrane is consistent with its role in conferring recognition of the extracellular Avr9 peptide.
Crofts AJ(1), Leborgne-Castel N, Hillmer S, Robinson DG, Phillipson B, Carlsson LE, Ashford DA, Denecke J. (1999). Saturation of the endoplasmic reticulum retention machinery reveals anterograde bulk flow Plant Cell. 11(11):2233-48.
Abstract
We have studied the possible mechanisms of endoplasmic reticulum (ER) export and retention by using natural residents of the plant ER. Under normal physiological conditions, calreticulin and the lumenal binding protein (BiP) are efficiently retained in the ER. When the ER retention signal is removed, truncated calreticulin is much more rapidly secreted than truncated BiP. Calreticulin carries two glycans of the typical ER high-mannose form. Both glycans are competent for Golgi-based modifications, as determined from treatment with brefeldin A or based on the deletion of the ER retention motif. In contrast to BiP, calreticulin accumulation is strongly dependent on its retention signal, thereby allowing us to test whether saturation of the retention mechanism is possible. Overexpression of calreticulin led to 100-fold higher levels in dilated globular ER cisternae as well as dilated nuclear envelopes and partial secretion of both BiP and calreticulin. This result shows that both molecules are competent for ER export and supports the concept that proteins are secreted by default. This result also supports previous data suggesting that truncated BiP devoid of its retention motif can be retained in the ER by association with calreticulin. Moreover, even under these saturating conditions, cellular calreticulin did not carry significant amounts of complex glycans, in contrast to secreted calreticulin. This result shows that calreticulin is rapidly secreted once complex glycans have been synthesized in the medial/trans Golgi apparatus and that the modified protein does not appear to recycle back to the ER.
Hinz G(1), Hillmer S, Baumer M, Hohl I I. (1999). Vacuolar storage proteins and the putative vacuolar sorting receptor BP-80 exit the golgi apparatus of developing pea cotyledons in different transport vesicles Plant Cell. 11(8):1509-24.
Abstract
In the parenchyma cells of developing legume cotyledons, storage proteins are deposited in a special type of vacuole, known as the protein storage vacuole (PSV). Storage proteins are synthesized at the endoplasmic reticulum and pass through the Golgi apparatus. In contrast to lysosomal acid hydrolases, storage proteins exit the Golgi apparatus in 130-nm-diameter electron-dense vesicles rather than in clathrin-coated vesicles. By combining isopycnic and rate zonal sucrose density gradient centrifugation with phase partitioning, we obtained a highly enriched dense vesicle fraction. This fraction contained prolegumin, which is the precursor of one of the major storage proteins. In dense vesicles, prolegumin occurred in a more aggregated form than it did in the endoplasmic reticulum. The putative vacuolar sorting receptor BP-80 was highly enriched in purified clathrin-coated vesicles, which, in turn, did not contain prolegumin. The amount of BP-80 was markedly reduced in the dense vesicle fraction. This result was confirmed by quantitative immunogold labeling of cryosections of pea cotyledons: whereas antibodies raised against BP-80 significantly labeled the Golgi stacks, labeling of the dense vesicles could not be detected. In contrast, 90% of the dense vesicles were labeled with antibodies raised against alpha-TIP (for tonoplast intrinsic protein), which is the aquaporin specific for the membrane of the PSV. These results lead to the conclusions that storage proteins and alpha-TIP are delivered via the same vesicular pathway into the PSVs and that the dense vesicles that carry these proteins in turn do not contain BP-80.
Steffens P(1), Van HN, Hillmer S, Saalbach I, Müntz K. (1997). Subcellular localization of the 2S globulin narbonin in seeds of Vicia narbonensis. Planta. 203(1):44-50.
Abstract
Narbonin is a 2S protein from the globulin fraction of narbon bean (Vicia narbonensis L.) cotyledons. Its amino acid composition and the pattern of its regulated accumulation in developing seeds led to the suggestion that narbonin could be a storage protein. Therefore, it was expected to be present in protein bodies of the storage tissue cells. Comparison of the cDNA-derived amino acid sequence with a directly determined partial N-terminal sequence revealed that the primary translation product of narbonin mRNA lacks a transient N-terminal signal peptide (V.H. Nong et al., 1995, Plant Mol Biol 28: 61 - 72). Narbonin polypeptides that had been synthesized in a cell-free translation system supplemented with dog pancreas microsomes were not protected against degradation by posttranslationally added proteases (protease protection assay). In accordance with the lack of a signal peptide this indicates that the polypeptide was not cotranslationally sequestered into the microsomes. The protein-body fraction that had been isolated from mature narbon bean cotyledons by a non-aqueous gradient centrifugation procedure was free of narbonin; this was found in the soluble cell fraction. In electron micrographs, narbonin could be localized in the cytoplasm using the immuno gold-labelling technique. Previously, it had already been shown that narbonin is too slowly degraded during narbon bean germination to act as a storage protein. From all these results it has to be concluded that narbonin is a cytoplasmic protein which does not belong to the storage proteins in the restricted sense. Other possible functions are discussed.
Bethke PC(1), Hillmer S, Jones RL. (1996). Isolation of Intact Protein Storage Vacuoles from Barley Aleurone (Identification of Aspartic and Cysteine Proteases). Plant Physiol. 110(2):521-529.
Abstract
Within the cereal aleurone reserve proteins are stored in specialized organelles, the protein storage vacuoles (PSV). We developed an aqueous method for the isolation of intact PSV. Barley (Hordeum vulgare L. cv Himalaya) aleurone protoplasts were gently lysed by passing them through a syringe needle. PSV were separated from cytoplasmic components by microfiltration and low-speed centrifugation. Isolated PSV appeared by light microscopy to be identical with those within barley aleurone protoplasts. Luminal contents were retained throughout the isolation procedure. We used isolated PSV to identify and characterize PSV-associated proteolytic activities. Isolated PSV contained cysteine proteases and aspartic proteases (APs). Gibberellic acid treatment of protoplasts increased cysteine protease activity. Protein blots probed with anti-H. vulgare aspartic proteinase (HvAP) indicated that one PSV-AP was HvAP. Immunocytochemical localization by electron microscopy confirmed the presence of HvAP within the lumen of PSV. We conclude that isolated barley aleurone PSV will be useful in further characterizing this organelle.
Hillmer S(1), Gilroy S, Jones RL. (1993). Visualizing Enzyme Secretion from Individual Barley (Hordeum vulgare) Aleurone Protoplasts. Plant Physiol. 102(1):279-286.
Abstract
A method was developed to detect [alpha]-amylase gene expression and [alpha]-amylase secretion from individual barley (Hordeum vulgare L. cv Himalaya) aleurone protoplasts. Protoplasts are incubated in liquid media with or without hormones and embedded in a thin film of agarose and starch, where they remain viable for up to 24 h. [alpha]-Amylase secreted by individual protoplasts digests the starch, and starch hydrolysis is visualized after 45 min by staining the preparation with I2KI. After I2KI staining, secreting protoplasts are surrounded by a clear, starch-free halo visible by light microscopy. The formation of starch-free halos is dependent on the synthesis and secretion of [alpha]-amylase and is not caused by carry-over of preformed enzyme from incubation media. Treating protoplasts with inhibitors of protein synthesis or exposing them to anaerobic conditions for 2 h before embedding them in agarose prevents the formation of halos. When [alpha]-amylase secretion is observed by counting the percentage of secreting protoplasts, the data are comparable to that obtained by measuring [alpha]-amylase secretion from a population of cells. The response of individual protoplasts to gibberellic acid (GA3) and abscisic acid measured by the thin-film method is almost identical to the response of populations of protoplasts to these hormones, validating the utility of this method. Although not generally practical for quantifying secretion, the thin-film method is uniquely useful in distinguishing secreting from nonsecreting protoplasts. In none of our experiments did more than 60% of the protoplasts secrete [alpha]-amylase when exposed to GA3, even though more than 95% of the protoplasts in the preparations were viable. Similar results were obtained when the response to GA3 was assayed at the level of gene transcription by visualizing the transient expression of a plasmid containing the promoter from [alpha]-amylase fused to the reporter gene glucuronidase in single protoplasts. The thin-film secretion assay also revealed that the response of a population of protoplasts to GA3 was not uniform with time. The effect of GA3 treatment was to gradually increase the percentage of responding protoplasts up to a maximum of 50 to 60%. Abscisic acid, which inhibits [alpha]-amylase secretion by GA3-treated protoplasts, reduced the proportion of protoplasts that secrete the enzyme.
Hillmer S(1), Joachim S, Robinson DG. ( 49. Histochemistry. 1991;95(3):315-8. ). Rapid polymerization of LR-white for immunocytochemistry. 49. Histochemistry. 1991;95(3):315-8.
Abstract
London Resin (LR) White a hydrophilic embedding medium for immunocytochemistry, can be polymerized in a commercial microwave oven in seven minutes using the chemical accelerator benzoyl peroxide. In order to minimize the effects of heating, the polymerization vessel was maintained in an ice bath. We demonstrate that this procedure does not affect the antigenicity of either barley aleurone nuclease or of the catalytic and regulatory subunits of rat parotid cAMP-dependent protein kinase.
Hillmer S(1), Bush DS, Robinson DG, Zingen-Sell I, Jones RL. (1990). Barley aleurone protoplasts are structurally and functionally similar to the walled cells of aleurone layers. Eur J Cell Biol. 52(1):169-73.
Abstract
PMID: 2387306 [PubMed - indexed for MEDLINE]

/var/www/cos/ / http://www.cos.uni-heidelberg.de/ Dr. Stefan Hillmer