Pancreatic acinar cells express vesicle-associated membrane protein 2- and 8-specific populations of zymogen granules with distinct and overlapping roles in secretion

Previous studies have demonstrated roles for vesicle-associated membrane protein 2 (VAMP 2) and VAMP 8 in Ca(2+)-regulated pancreatic acinar cell secretion, however, their coordinated function in the secretory pathway has not been addressed. Here we provide evidence using immunofluorescence microscopy, cell fractionation, and SNARE protein interaction studies that acinar cells contain two distinct populations of zymogen granules (ZGs) expressing either VAMP 2 or VAMP 8. Further, VAMP 8-positive granules also contain the synaptosome-associated protein 29, whereas VAMP 2-expressing granules do not. Analysis of acinar secretion by Texas red-dextran labeling indicated that VAMP 2-positive ZGs mediate the majority of exocytotic events during constitutive secretion and also participate in Ca(2+)-regulated exocytosis, whereas VAMP 8-positive ZGs are more largely involved in Ca(2+)-stimulated secretion. Previously undefined functional roles for VAMP and syntaxin isoforms in acinar secretion were established by introducing truncated constructs of these proteins into permeabilized acini. VAMP 2 and VAMP 8 constructs each attenuated Ca(2+)-stimulated exocytosis by 50%, whereas the neuronal VAMP 1 had no effects. In comparison, the plasma membrane SNAREs syntaxin 2 and syntaxin 4 each inhibited basal exocytosis, but only syntaxin 4 significantly inhibited Ca(2+)-stimulated secretion. Syntaxin 3, which is expressed on ZGs, had no effects. Collectively, these data demonstrate that individual acinar cells express VAMP 2- and VAMP 8-specific populations of ZGs that orchestrate the constitutive and Ca(2+)-regulated secretory pathways.     

Cytosolic hydroxymethyldihydropterin pyrophosphokinase/dihydropteroate synthase from Arabidopsis thaliana: a specific role in early development and stress response

In plants, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase (mitHPPK/DHPS) is a bifunctional mitochondrial enzyme, which catalyzes the first two consecutive steps of tetrahydrofolate biosynthesis. Mining the Arabidopsis genome data base has revealed a second gene encoding a protein that lacks a potential transit peptide, suggesting a cytosolic localization of the isoenzyme (cytHPPK/DHPS). When the N-terminal part of the cytHPPK/DHPS was fused to green fluorescent protein, the fusion protein appeared only in the cytosol, confirming the above prediction. Functionality of cytHPPK/DHPS was addressed by two parallel approaches: first, the cytHPPK/DHPS was able to rescue yeast mutants lacking corresponding activities; second, recombinant cytHPPK/DHPS expressed and purified from Escherichia coli displayed both HPPK and DHPS activities in vitro. In contrast to mitHPPK/DHPS, which was ubiquitously expressed, the cytHPPK/DHPS gene was exclusively expressed in reproductive tissue, more precisely in developing seeds as revealed by histochemical analysis of a transgenic cytHPPK/DHPS promoter-GUS line. In addition, it was observed that expression of cytHPPK/DHPS mRNA was induced by salt stress, suggesting a potential role of the enzyme in stress response. This was supported by the phenotype of a T-DNA insertion mutant in the cytHPPK/DHPS gene, resulting in lower germination rates as compared with the wild-type upon application of oxidative and osmotic stress.     

Hepatitis C virus NS5A is a direct substrate of casein kinase I-alpha, a cellular kinase identified by inhibitor affinity chromatography using specific NS5A hyperphosphorylation inhibitors

The hepatitis C virus encodes a single polyprotein that is processed by host and viral proteases to yield at least 10 mature viral proteins. The nonstructural (NS) protein 5A is a phosphoprotein, and experimental data indicate that the phosphorylation state of NS5A is important for the outcome of viral RNA replication. We were able to identify kinase inhibitors that specifically inhibit the formation of the hyperphosphorylated form of NS5A (p58) in cells. These kinase inhibitors were used for inhibitor affinity chromatography in order to identify the cellular targets of these compounds. The kinases casein kinase I (CKI), p38 MAPK, CIT (Citron Rho-interacting kinase), GAK, JNK2, PKA, RSK1/2, and RIPK2 were identified in the high affinity binding fractions of two NS5A hyperphosphorylation inhibitors (NS5A-p58-i). Even though these kinases are targets of the NS5A-p58-i, the only kinase showing an effect on NS5A hyperphosphorylation was confirmed to be CKI-alpha. Although this finding does not exclude the possibility that other kinase(s) might be involved in basal or regulatory phosphorylation of NS5A, we show here that NS5A is a direct substrate of CKI-alpha. Moreover, in vitro phosphorylation of NS5A by CKI-alpha resulted for the first time in the production of basal and hyperphosphorylated forms resembling those produced in cells. In vitro kinase reactions performed with NS5A peptides show that Ser-2204 is a preferred substrate residue for CKI-alpha after pre-phosphorylation of Ser-2201.     

Estimation of the deformations induced by articulated bodies: Registration of the spinal column

We present a new non-rigid registration algorithm estimating the displacement field generated by articulated bodies. Indeed the bony structures between different patient images may rigidly move while other tissues may deform in a more complex way. Our algorithm tracks the displacement induced in the column by a movement of the patient between two acquisitions. The volumetric deformation field in the whole body is then inferred from those displacements using a linear elastic biomechanical finite element model. We demonstrate in this paper that this method provides accurate results on 3D sets of computed tomography (CT), MR and positron emission tomography (PET) images and that the results of the registration algorithm show significant decreases in the mean, min and max errors.     

Effect of botanical composition of silages on rumen fatty acid metabolism and fatty acid composition in longissimus muscle and subcutaneous fat of lambs

To study the effect of feeding silages with different botanical composition, on rumen and lamb fat, 30 male lambs were assigned to five different silage groups for 11 weeks: botanically diverse silage (BDS); white clover silage (WCS); red clover silage (RCS), intensive English ryegrass silage (IRS) and crushed linseed and maize silage (MSL). Besides the silages, animals received organic wheat and barley and the MSL group additionally received bicarbonate (15 g/day). Silages were sampled when the bales were opened and analysed for fatty acid (FA) content and chemical composition. At slaughter, ruminal contents were sampled and 24 h after slaughter, longissimus muscle and subcutaneous (SC) fat were sampled. All samples were analysed for FA composition. The MSL group ingested the highest amount of FA (35.8 g/day v. 13.5, 19.4, 17.2 and 30.4 g/day for MSL v. BDS, WCS, RCS and IRS, respectively) and the sum of the major polyunsaturated FA, C18:2 n-6 and C18:3 n-3, was similar for groups BDS, WCS, RCS and MSL (61.3 g/100 g, 62.3 g/100 g, 62.3 g/100 g, 63.7 g/100 g of FA methylesters (FAME), respectively), while group IRS ingested higher proportions of these FA (74.5 g/100 g of FAME). Rumen data showed that animals fed BDS presented higher proportions of biohydrogenation intermediates, particularly C18:1 t11 and CLA c9t11, suggesting partial inhibition of rumen biohydrogenation. In the MSL group, the content of C18:3 n-3 in the rumen was highest, most probably due to reduced lipolysis and hence biohydrogenation through the combined effect of esterified C18:3 n-3 and seed protection. Additionally, C18:3 n-3 proportions were higher in rumen contents of RCS animals compared with WCS animals, which could be due to the activity of the polyphenol oxidase enzyme in the RC silages. Proportions of C18:3 n-3 were similar between treatments both for SC and intramuscular (IM) fat, whereas CLA c9t11 content was higher in the SC fat of BDS animals and lower in the IM fat of IRS animals compared with the other forage groups. No differences were found for C20:4 n-6, C20:5 n-3, C22:5 n-3 and C22:6 n-3 in the IM fat of the animals. Nevertheless, indices for desaturation and elongation activity in muscle of BDS animals suggest some stimulation of the first three steps of desaturation and elongation (Δ6-desaturase, elongase and Δ5-desaturase) of long-chain FA.     

Incidence and tracking of Escherichia coli O157:H7 in a major produce production region in California

Fresh vegetables have become associated with outbreaks caused by Escherichia coli O157:H7 (EcO157). Between 1995-2006, 22 produce outbreaks were documented in the United States, with nearly half traced to lettuce or spinach grown in California. Outbreaks between 2002 and 2006 induced investigations of possible sources of pre-harvest contamination on implicated farms in the Salinas and San Juan valleys of California, and a survey of the Salinas watershed. EcO157 was isolated at least once from 15 of 22 different watershed sites over a 19 month period. The incidence of EcO157 increased significantly when heavy rain caused an increased flow rate in the rivers. Approximately 1000 EcO157 isolates obtained from cultures of>100 individual samples were typed using Multi-Locus Variable-number-tandem-repeat Analysis (MLVA) to assist in identifying potential fate and transport of EcO157 in this region. A subset of these environmental isolates were typed by Pulse Field Gel Electrophoresis (PFGE) in order to make comparisons with human clinical isolates associated with outbreak and sporadic illness. Recurrence of identical and closely related EcO157 strains from specific locations in the Salinas and San Juan valleys suggests that transport of the pathogen is usually restricted. In a preliminary study, EcO157 was detected in water at multiple locations in a low-flow creek only within 135 meters of a point source. However, possible transport up to 32 km was detected during periods of higher water flow associated with flooding. During the 2006 baby spinach outbreak investigation, transport was also detected where water was unlikely to be involved. These results indicate that contamination of the environment is a dynamic process involving multiple sources and methods of transport. Intensive studies of the sources, incidence, fate and transport of EcO157 near produce production are required to determine the mechanisms of pre-harvest contamination and potential risks for human illness.     

Differences in variation of human immunodeficiency virus type 1 sequences from Henan and Shanghai regions of China

Illegally paid blood donation was a risk factor for HIV acquisition exclusively in Henan and Hubei Provinces of China, and not in Shanghai. Nucleotide sequences in the gag and env genes of HIV-1 were compared between isolates from Henan and Shanghai regions of China to test whether an expected higher degree of a common source of infections from this unique blood donation transmission risk would be evident as decreased variation among Henan isolates in an exploratory cross-sectional analysis. Among 38 isolates studied, 23 of 23 (100%) from Henan and 8 of 15 (54%) from Shanghai were subtype B. In addition, fewer sequence differences were found in gp41 of subtype B isolates from Henan than from Shanghai isolates. Further studies with additional controls are therefore warranted to confirm the role of the degree of a common source of infections in differences in HIV variation across populations. Fundation items: The Vanderbilt-Meharry Center for AIDS Research (P30 AI 54999); R.T.D (R01 AI 29193); Start Fund of Ministry of Education of China (for Hong-zhou LU, 2004BA719A10).     

Quantitative profiling of the pathological prion protein allotypes in bank voles by liquid chromatography-mass spectrometry

The conversion of the cellular prion protein (PrP(C)) into a misfolded isoform (PrP(TSE)) that accumulates in the brain of affected individuals is the key feature of transmissible spongiform encephalopaties (TSEs). Susceptibility to TSEs is influenced by polymorphisms of the prion gene suggesting that the presence of certain amino acid residues may facilitate the pathological conversion. In this work, we describe a quantitative, fast and reliable HPLC-MS method that allowed to demonstrate that in the brain of 109(Met/Ile) heterozygous bank voles infected with the mouse adapted scrapie strain 139A, there are comparable amounts of PrP(TSE) with methionine or isoleucine in position 109, suggesting that in this TSE model the two allotypes have similar rates of accumulation. This method can be easily adapted for the quantitative determination of PrP allotypes in the brain of other natural or experimental TSE models.