A novel multiprotein complex is required to generate the prechylomicron transport vesicle from intestinal ER

Dietary lipid absorption is dependent on chylomicron production whose rate-limiting step across the intestinal absorptive cell is the exit of chylomicrons from the endoplasmic reticulum (ER) in its ER-to-Golgi transport vesicle, the prechylomicron transport vesicle (PCTV). This study addresses the composition of the budding complex for PCTV. Immunoprecipitation (IP) studies from rat intestinal ER solubilized in Triton X-100 suggested that vesicle-associated membrane protein 7 (VAMP7), apolipoprotein B48 (apoB48), liver fatty acid-binding protein (L-FABP), CD36, and the COPII proteins were associated on incubation of the ER with cytosol and ATP. This association was confirmed by chromatography of the solubilized ER over Sephacryl S400-HR in which these constituents cochromatographed with an apparent kDa of 630. No multiprotein complex was detected when the ER was chromatographed in the absence of PCTV budding activity (resting ER or PKCζ depletion of ER and cytosol). Treatment of the ER with anti-apoB48 or anti-VAMP7 antibodies or using gene disrupted L-FABP or CD36 mice all significantly inhibited PCTV generation. A smaller complex (no COPII proteins) was formed when only rL-FABP was used to bud PCTV. The data support the conclusion that the PCTV budding complex in intestinal ER is composed of VAMP7, apoB48, CD36, and L-FABP, plus the COPII proteins.     

Cell organisation,sulphur metabolism and ion transport-related genes are differentially expressed in Paracoccidioides brasiliensis mycelium and yeast cells

Background

Bread wheat (Triticum aestivum) is an important staple food. However, wheat gluten proteins cause celiac disease (CD) in 0.5 to 1% of the general population. Among these proteins, the α-gliadins contain several peptides that are associated to the disease.

Results

We obtained 230 distinct α-gliadin gene sequences from severaldiploid wheat species representing the ancestral A, B, and D genomes of the hexaploid bread wheat. The large majority of these sequences (87%) contained an internal stop codon. All α-gliadin sequences could be distinguished according to the genome of origin on the basis of sequence similarity, of the average length of the polyglutamine repeats, and of the differences in the presence of four peptides that have been identified as T cell stimulatory epitopes in CD patients through binding to HLA-DQ2/8. By sequence similarity, α-gliadins from the public database of hexaploid T. aestivum could be assigned directly to chromosome 6A, 6B, or 6D. T. monococcum (A genome) sequences, as well as those from chromosome 6A of bread wheat, almost invariably contained epitope glia-α9 and glia-α20, but never the intact epitopes glia-α and glia-α2. A number of sequences from T. speltoides, as well as a number of sequences fromchromosome 6B of bread wheat, did not contain any of the four T cell epitopes screened for. The sequences from T. tauschii (D genome), as well as those from chromosome 6D of bread wheat, were found to contain all of these T cell epitopes in variable combinations per gene. The differences in epitope composition resulted mainly from point mutations. These substitutions appeared to be genome specific.

Conclusion

Our analysis shows that α-gliadin sequences from the three genomes of bread wheat form distinct groups. The four known T cell stimulatory epitopes are distributed non-randomly across the sequences, indicating that the three genomes contribute differently to epitope content. A systematic analysis of all known epitopes in gliadins and glutenins will lead to better understanding of the differences in toxiCity among wheat varieties. On the basis of such insight, breeding strategies can be designed to generate less toxic varieties of wheat which may be tolerated by at least part of the CD patient population.     

Simultaneous determination of three isomeric metabolites of tacrolimus (FK506) in human whole blood and plasma using high performance liquid chromatography-tandem mass spectrometry

An ammonium-adduct based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of three isomeric metabolites of tacrolimus (FK506), 13-O-demethylated (M1), 31-O-demethylated (M2) and 15-O-demethylated (M3) tacrolimus in human whole blood and plasma. These metabolites and the internal standards were extracted from biological matrix by methylbutyl ether (MTBE). Separation was achieved on a Genesis C(18) column with a gradient mobile phase elution. Ammonium-adduct ions formed by a Turbo Ionspray in positive ion mode were used to detect each analyte and internal standard. The MS/MS detection was by monitoring the fragmentation of 807.5-->772.4 (m/z) for M1, 807.5-->754.5 (m/z) for both M2 and M3, 795.5-->760.5 (m/z) for IS1 (FR298701) and 961.5-->908.5 (m/z) for IS2 (FR290198) on a triple quadrupole mass spectrometer (Sciex API 3000). The retention times were approximately 4.1 min for M1, 6.8 min for M2, 6.0 min for M3, and 3.9 min for IS1 and 6.4 min for IS2, respectively. The validated dynamic range was 0.2-20 ng/ml for all three metabolites based on a sample volume of 0.25-ml. The linearity of calibration curves for M1, M2, and M3 in both matrices had a correlation coefficient of >/=0.9984. In whole blood, validation data showed intra-batch (n=6) CVs of 相似文献   

Light-to-dark transitions trigger a transient increase in intracellular Ca2+ modulated by the redox state of the photosynthetic electron transport chain in the cyanobacterium Anabaena sp. PCC7120

Light‐to‐dark transitions represent one of the most crucial environmental stresses that photosynthetic organisms must cope with, since substantial metabolism adaptations are required in order to utilize alternative energy and carbon sources. Although signal transduction systems for changing light regimes are not sufficiently understood, calcium has been implicated in plants as a second messenger in light‐on and light‐off events. Much less is known about light signalling in cyanobacteria, but it has been shown that calcium probably performs similar signalling roles in these organisms and other prokaryotes. Herein it is reported that light‐to‐dark transitions trigger a calcium transient in aequorin expressing Anabaena sp. PCC7120. The magnitude of this transient depends on the fluence rate previously irradiated and can reach a peak height over 2 µm free calcium when the fluence rate of light is around 400 µmol photons s^?1 m^?2. The use of increasing calcium concentration, ethylene glycol‐bis (β‐aminoethylether) N,N,N′,N′‐tetraacetic acid (EGTA), verapamil and trifluoperazine indicated that these transients are originated by a calcium influx probably through verapamil‐sensitive Ca²⁺ channels and are probably modulated by calcium‐binding proteins. Experiments with different light spectral qualities and the photosynthetic inhibitors 3‐(3,4 dichlorophenyl)1,1,dimelthylurea (DCMU) and 3,5‐dibromo‐3‐methyl‐b‐isopropyl‐p‐benzoquinone (DBMIB) indicate that the calcium transient triggered by the light‐to‐dark transition is not coupled to a specific photoreceptor but rather to changes in the redox state of photosynthetic electron transport chain components other than the plastoquinone pool.     

Proteomics analysis of sensitive and tolerant barley genotypes under drought stress

Drought is a severe environmental constraint to plant productivity and an important factor limiting barley yield. To investigate the initial response of barley to drought stress, changes in protein profile were analyzed using a proteomics technique. Three-day-old barley seedlings of sensitive genotype 004186 and tolerant genotype 004223 were given two treatments, one with 20 % polyethylene glycol and the second with drought induced by withholding water. After 3 days of treatments, proteins were extracted from shoots and separated by 2-dimensional polyacrylamide gel electrophoresis. Metabolism related proteins were decreased in the sensitive genotype under drought; however, they were increased in the tolerant genotype. Photosynthetic related proteins were decreased and increased among the three sensitive and three tolerant genotypes, respectively. In addition, amino acid synthesis and degradation related proteins were increased and decreased among the three tolerant genotypes. These results suggest that chloroplastic metabolism and energy related proteins might play a significant role in the adaptation process of barley seedlings under drought stress.     

The Effect of Hospital Volume on Mortality in Patients Admitted with Severe Sepsis

Importance

The association between hospital volume and inpatient mortality for severe sepsis is unclear.

Objective

To assess the effect of severe sepsis case volume and inpatient mortality.

Design Setting and Participants

Retrospective cohort study from 646,988 patient discharges with severe sepsis from 3,487 hospitals in the Nationwide Inpatient Sample from 2002 to 2011.

Exposures

The exposure of interest was the mean yearly sepsis case volume per hospital divided into tertiles.

Main Outcomes and Measures

Inpatient mortality.

Results

Compared with the highest tertile of severe sepsis volume (>60 cases per year), the odds ratio for inpatient mortality among persons admitted to hospitals in the lowest tertile (≤10 severe sepsis cases per year) was 1.188 (95% CI: 1.074–1.315), while the odds ratio was 1.090 (95% CI: 1.031–1.152) for patients admitted to hospitals in the middle tertile. Similarly, improved survival was seen across the tertiles with an adjusted inpatient mortality incidence of 35.81 (95% CI: 33.64–38.03) for hospitals with the lowest volume of severe sepsis cases and a drop to 32.07 (95% CI: 31.51–32.64) for hospitals with the highest volume.

Conclusions and Relevance

We demonstrate an association between a higher severe sepsis case volume and decreased mortality. The need for a systems-based approach for improved outcomes may require a high volume of severely septic patients.     

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

Descemet’s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, along with its basement membrane (Descemet''s membrane) is introduced onto the recipient''s posterior stroma³. Unlike Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK), where additional donor stroma is introduced, no unnatural stroma-to-stroma interface is created. As a result, the natural anatomy of the cornea is preserved as much as possible allowing for improved recovery time and visual acuity⁴. Endothelial Keratoplasty (EK) is the procedure of choice for treatment of endothelial dysfunction. The advantages of EK include rapid recovery of vision, preservation of ocular integrity and minimal refractive change due to use of a small, peripheral incision¹. DSAEK utilizes donor tissue prepared with partial thickness stroma and endothelium. The rapid success and utilization of this procedure can be attributed to availability of eye-bank prepared precut tissue. The benefits of eye-bank preparation of donor tissue include elimination of need for specialized equipment in the operating room and availability of back up donor tissue in case of tissue perforation during preparation. In addition, high volume preparation of donor tissue by eye-bank technicians may provide improved quality of donor tissue. DSAEK may have limited best corrected visual acuity due to creation of a stromal interface between the donor and recipient cornea. Elimination of this interface with transplantation of only donor Descemet''s membrane and endothelium in DMEK may improve visual outcomes and reduce complications after EK⁵. Similar to DSAEK, long term success and acceptance of DMEK is dependent on ease of availability of precut, eye-bank prepared donor tissue. Here we present a stepwise approach to donor tissue preparation which may reduce some barriers eye-banks face in providing DMEK grafts.