Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

Background

Protein translocation across the membrane of the Endoplasmic Reticulum (ER) is the first step in the biogenesis of secretory and membrane proteins. Proteins enter the ER by the Sec61 translocon, a proteinaceous channel composed of three subunits, α, β and γ. While it is known that Sec61α forms the actual channel, the function of the other two subunits remains to be characterized.

Results

In the present study we have investigated the function of Sec61β in Drosophila melanogaster. We describe its role in the plasma membrane traffic of Gurken, the ligand for the Epidermal Growth Factor (EGF) receptor in the oocyte. Germline clones of the mutant allele of Sec61β show normal translocation of Gurken into the ER and transport to the Golgi complex, but further traffic to the plasma membrane is impeded. The defect in plasma membrane traffic due to absence of Sec61β is specific for Gurken and is not due to a general trafficking defect.

Conclusion

Based on our study we conclude that Sec61β, which is part of the ER protein translocation channel affects a post-ER step during Gurken trafficking to the plasma membrane. We propose an additional role of Sec61β beyond protein translocation into the ER.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Comparison of intrinsic dynamics of cytochrome p450 proteins using normal mode analysis

Cytochrome P450 enzymes are hemeproteins that catalyze the monooxygenation of a wide‐range of structurally diverse substrates of endogenous and exogenous origin. These heme monooxygenases receive electrons from NADH/NADPH via electron transfer proteins. The cytochrome P450 enzymes, which constitute a diverse superfamily of more than 8,700 proteins, share a common tertiary fold but < 25% sequence identity. Based on their electron transfer protein partner, cytochrome P450 proteins are classified into six broad classes. Traditional methods of protein classification are based on the canonical paradigm that attributes proteins’ function to their three‐dimensional structure, which is determined by their primary structure that is the amino acid sequence. It is increasingly recognized that protein dynamics play an important role in molecular recognition and catalytic activity. As the mobility of a protein is an intrinsic property that is encrypted in its primary structure, we examined if different classes of cytochrome P450 enzymes display any unique patterns of intrinsic mobility. Normal mode analysis was performed to characterize the intrinsic dynamics of five classes of cytochrome P450 proteins. The present study revealed that cytochrome P450 enzymes share a strong dynamic similarity (root mean squared inner product > 55% and Bhattacharyya coefficient > 80%), despite the low sequence identity (< 25%) and sequence similarity (< 50%) across the cytochrome P450 superfamily. Noticeable differences in C_α atom fluctuations of structural elements responsible for substrate binding were noticed. These differences in residue fluctuations might be crucial for substrate selectivity in these enzymes.     

Immunohistochemical staining of slit2 in primary and metastatic prostatic adenocarcinoma

BACKGROUND: Conflicting roles for Slit2, a protein involved in mediating the processes of cell migration and chemotactic response, have been previously described in prostate cancer. Here we use immunohistochemistry to evaluate the expression of Slit2 in normal donor prostate (NDP), benign prostatic hyperplasia (BPH), high-grade prostatic intraepithelial neoplasia (HGPIN), normal tissue adjacent to prostatic adenocarcinoma (NAC), primary prostatic adenocarcinoma (PCa), and metastatic prostatic adenocarcinoma (Mets). METHODS: Tissue microarrays were immunostained for Slit2. The staining intensities were quantified using automated image analysis software. The data was statistically analyzed using one-way analysis of variance with subsequent Tukey tests for multiple comparisons or a nonparametric equivalent. Eleven cases of NDP, 35 cases of NAC, 15 cases of BPH, 35 cases of HGPIN, 106 cases of PCa, and 37 cases of Mets were analyzed. RESULTS: Specimens of PCa and HGPIN had the highest absolute staining for Slit2. Significant differences were seen between PCa and NDP (P < .05), PCa and NAC (P < .05), HGPIN and NDP (P < .05), and HGPIN and NAC (P < .05). Whereas the average Mets staining was not significantly different from NDP or NAC, several individual Mets cases featured intense staining. CONCLUSIONS: To our knowledge, this represents the first study comparing the immunohistochemical profiles of Slit2 in PCa and Mets to specimens of HGPIN, BPH, NDP, and NAC. These findings suggest that Slit2 expression can be increased in HGPIN, PCa, and Mets, making it a potentially important biomarker for prostate cancer.     

Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane

In order to examine the widely held hypothesis that the reticulum of proteins which covers the cytoplamsic surface of the human erythrocyte membrane controls cell stability and shape, we have assessed some of its properties. The reticulum, freed of the bilayer by extraction with Triton X-100, was found to be mechanically stable at physiological ionic strength but physically unstable at low ionic strength. The reticulum broke down after a characteristic lag period which decreased 500-fold between 0 degrees and 37 degrees C. The release of polypeptide band 4.1 from the reticulum preceded that of spectrin and actin, suggesting that band 4.1 might stabilize the ensemble but is not essential to its integrity. The time-course of breakdown was similar for ghosts, the reticulum inside of ghosts, and the isolated reticulum. However, at very low ionic strength, the reticulum was less stable within the ghost than when free; at higher ionic strength, the reverse was true. Over a wide range of conditions the membrane broke down to vesicles just as the reticulum disintegrated, presumably because the bilayer was mechanically stabilized by this network. The volume of both ghosts and naked reticula varied inversely and reversibly with ionic strength. The volume of the naked reticulum varied far more widely than the ghost, suggesting that its deformation was normally limited by the less extensible bilayer. The contour of the isolated reticulum was discoid and often dimpled or indented, as visualized in the fluorescence microscope after labeling of the ghosts with fluoroscein isothiocyanate. Reticula derived from ghosts which had lost the ability to crenate in isotonic saline were shriveled, even though the bilayer was smooth and expanded. Conversly, ghosts crenated by dinitrophenol yielded smooth, expanded reticula. We conclude that the reticulum is a durable, flexible, and elastic network which assumes and stabilizes the contour of the membrane but is not responsible for its crenation.     

Molecular evolution of a multigene family in group A streptococci

The emm genes are members of a gene family in group A streptococci (GAS) that encode for antiphagocytic cell-surface proteins and/or immunoglobulin-binding proteins. Previously sequenced genes in this family have been named "emm," "fcrA," "enn," "arp," "protH," and "mrp"; herein they will be referred to as the "emm gene family." The genes in the emm family are located in a cluster occupying 3-6 kb between the genes mry and scpA on the chromosome of Streptococcus pyogenes. Most GAS strains contain one to three tandemly arranged copies of emm-family genes in the cluster, but the alleles within the cluster vary among different strains. Phylogenetic analysis of the conserved sequences at the 3' end of these genes differentiates all known members of this family into four evolutionarily distinct emm subfamilies. As a starting point to analyze how the different subfamilies are related evolutionarily, the structure of the emm chromosomal region was mapped in a number of diverse GAS strains by using subfamily-specific primers in the polymerase chain reaction. Nine distinct chromosomal patterns of the genes in the emm gene cluster were found. These nine chromosomal patterns support a model for the evolution of the emm gene family in which gene duplication followed by sequence divergence resulted in the generation of four major-gene subfamilies in this locus.      

Diversity of lactic acid bacteria of the bioethanol process

Background  

Bacteria may compete with yeast for nutrients during bioethanol production process, potentially causing economic losses. This is the first study aiming at the quantification and identification of Lactic Acid Bacteria (LAB) present in the bioethanol industrial processes in different distilleries of Brazil.     

High and persistent HIV seroincidence in men who have sex with men across 47 U.S. cities

Objective

To provide HIV seroincidence data among men who have sex with men (MSM) in the United States and to identify predictive factors for seroconversion.

Methods

From 1998–2002, 4684 high-risk MSM, age 18–60 years, participated in a randomized, placebo-controlled HIV vaccine efficacy trial at 56 U.S. clinical trial sites. Demographics, behavioral data, and HIV status were assessed at baseline and 6 month intervals. Since no overall vaccine efficacy was detected, data were combined from both trial arms to calculate HIV incidence based on person-years (py) of follow-up. Predictors of seroconversion, adjusted hazards ratio (aHR), were evaluated using a Cox proportional hazard model with time-varying covariates.

Results

Overall, HIV incidence was 2.7/100 py and was relatively uniform across study sites and study years. HIV incidence was highest among young men and men reporting unprotected sex, recreational drug use, and a history of a sexually transmitted infection. Independent predictors of HIV seroconversion included: age 18–30 years (aHR = 2.4; 95% CI 1.4,4.0), having >10 partners (aHR = 2.4; 95% CI 1.7,3.3), having a known HIV-positive male sex partner (aHR = 1.6; 95% CI 1.2, 2.0), unprotected anal intercourse with HIV positive/unknown male partners (aHR = 1.7; 95% CI 1.3, 2.3), and amphetamine (aHR = 1.6; 95% CI 1.1, 2.1) and popper (aHR = 1.7; 95% CI 1.3, 2.2) use.

Conclusions

HIV seroincidence was high among MSM despite repeated HIV counseling and reported declines in sexual risk behaviors. Continuing development of new HIV prevention strategies and intensification of existing efforts will be necessary to reduce the rate of new HIV infections, especially among young men.     

Prevalence,Awareness, Treatment and Influence of Socioeconomic Variables on Control of High Blood Pressure: Results of the ELSA-Brasil Study

High blood pressure (HBP) is the leading risk factor for years of life lost in Brazil. Factors associated with HBP awareness, treatment and control need to be understood better. Our aim is to estimate prevalence, awareness, and types of anti-hypertensive treatment and to investigate the association of HBP control with social position. Data of 15,103 (54% female) civil servants in six Brazilian state capitals collected at the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil) baseline (2008-2010) were used to estimate prevalence and cross-sectional association of HBP control with education, per capita family income and self-reported race, using multiple logistic regression. Blood pressure was measured by the oscillometric method. 35.8% were classified as presenting HBP; 76.8% of these used anti-hypertensive medication. Women were more aware than men (84.8% v. 75.8%) and more often using medication (83.1% v. 70.7%). Adjusted HBP prevalence was, in ascending order, Whites (30.3%), Browns (38.2%) and Blacks (49.3%). The therapeutic schemes most used were angiotensin-converting enzyme inhibitors, in isolation (12.4%) or combined with diuretics (13.3%). Among those in drug treatment, controlled blood pressure was more likely in the (postgraduate) higher education group than among participants with less than secondary school education (PR = 1.21; 95% CI: 1.14–1.28), and among Asian (PR = 1.21; 95% CI: 1.12–1.32) and ‘Whites (PR = 1.19; 95% CI: 1.12–1.26) compared to Blacks. Socioeconomic and racial inequality—as measured by different indicators—are strongly associated with HBP control, beyond the expected influence of health services access.