Somatostatin-detergent interaction

The effect of cationic, anionic and nonionic detergents on the EPR spectrum of spin-labeled somatostatin has been studied. At detergent concentrations well above the critical micelle concentration, nonionic detergents do not alter the EPR spectrum. Sodium dodecyl sulfate markedly alters both the line height ratio and the hyperfine splitting constant, whilst dodecyltrimethylammonium bromide alters only slightly the hyperfine splitting constant and line height ratio. The somatostatin-sodium dodecyl sulfate complex appeared monodisperse by sedimentation equilibrium with about 17 g bound detergent per g peptide. Circular dichroic and difference spectra of the dodecyl sulfate-somatostatin complex show that the tryptophanyl residue is buried in a nonpolar environment and that the secondary and tertiary structure of the peptide is markedly altered. Sedimentation equilibrium studies suggest that two types of dodecyltrimethylammonium-somatostatin complex exist. One type resembles the dodecyl sulfate-peptide complex, whilst the other appears to include several peptide units with only about one gram bound detergent per gram peptide.     

DNA-Launched Alphavirus Replicons Encoding a Fusion of Mycobacterial Antigens Acr and Ag85B Are Immunogenic and Protective in a Murine Model of TB Infection

There is an urgent need for effective prophylactic measures against Mycobacterium tuberculosis (Mtb) infection, particularly given the highly variable efficacy of Bacille Calmette-Guerin (BCG), the only licensed vaccine against tuberculosis (TB). Most studies indicate that cell-mediated immune responses involving both CD4+ and CD8+ T cells are necessary for effective immunity against Mtb. Genetic vaccination induces humoral and cellular immune responses, including CD4+ and CD8+ T-cell responses, against a variety of bacterial, viral, parasitic and tumor antigens, and this strategy may therefore hold promise for the development of more effective TB vaccines. Novel formulations and delivery strategies to improve the immunogenicity of DNA-based vaccines have recently been evaluated, and have shown varying degrees of success. In the present study, we evaluated DNA-launched Venezuelan equine encephalitis replicons (Vrep) encoding a novel fusion of the mycobacterial antigens α-crystallin (Acr) and antigen 85B (Ag85B), termed Vrep-Acr/Ag85B, for their immunogenicity and protective efficacy in a murine model of pulmonary TB. Vrep-Acr/Ag85B generated antigen-specific CD4+ and CD8+ T cell responses that persisted for at least 10 wk post-immunization. Interestingly, parenterally administered Vrep-Acr/Ag85B also induced T cell responses in the lung tissues, the primary site of infection, and inhibited bacterial growth in both the lungs and spleens following aerosol challenge with Mtb. DNA-launched Vrep may, therefore, represent an effective approach to the development of gene-based vaccines against TB, particularly as components of heterologous prime-boost strategies or as BCG boosters.     

Lower Neighborhood Socioeconomic Status Associated with Reduced Diversity of the Colonic Microbiota in Healthy Adults

In the United States, there are persistent and widening socioeconomic gaps in morbidity and mortality from chronic diseases. Although most disparities research focuses on person-level socioeconomic-status, mounting evidence suggest that chronic diseases also pattern by the demographic characteristics of neighborhoods. Yet the biological mechanisms underlying these associations are poorly understood. There is increasing recognition that chronic diseases share common pathogenic features, some of which involve alterations in the composition, diversity, and functioning of the gut microbiota. This study examined whether socioeconomic-status was associated with alpha-diversity of the colonic microbiota. Forty-four healthy adults underwent un-prepped sigmoidoscopy, during which mucosal biopsies and fecal samples were collected. Subjects’ zip codes were geocoded, and census data was used to form a composite indicator of neighborhood socioeconomic-status, reflecting household income, educational attainment, employment status, and home value. In unadjusted analyses, neighborhood socioeconomic-status explained 12–18 percent of the variability in alpha-diversity of colonic microbiota. The direction of these associations was positive, meaning that as neighborhood socioeconomic-status increased, so did alpha-diversity of both the colonic sigmoid mucosa and fecal microbiota. The strength of these associations persisted when models were expanded to include covariates reflecting potential demographic (age, gender, race/ethnicity) and lifestyle (adiposity, alcohol use, smoking) confounds. In these models neighborhood socioeconomic-status continued to explain 11–22 percent of the variability in diversity indicators. Further analyses suggested these patterns reflected socioeconomic variations in evenness, but not richness, of microbial communities residing in the sigmoid. We also found indications that residence in neighborhoods of higher socioeconomic-status was associated with a greater abundance of Bacteroides and a lower abundance of Prevotella, suggesting that diet potentially underlies differences in microbiota composition. These findings suggest the presence of socioeconomic variations in colonic microbiota diversity. Future research should explore whether these variations contribute to disparities in chronic disease outcomes.     

Simple and selective high-performance liquid chromatographic method for estimating plasma quinidine levels

A reversed-phase, high-performance liquid chromatographic method employing fluorescence detection is described for the rapid quantification of plasma levels of quinidine, dihydroquinidine and 3-hydroxyquinidine. It involves protein precipitation with acetonitrile followed by direct injection of the supernatant into the chromatograph. For the preparation of plasma standards, pure 3-hydroxyquinidine was isolated from human urine by a simplified thin-layer chromatographic procedure. The mobile phase for the chromatography was a mixture of 1.5 mM aqueous phosphoric acid and acetonitrile (90:10) at a flow-rate of 2 ml/min. The intra-assay coefficient of variation for the assay of quinidine and 3-hydroxyquinidine over the concentration range 2.5–20 μmole/l was < 1% for both. Interassay coefficients of variation for quinidine (10 μmole/l) and 3-hydroxyquinidine (5 μmole/l) were 3.5% and 4.0% with detection limits of 50 and 25 μmole/l respectively. The method correlated well (r² = 0.96) with an independently developed gas—liquid chromatographic—nitrogen detection assay for quinidine which also possessed a high degree of precision. (Intra-assay coefficient of variation 3.6% at 20 μmole/l). As expected, comparison of the high-performance liquid chromatographic assay with a published protein precipitation—fluorescence assay showed poor correlation (r² = 0.78).     

Cell culture and in vitro studies of fresh and cryopreserved human insulinoma

Summary Dispersed cells from both fresh and cryopreserved human insulinoma have been maintained in cell culture. Initial yield of viable cells was 50% for fresh and 25% for cryopreserved tissue. Viability of cells in culture was documented by increasing numbers of cells (doubling time approximately 5 d initially and 2 d at the sixth subculture for both fresh and cryopreserved tissue) and continued release of insulin over time (approximately 100 ng/ml per 10⁵ cells at 10 d and 175 ng/ml per 10⁵ cells at 30 d of culture for both fresh and cryopreserved tissue). Evidence that cells growing in culture were beta cells was provided by: (a) recovery of intracellular and extracellular immunoreactive insulin (IRI), (b) electron microscopic morphology, and (c) immunohistochemical staining. Cells from fresh insulinoma incubated with increasing concentrations of extracellular glucose released increasing amounts of IRI up to approximately 15 mM glucose, which paralleled changes in plasma insulin obtained during a preoperative glucose tolerance test. Under an Intergovernmental Personnel Act Exchange from the Department of Surgery, University of California, Davis, Sacramento Medical Center.