Quantitative assessment of the tetracycline resistance gene pool in cheese samples by real-time TaqMan PCR

A TaqMan real-time PCR assay was developed to quantify the tetS gene pool present in retail cheeses. This protocol offers a rapid, specific, sensitive, and culture-independent method for assessing antibiotic resistance genes in food samples rich in fats and proteins.     

Effect of indomethacin on prostaglandin content of several rabbit tissues

The prostaglandin (PG) content of several tissues and fluids from 6 day pregnant rabbits was evaluated following treatment with indomethacin or vehicle . PGE and PGF were measured by radioimmunoassay. More complete depletion of PGE and PGF was accomplished by 3 injections of indomethacin (s.c.) given during the 18 h before sacrifice at a dose of 10 mg indomethacin per kg body weight than was accomplished by 1 injection of the same amount of indomethacin (i.v.) 1.5 h before sacrifice. Levels of PGF were more easily depressed by indomethacin than were those of PGE. PG levels in the kidney and blastocysts were depressed to a greater extent by indomethacin than were those in the uterus, uterine fluid or peritoneal fluid. Evaluation of the effect of indomethacin on a particular physiological function should be interpreted with caution unless the extent of PG depletion in that tissue is also measured.     

Temporal and spatial archaeal colonization of hydrothermal vent deposits

Thermocouple arrays were deployed on two deep-sea hydrothermal vents at Guaymas Basin (27 degrees 0.5'N, 111 degrees 24.5'W) in order to measure in situ temperatures at which microorganisms colonize the associated mineral deposits. Intact sections of three structures that formed around the arrays were collected after 4 and 72 day deployments (named BM4, BM72 and TS72). Archaeal diversity associated with discreet subsamples collected across each deposit was determined by polymerase chain reaction amplification of 16S rRNA genes. Spatial differences in archaeal diversity were observed in all deposits and appeared related to in situ temperature. In BM4, no 16S rRNA genes were detected beyond about 1.5 cm within the sample (> 200 degrees C). Phylotypes detected on the outside of this deposit belong to taxonomic groups containing mesophiles and (hyper)thermophiles, whereas only putative hyperthermophiles were detected 1.5 cm inside the structure (approximately 110 degrees C). In contrast, the more moderate thermal gradient recorded across TS72 was associated with a deeper colonization (2-3 cm inside the deposit) of putative hyperthermophilic phylotypes. Although our study does not provide a precise assessment of the highest temperature for the existence of microbial habitats inside the deposits, archaeal 16S rRNA genes were detected directly next to thermocouples that measured 110 degrees C (Methanocaldococcus spp. in BM4) and 116 degrees C (Desulfurococcaceae in TS72). The successive array deployments conducted at the Broken Mushroom (BM) site also revealed compositional differences in archaeal communities associated with immature (BM4) and mature chimneys (BM72) formed by the same fluids. These differences suggest a temporal transition in the primary carbon sources used by the archaeal communities, with potential CO(2)/H(2) methanogens prevalent in BM4 being replaced by possible methylotroph or acetoclastic methanogens and heterotrophs in BM72. This study is the first direct assessment of in situ conditions experienced by microorganisms inhabiting actively forming hydrothermal deposits at different stages of structure development.     

Study on the dehydrating effect of the red cell Na+/K+-pump in nystatin-treated cells with varying Na+ and water contents

Using the antibiotic Nystatin, we have developed a systematic method for the preparation of red blood cells with independently selected levels of intracellular Na⁺ concentrations and water content. Such cells provided an experimental model to study the effect of Na⁺/K⁺ pump stimulation on red cell water content. Even in initially dehydrated cells, stimulation of the Na⁺/K⁺ pump by elevated intracellular Na⁺ caused subsequent further loss of cell water. Cell water loss was reflected in decreased monovalent cation content per unit mass of hemoglobin and by a shift in the density distribution of the cell populations to higher densities on discontinuous Stractan gradients. We conclude that the 3 Na_out⁺ : 2 K_in⁺ stoichiometry of the Na⁺/K⁺ pump results in a net desalting effect with increased pump activity. Under the conditions of these experiments, the cell appears to have no effective mechanism to compensate for a net loss of ions and water.     

Calcium dynamics and endoplasmic reticular function in the regulation of protein synthesis: implications for cell growth and adaptability

The endoplasmic reticulum (ER) possesses the structural and functional features expected of an organelle that supports the integration and coordination of major cellular processes. Ca(2+) sequestered within the ER sustains lumenal protein processing while providing a reservoir of the cation to support stimulus-response coupling in the cytosol. Release of ER Ca(2+) sufficient to impair protein processing promotes ER stress and signals the "unfolded protein response" (UPR). The association of the UPR with an acute suppression of mRNA translational initiation and a longer term up-regulation of ER chaperones and partial translational recovery is discussed. Regulatory sites in mRNA translation and the mechanisms responsible for the early and later phases of the UPR are reviewed. The regulatory significance of GRP78/BiP, a multifunctional, broad-specificity ER chaperone, in the coordination of ER protein processing with mRNA translation during acute and chronic ER stress is addressed. The relationship of ER stress to protein misfolding in the cytoplasm is examined. Translational alterations in embryonic cardiomyocytes during treatments with various Ca(2+)-mobilizing, growth-promoting stimuli are described. The importance of ER Ca(2+) stores, ER chaperones, and cytosolic-free Ca(2+) in translational control and growth promotion by these stimuli is assessed. Some perspectives are provided regarding Ca(2+) as an integrating factor in the generation or diversion of metabolic energy. Circumstances impacting upon cellular adaptability during exposure to growth stimuli or during stressful conditions that require rapid adjustments in ATP for continued viability are considered.     

From histamine to imidazolylalkyl-sulfonamides: the design of a novel series of histamine H3-receptor antagonists.

Histamine was converted to a selective histamine H3-receptor antagonist by capping the primary amine with 2-naphthalenesulfonyl chloride. Higher receptor affinity and lower variability in the data from the various bioassays were achieved with the 2-naphthalensulfonamides of histamine homologues.     

fus-1, a pH Shift Mutant of Semliki Forest Virus, Acts by Altering Spike Subunit Interactions via a Mutation in the E2 Subunit

Semliki Forest virus (SFV), an enveloped alphavirus, is a well-characterized paradigm for viruses that infect cells via endocytic uptake and low-pH-triggered fusion. The SFV spike protein is composed of a dimer of E1 and E2 transmembrane subunits, which dissociate upon exposure to low pH, liberating E2 and the fusogenic E1 subunit to undergo independent conformational changes. SFV fusion and infection are blocked by agents such as ammonium chloride, which act by raising the pH in the endosome and inhibiting the low-pH-induced conformational changes in the SFV spike protein. We have previously isolated an SFV mutant, fus-1, that requires more acidic pH to trigger its fusion activity and is therefore more sensitive to inhibition by ammonium chloride. The acid shift in the fusion activity of fus-1 was here shown to be due to a more acidic pH threshold for the initial dissociation of the fus-1 spike dimer, thereby resulting in a more acidic pH requirement for the subsequent conformational changes in both fus-1 E1 and fus-1 E2. Sequence analysis demonstrated that the fus-1 phenotype was due to a mutation in the E2 spike subunit, threonine 12 to isoleucine. fus-1 revertants that have regained the parental fusion phenotype and ammonium chloride sensitivity were shown to have also regained E2 threonine 12. Our results identify a region of the SFV E2 spike protein subunit that regulates the pH dependence of E1-catalyzed fusion by controlling the dissociation of the E1/E2 dimer.     

Hepatitis C Virus Phylogenetic Clustering Is Associated with the Social-Injecting Network in a Cohort of People Who Inject Drugs

It is hypothesized that social networks facilitate transmission of the hepatitis C virus (HCV). We tested for association between HCV phylogeny and reported injecting relationships using longitudinal data from a social network design study. People who inject drugs were recruited from street drug markets in Melbourne, Australia. Interviews and blood tests took place three monthly (during 2005–2008), with participants asked to nominate up to five injecting partners at each interview. The HCV core region of individual isolates was then sequenced and phylogenetic trees were constructed. Genetic clusters were identified using bootstrapping (cut-off: 70%). An adjusted Jaccard similarity coefficient was used to measure the association between the reported injecting relationships and relationships defined by clustering in the phylogenetic analysis (statistical significance assessed using the quadratic assignment procedure). 402 participants consented to participate; 244 HCV infections were observed in 238 individuals. 26 genetic clusters were identified, with 2–7 infections per cluster. Newly acquired infection (AOR = 2.03, 95% CI: 1.04–3.96, p = 0.037, and HCV genotype 3 (vs. genotype 1, AOR = 2.72, 95% CI: 1.48–4.99) were independent predictors of being in a cluster. 54% of participants whose infections were part of a cluster in the phylogenetic analysis reported injecting with at least one other participant in that cluster during the study. Overall, 16% of participants who were infected at study entry and 40% of participants with newly acquired infections had molecular evidence of related infections with at least one injecting partner. Likely transmission clusters identified in phylogenetic analysis correlated with reported injecting relationships (adjusted Jaccard coefficient: 0.300; p<0.001). This is the first study to show that HCV phylogeny is associated with the injecting network, highlighting the importance of the injecting network in HCV transmission.