Enzymic comparisons of the inorganic sulfur metabolism in autotrophic and heterotrophic Thiobacillus ferrooxidans.

Activities of enzymes which mediate the oxidation of thiosulfate to sulfate and the assimilation of sulfate to sulfide were assayed in various cell-free fractions of Thiobacillus ferrooxidans grown autotrophically on either ferrous iron or thiosulfate or heterotrophically on glucose. There was no activity of the thiosulfate-oxidizing enzyme in extracts of bacteria grown with ferrous iron. Comparable activities for ATP-sulfurylase (EC 2.7.7.4), ADP-sulfurylase (EC 2.7.7.5), and adenylate kinase (EC 2.7.4.3) were found in the bacteria grown autotrophically with either Fe2+ or S2O32- or heterotrophically with glucose.     

DNA Duplexes Containing Alpha Anomeric Nucleotides and Polarity Reversals: Coexistence of Parallel and Antiparallel DNA

Abstract

Oligodeoxynucleotides that possess alpha anomeric nucleotides and polarity reversals show promise for application in the area of antisense therapy. Here we provide a survey of the spectroscopic, thermodynamic, and enzymatic techniques used in our laboratories to investigate model systems containing such unnatural features with the ultimate goal of designing a new class of more potent and effective antisense therapeutics.     

Inhibitory effects of proopiomelanocortin on cortical and medullary activity in the calf adrenal.

The effects of the infusion of ACTH1-24 and proopiomelanocortin on the denervated adrenal gland have been investigated in conscious 3-6 weeks-old calves by means of the adrenal-clamp technique. To prevent variation in the release of endogenous ACTH the pituitary stalk was cauterized during preparatory surgery. ACTH1-24 (5 ng/min per kg, i.v.) increased the output of cortisol from the adrenal by about 500 ng/min per kg body weight and this effect was rapidly reduced by simultaneous infusion of ovine proopiomelanocortin at 5 ng/min per kg. Release of met5-enkephalin and leu-enkephalin from the adrenal was reduced by ACTH1-24 (P less than 0.05) and this effect was enhanced significantly by additional infusion of proopiomelanocortin (P less than 0.02). However met5-enkephalin represented less than one-third of the met-enkephalin containing peptide released and the output of this pool was unaffected by infusion of ACTH. Proopiomelanocortin had no effect on met5-enkephalin production nor that of the total pool of met5-enkephalin containing peptides. It is concluded that ACTH reduced the quantity of pro-enkephalin processed to met5-enkephalin.     

Symptom prescription: Inducing anxiety by 70% exhalation

This study investigates the effects of partial exhalation to feelings of anxiety. Thirty five volunteer subjects (14 male, 21 female, mean age 40.6) were first trained in slow diaphragmatic breathing (SDB). Then subjects rated their anxiety levels on a scale from 1 (none) to 5 (extreme) in sequential conditions of SDB, 70% subjective exhalation, and SDB. During the 70% subjective exhalation phase, subjects were instructed to breathe and limit their exhalation to 70% of the inhaled volume during each consecutive breath. The 70% subjective condition significantly (P<.0005) increased=" subjects'=" anxiety=" levels=" as=" compared=" to=" the=" initial=" sdb=" baseline,=" while=" a=" return=" to=" sdb=" significantly=" reduced=" the=" anxiety=" levels.=" the=" 70%=" approach=" appears=" useful=" in=" demonstrating=" to=" the=" client=" that=" possible=" changes=" in=" breathing=" patterns=" can=" affect=">We gratefully acknowledge the assistance of Richard Steiner, Ph.D. for his comments and help with the statistical analysis in this article. The preliminary findings of this article were presented at the Twenty-first Annual Meeting of the Association for Applied Psychophysiology and Biofeedback.     

Neonatal DNA immunization with a plasmid encoding an internal viral protein is effective in the presence of maternal antibodies and protects against subsequent viral challenge.

Conventional vaccines are remarkably effective in adults but are much less successful in the very young, who are less able to initiate a mature immune response and who may carry maternal antibodies which inactivate standard vaccines. We set out to determine whether DNA immunization might circumvent these problems. We have previously shown that intramuscular injection of plasmid DNA encoding the nucleoprotein (NP) gene of lymphocytic choriomeningitis virus (LCMV) is capable of inducing immune responses and protecting 50% of adult mice against lethal and sublethal challenge with LCMV. Here we demonstrate that mouse pups injected with the same plasmid hours or days after birth produce major histocompatibility complex-restricted, NP-specific cytotoxic T lymphocytes (CTL) that persist into adulthood; 48% of vaccinated pups responded to subsequent sublethal viral challenge by the accelerated production of anti-NP LCMV-specific CTL, indicating that these animals had been successfully immunized by the plasmid DNA. In addition, these mice showed a >95% reduction in splenic viral titers 4 days postinfection compared to control mice, demonstrating a more rapid control of infection in vivo. Furthermore, pups born of and suckled on LCMV-immune dams (and therefore containing passively acquired anti-LCMV antibodies at the time of DNA inoculation) responded to the DNA vaccine in a similar manner, showing that maternally derived anti-LCMV antibodies do not significantly inhibit the generation of protective immune responses following DNA vaccination. These findings suggest that, at least in this model system, DNA immunization circumvents many of the problems associated with neonatal immunization.     

First-in-Man Open Clinical Trial of a Combined rdESAT-6 and rCFP-10 Tuberculosis Specific Skin Test Reagent

Background

Tuberculin is still the only available skin test reagent for the diagnosis of mycobacterial infection. The product has a remarkable sensitivity, but poor specificity. Previous studies, including two human phase I clinical trials, have indicated that rdESAT-6 has a potential as an improved skin test reagent. Animal studies have shown that the sensitivity may be increased by inclusion of the genetically related CFP-10 antigen in the preparation without loosing specificity.

Methodology

In this study a Lactococcus fermented, recombinant skin test reagent consisting of a 1∶1 wt/wt of rdESAT-6 and CFP-10 was manufactured according to GMP standards and tested for the first time in 42 healthy adult volunteers. The two doses of 0.01 µg or 0.1 µg were injected intradermally by the Mantoux technique with 6 or 12 weeks interval. No serious adverse events and only mild adverse reactions were reported. The reagent elicited a positive skin test reaction after the first injection in one participant, who most likely was latently infected with M. tuberculosis as indicated by an appreciable IFN γ response just below the Quantiferon® cut-off level at the screening visit. None of the remaining participants in the four groups had any skin test reactions and sensitisation by the reagent could therefore be excluded.

Conclusion

The investigational skin test reagent rdESAT-6 and CFP-10 appeared safe and non-sensitising in this first-in-man clinical trial in human volunteers and can now be tested in larger clinical trials involving individuals with latent M. tuberculosis infection or active TB disease.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00793702","term_id":"NCT00793702"}}NCT00793702     

Regulated specific proteolysis of the Cajal body marker protein coilin

Cajal bodies (CB) are subnuclear domains that contain various proteins with diverse functions including the CB marker protein coilin. In this study, we investigate the proteolytic activity of calpain on coilin. Here, we report a 28-kDa cleaved coilin fragment detected by two coilin antibodies that is cell cycle regulated, with levels that are consistently reduced during mitosis. We further show that an in vitro calpain assay with full-length or C-terminal coilin recombinant protein releases the same size cleaved fragment. Furthermore, addition of exogenous RNA to purified coilin induces proteolysis by calpain. We also report that the relative levels of this cleaved coilin fragment are susceptible to changes induced by various cell stressors, and that coilin localization is affected by inhibition or knockdown of calpain both under normal and stressed conditions. Collectively, our data suggest that coilin is subjected to regulated specific proteolysis by calpain, and this processing may play a role in the regulation of coilin activity and CB formation.     

In vitro glycosidation potential towards olomoucine-type cyclin-dependent kinase inhibitors in rodent and primate microsomes

Interspecies differences in glycosidation potential in mammalian tissues represent a factor contributing to ambiguity when endobiotic and/or xenobiotic metabolic pathways are extrapolated from animals to man. Using the TLC/autoradiographic technique, we conducted an in vitro investigation involving mouse, rat, monkey, as well as human liver and kidney microsomes to evaluate their glycoconjugation potential towards (3)H-labeled, purine-derived selective inhibitors of cyclin-dependent kinases such as olomoucine, bohemine, roscovitine, 6-(2-hydroxybenzyl)amino-2-(1-hydroxymethyl-2-methylpropyl)amino-9-isopropylpurine (compound A-4), and 6-(3-hydroxybenzyl)amino-2-[(1(R/S)-hydroxymethyl)propyl]amino-9-isopropylpurine (compound A-5) as aglycones. Principally, this study confirmed the aliphatic hydroxyl group of olomoucine-type inhibitors as a relatively suitable target for glucuronide, glucoside, xyloside, galactoside, and/or N-acetylaminoglucoside conjugation. Of the tissues examined, only the mouse microsomes were able to perform glucosidation and galactosidation reactions with the aglycones. On the other hand, monkey microsomes were superior to the mouse microsomes in a variety of glucuronide conjugates produced with compounds A-4 and A-5.