The origin of chloroplastic acetyl coenzyme A

Pyruvic dehydrogenase activity has been examined in a number of highly purified leaf organelles. In spinach leaf cell, the major activity is in the mitochrondrion with low activity in isolated chloroplasts. The major source of CO₂ derived from pyruvic acid metabolism in the isolated chloroplast is via the acetolactic synthase reaction localized in the chloroplast. Evidence is presented that the leaf mitochondrion contains both the pyruvic acid dehydrogenase and an acetyl coenzyme A hydrolase. It is suggested that free acetic acid is generated in the mitochrondrion and then moves to the chloroplast where acetyl coenzyme synthetase converts it from the metabolically inert acid to the very metabolically active acetyl coenzyme A.     

Vitek 2 ANC card versus BBL Crystal Anaerobe and RapID ANA II for identification of clinical anaerobic bacteria

The Vitek 2 Anaerobe and Corynebacterium Identification Card (ANC) was recently evaluated in a multicentre study. In the present work, this system was compared with the BBL Crystal Anaerobe and RapID ANA II panels. These kits were tested using 196 strains of anaerobes that had been previously identified by gas–liquid chromatography. Identification to the species or to the genus level was 75.0%, 81.1% and 70.9% for Crystal, RapID and Vitek, respectively. Vitek ANC failed to provide any identification in 20.4% of the strains, but it had fewer misidentifications than RapID. The confidence factors provided on the results report of each kit were not always correlated with a lower risk of major errors, with the exception of Vitek 2 in which a confidence factor higher than 0.86 excluded the risk of misidentification in more than 87% of isolates. The lower rate of identification by the Vitek and Crystal panels is mostly due the lower ability of these systems to identify the Clostridia. Overall, the three panels are comparable but need improvement to a better accuracy.     

In vivo simulation of human pharmacokinetics in the rabbit

The evaluation of drugs in vivo is often based on experimental models using small animals such as mice, rats and rabbits. However, these models could be improved to correspond more closely to the human situation if the pharmacokinetics of the drugs tested in animals were similar to that observed in humans. The use of a computer-controlled pump allowing an adequate flow of tobramycin and amikacin to be infused into rabbits enabled us to simulate the human pharmacokinetics of these antibiotics in vivo in this study. The function defining the rate of infusion required to perform the simulation of an intravenous bolus was first determined generally and symbolically for linear pharmacokinetic models independently from the number of compartments involved. The practical simulation of a decreasing monoexponential serum profile with a half-life of 2 h (one-compartment model for the human pharmacokinetics of aminoglycosides) was then studied for tobramycin and amikacin on the basis of a two-compartment model in the animal. The kinetics obtained had an apparent elimination half-life of 1.97 and 1.86 h, respectively. Linearity of the semilogarithmic regressions of the profiles obtained was quite sound. Finally, an a posteriori analysis of the pharmacokinetic model and its parameters is proposed on the basis of the results obtained after simulation.     

Treatment of HeLa cells with bacterial water extracts inhibits Shigella flexneri invasion

Abstract Pathogenesis mediated by Shigella flexneri requires invasion of the gastrointestinal epithelium. It has been previously shown that HeLa cells challenged with S. flexneri show alterations in their phosphotyrosine-containing protein profile. In this report, we demonstrated that bacterial water extracts (WE) abrogated the invasion of HeLa cells by S. flexneri in a dose-dependent manner. A proteinaceous component of S. flexneri was shown to be responsible for this inhibitory activity. Proteins encoded on the 140-MDa plasmid were not responsible for the observed inhibition. WE from other Gram-negative bacteria also inhibited Shigella invasion of HeLa cells. HeLa cells pretreated with WE showed changes in the profile and the intensity of phosphotyrosine-containing protein bands. These data were consistent with a surface protein component in WE which initiated aberrant host cell signaling at the membrane which may account for the inhibition of bacterial entry.     

A New HPLC Analytical Method to Study Fungal Caffeine Metabolism

A simple and rapid HPLC method has been developed to analyse all the methylxanthines that can be produced by N-demethylation of 1,3,7-trimethylxanthine (caffeine). This method is particularly suitable to study caffeine metabolism of a filamentous fungus (Aspergillus sp V12A25) cultivated in a synthetic liquid medium containing caffeine as the sole source of nitrogen.     

Construction of a Vibrio splendidus mutant lacking the metalloprotease gene vsm by use of a novel counterselectable suicide vector

Vibrio splendidus is a dominant culturable Vibrio in seawater, and strains related to this species are also associated with mortality in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly understood; however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, provides an opportunity to decipher the basis of the virulence properties by disruption of candidate genes. We developed a novel suicide vector based on the pir-dependent R6K replicative origin, which potentially can be transferred by RP4-based conjugation to any Vibrio strain and which also carries the plasmid F toxin ccdB gene under control of the PBAD promoter. We demonstrated that this genetic system allows efficient counterselection of integrated plasmids in the presence of arabinose in both V. splendidus and Vibrio cholerae and thus permits efficient markerless allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative with a secreted metalloprotease gene, vsm, deleted. We found that this gene is essential for LGP32 extracellular product toxicity when the extracellular products are injected into oysters but is not necessary for virulence of bacteria in the oyster infection model when bacteria are injected.     

Noninvasive monitoring of fecal cortisol metabolites in the eastern chipmunk (Tamias striatus): validation and comparison of two enzyme immunoassays

Monitoring fecal glucocorticoid metabolites in wild animals, using enzyme immunoassays, enables the study of endocrinological patterns relevant to ecology and evolution. While some researchers use antibodies against the parent hormone (which is typically absent from fecal samples), others advocate the use of antibodies designed to detect glucocorticoid metabolites. We validated two assays to monitor fecal cortisol metabolites in the eastern chipmunk (Tamias striatus). We compared an antibody produced against cortisol and one produced against 5α-pregnane-3β, 11β, 21-triol-20-one using a radiometabolism study and an injection with adrenocorticotropic hormone (ACTH). Most cortisol metabolites were excreted in the urine (～83%). Peak excretion in the feces occurred 8 h after injection. Both assays detected an increase in fecal cortisol metabolite levels after injection of ACTH. Males, but not females, exhibited a circadian variation in metabolite levels. The sexes did not exhibit any difference over the time course and route of excretion or the relative increase in fecal cortisol metabolite levels after ACTH injection. The cortisol assay displayed higher reactivity to ACTH injection relative to baseline than did the metabolite assay. While both antibodies gave comparable results, the cortisol antibody was more sensitive to changes in plasma cortisol levels in eastern chipmunks.