Comparison of a multiplex-PCR assay with mycolic acids analysis and conventional methods for the identification of mycobacteria

A fast, sensitive and cost-effective multiplex-PCR assay for Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium (M. avium) identification for routine diagnosis was evaluated. A total of 158 isolates of mycobacteria from 448 clinical specimens from patients with symptoms of mycobacterial disease were analyzed. By conventional biochemical methods 151 isolates were identified as M. tuberculosis, five as M. avium and two as Mycobacterium chelonae (M. chelonae). Mycolic acid patterns confirmed these results. Multiplex-PCR detected only IS6110 in isolates identified as MTC, and IS1245 was found only in the M. avium isolates. The method applied to isolates from two patients, identified by conventional methods and mycolic acid analysis, one as M. avium and other as M. chelonae, resulted positive for IS6110, suggesting co-infection with M. tuberculosis. These patients were successfully submitted to tuberculosis treatment. The multiplex-PCR method may offer expeditious identification of MTC and M. avium, which may minimize risks for active transmission of these organisms and provide useful treatment information.     

Terpenoids in Buddleja: relevance to chemosystematics,chemical ecology and biological activity

The terpenoids reported from Buddleja species are described. The antifungal activity of chloroform extracts of B. cordata and B. davidii stembark against the soil fungi Fusarium culmorum and Sordari fimicola is reported, with buddledin A shown to be the major compound responsible. The terpenoids present support the view that the Buddlejaceae should be classified in a taxon with Scrophulariaceae rather than Loganiaceae. Ecological aspects of the terpenoids are considered in relation to insects and soil fungi and the role of terpenoids in the chemical basis of the use of Buddleja in traditional medicine is also discussed, especially with regard to their anti-inflammatory properties.     

Random mutagenesis of the M3 muscarinic acetylcholine receptor expressed in yeast. Identification of point mutations that "silence" a constitutively active mutant M3 receptor and greatly impair receptor/G protein coupling

The M3 muscarinic receptor is a prototypical member of the class I family of G protein-coupled receptors (GPCRs). To facilitate studies on the structural mechanisms governing M3 receptor activation, we generated an M3 receptor-expressing yeast strain (Saccharomyces cerevisiae) that requires agonist-dependent M3 receptor activation for cell growth. By using receptor random mutagenesis followed by a genetic screen in yeast, we initially identified a point mutation at the cytoplasmic end of transmembrane domain (TM) VI (Q490L) that led to robust agonist-independent M3 receptor signaling in both yeast and mammalian cells. To explore further the molecular mechanisms by which point mutations can render GPCRs constitutively active, we subjected a region of the Q490L mutant M3 receptor that included TM V-VII to random mutagenesis. We then applied a yeast genetic screen to identify second-site mutations that could suppress the activating effects of the Q490L mutation and restore wild-type receptor-like function to the Q490L mutant receptor. This analysis led to the identification of 12 point mutations that allowed the Q490L mutant receptor to function in a fashion similar to the wild-type receptor. These amino acid substitutions mapped to two distinct regions of the M3 receptor, the exofacial segments of TM V and VI and the cytoplasmic ends of TM V-VII. Strikingly, in the absence of the activating Q490L mutation, all recovered point mutations severely reduced the efficiency of receptor/G protein coupling, indicating that the targeted residues play important roles in receptor activation and/or receptor/G protein coupling. This strategy should be generally applicable to identify sites in GPCRs that are critically involved in receptor function.     

Studies on the production and regulation of interleukin,IL-13, IL-4 and interferon-gamma in human Schistosomiasis mansoni

The production and regulation of interleukin (IL) IL-13, IL-4 and interferon-gamma was evaluated in different clinical forms of human schistosomiasis. The mechanisms of immune regulation are apparently different in the various clinical stages of the disease, some of them being antigen specific.     

Analysis of clustered binary outcomes using within-cluster paired resampling

Conditional logistic regression (CLR) is useful for analyzing clustered binary outcome data when interest lies in estimating a cluster-specific exposure parameter while treating the dependency arising from random cluster effects as a nuisance. CLR aggregates unmeasured cluster-specific factors into a cluster-specific baseline risk and is invalid in the presence of unmodeled heterogeneous covariate effects or within-cluster dependency. We propose an alternative, resampling-based method for analyzing clustered binary outcome data, within-cluster paired resampling (WCPR), which allows for within-cluster dependency not solely due to baseline heterogeneity. For example, dependency may be in part caused by heterogeneity in response to an exposure across clusters due to unmeasured cofactors. When both CLR and WCPR are valid, our simulations suggest that the two methods perform comparably. When CLR is invalid, WCPR continues to have good operating characteristics. For illustration, we apply both WCPR and CLR to a periodontal data set where there is heterogeneity in response to exposure across clusters.     

Adsorptive control of water in esterification with immobilized enzymes: I. Batch reactor behavior

Reducing the influence of an undesired product in an enzymatic reaction could have a significant impact on the productivity of such systems. Here, we focus on the removal of water formed during an enzymatic esterification in a batch reactor. A commercial immobilized lipase preparation, known as Lipozyme, is used as the biocatalyst and propionic acid and isoamyl alcohol dissolved in hexane are the substrates. In this system, the water formed will partition between the catalyst and the medium. As the more polar reactants are converted into the less polar ester product, the water is partitioned more towards the biocatalyst and the accumulation of water eventually causes lower reaction rates. Addition of a strong-acid cation exchange resin in sodium form is found to control the water accumulation on the biocatalyst without stripping the essential water needed for the enzyme to function and substantial improvements in conversion are achieved. A mathematical model is developed to describe the batch reaction behavior with and without added absorbent, which successfully predicts the behavior of water and its effects.     

COEXLSTENCE OF TOXIC AND NONTOXIC DINOKLAGELLAIES RESEMBLING GQNYAULAX TAMARENSIS IN NEW ENCILAND COASTAL WATERS (NW ATLANTIC)1

Two forms resembling Gonyaulax tamarrensis Lebour coexist in Maine cosat plankton; one is toxic, the other is nontoxic. At times, red patches of dinoflagellates were identified as G. tamarensis, yet only presumed to he toxic. Toxic froms were found in June, July 1975 and 1976. The nontoxic form was found in a more esraurine area, e.g., mid-July 1975, early July 1976. These two forms are not easily distinguished by conventional microncopic ohservation, nor by pigment analysis. Preliminary observations suggest that the nontoxic form is smaller than G. excavata (Braarud) Balech and tacks the “excavated” ventrat region. Characteristic G. excavata resting cysts were found in sediment from the shellfish toxic area off Newagen and Monhegan (Maine) and were not found in sediments from areas where the nontoxic form bloomed. There appear to be a minimum of two G. tamarensis-like organisms. Full systematic treatment of these must await further information.     

beta-Synuclein reduces proteasomal inhibition by alpha-synuclein but not gamma-synuclein

The accumulation of aggregated alpha-synuclein is thought to contribute to the pathogenesis of Parkinson's disease. Recent studies indicate that aggregated alpha-synuclein binds to S6', a component of the 19 S subunit in the 26 S proteasome and inhibits 26 S proteasomal degradation, both ubiquitin-independent and ubiquitin-dependent. The IC(50) of aggregated alpha-synuclein for inhibition of the 26 S ubiquitin-independent proteasomal activity is approximately 1 nm. alpha-Synuclein has two close homologues, termed beta-synuclein and gamma-synuclein. In the present study we compared the effects of the three synuclein homologues on proteasomal activity. The proteasome exists as a 26 S and a 20 S species, with the 26 S proteasome containing the 20 S core and 19 S cap. Monomeric alpha- and beta-synucleins inhibited the 20 S and 26 S proteasomal activities only weakly, but monomeric gamma-synuclein strongly inhibited ubiquitin-independent proteolysis. The IC(50) of monomeric gamma-synuclein for the 20 S proteolysis was 400 nm. In monomeric form, none of the three synuclein proteins inhibited 26 S ubiquitin-dependent proteasomal activity. Although beta-synuclein had no direct effect on proteasomal activity, co-incubating monomeric beta-synuclein with aggregated alpha-synuclein antagonized the inhibition of the 26 S ubiquitin-independent proteasome by aggregated alpha-synuclein when added before the aggregated alpha-synuclein. Co-incubating beta-synuclein with gamma-synuclein had no effect on the inhibition of the 20 S proteasome by monomeric gamma-synuclein. Immunoprecipitation and pull-down experiments suggested that antagonism by beta-synuclein resulted from binding to alpha-synuclein rather than binding to S6'. Pull-down experiments demonstrated that recombinant monomeric beta-synuclein does not interact with the proteasomal subunit S6', unlike alpha-synuclein, but beta-synuclein does bind alpha-synuclein and competes with S6' for binding to alpha-synuclein. Based on these data, we hypothesize that the alpha- and gamma-synucleins regulate proteasomal function and that beta-synuclein acts as a negative regulator of alpha-synuclein.     

Molecular characterization of antibiotic resistance in clinical Salmonella typhi isolated in Ghana

Fifty-eight clinical Salmonella typhi strains isolated from patients suspected of suffering from typhoid fever were obtained at the Korle-Bu Teaching Hospital and the Noguchi Memorial Institute for Medical Research, both located in Ghana, Africa. Each isolate was examined for susceptibility to ampicillin, chloramphenicol, streptomycin, tetracycline, and trimethoprim/sulfamethoxazole by the disk diffusion assay. Five of the isolates were resistant to all five antibiotics while 10 isolates were resistant to ampicillin, chloramphenicol, and trimethoprim/sulfamethoxazole, which are considered 'first line' antibiotics in the treatment of typhoid fever. Thirty-four isolates were resistant to at least one of the antibiotics tested and 62% of these isolates possessed conjugable plasmids belonging to incompatibility group IncHI. Ninety percent of the conjugable plasmids conferred a multiple drug-resistant phenotype on the strains harboring them. Additionally, 14 strains contained plasmids that were transformable and six of them encoded multiple drug resistance. Our findings indicate that multiple drug resistance to the 'first line' antibiotics in S. typhi may be more prevalent in Africa than previously thought.