Enantioselective determination of metoprolol and major metabolites in human urine by capillary electrophoresis

The enantiomeric separation of metoprolol and its metabolites in human urine was undertaken using capillary electrophoresis (CE). Resolution of the enantiomers was achieved using carboxymethyl-β-cyclodextrin (CM-β-CD) as the chiral selector. A 100-mM acetate buffer (pH 4.0) containing 5% 2-propanol and 10 mM CM-β-CD resulted in the optimum separation of the metoprolol enantiomers and its acidic metabolite in human urine. Following a single metoprolol oral administration of 100 mg racemic metoprolol tartrate, stereoselective pharmacokinetic analysis showed that urinary acidic metabolite 3 of metoprolol accounted for 62.3% of the dose with an R/S ratio of 1.23 and urinary unchanged metoprolol 1 accounted for 6.3% of the dose with an R/S ratio of 0.72.     

The delta and delta ' subunits of the DNA polymerase III holoenzyme are essential for initiation complex formation and processive elongation.

delta and delta' are required for assembly of the processivity factor beta(2) onto primed DNA in the DNA polymerase III holoenzyme-catalyzed reaction. We developed protocols for generating highly purified preparations of delta and delta'. In holoenzyme reconstitution assays, delta' could not be replaced by delta, tau, or gamma, even when either of the latter were present at a 10,000-fold molar excess. Likewise, delta could not be replaced by delta', tau, or gamma. Bacterial strains bearing chromosomal knockouts of either the holA(delta) or holB(delta') genes were not viable, demonstrating that both delta and delta' are essential. Western blots of isolated initiation complexes demonstrated the presence of both delta and delta'. However, in the absence of chipsi and single-stranded DNA-binding protein, a stable initiation complex lacking deltadelta' was isolated by gel filtration. Lack of delta-delta' decreased the rate of elongation about 3-fold, and the extent of processive replication was significantly decreased. Adding back delta-delta' but not chipsi, delta, or delta' alone restored the diminished activity, indicating that in addition to being key components required for the beta loading activity of the DnaX complex, deltadelta' is present in initiation complex and is required for processive elongation.     

Fate and Dissemination of Bacillus subtilis Spores in a Murine Model

Bacterial spores are being consumed as probiotics, although little is known about their efficacy or mode of action. As a first step in characterizing spore probiotics, we have studied the persistence and dissemination of Bacillus subtilis spores given orally to mice. Our results have shown that spores do not appear to disseminate across the mucosal surfaces. However, we found that the number of spores excreted in the feces of mice was, in some experiments, larger than the original inoculum. This was an intriguing result and might be explained by germination of a proportion of the spore inoculum in the intestinal tract, followed by limited rounds of cell growth and then sporulation again. This result raises the interesting question of whether it is the spore or the germinated spore that contributes to the probiotic effect of bacterial spores.     

Direct whole plant regeneration of cowpea [Vigna unguiculata (L.) Walp] from cotyledonary node thin cell layer explants

A simple protocol was established for high frequency direct shoot regeneration of cowpea [Vigna unguiculata (L.) cv. EPACE-1]. Bud proliferation occurred at the cotyledonary nodes of cowpea seedlings three weeks after culture on a medium containing Murashige and Skoog salts (1962) and B5 vitamins (Gamborg et al. 1968) supplemented with TDZ. A 10 μmol/L TDZ pre-treatment, shoot tip removal and excision of longitudinal thin cell layers (TCL) at the level of the cotyledonary nodes with subsequent culture on a MSB5 medium supplemented with 1 μmol/L IBA and 1 μmol/L TDZ were the optimal conditions for maximum bud proliferation. Up to 32.5 regenerated shoot buds were produced per TCL. The regenerated plants (R₀) were true-to-type and successfully transferred to soil.     

A Software Suite for High-Performance Communications on Clusters of SMPs

A cluster, by opposition to a parallel computer, is a set of separate workstations interconnected by a high-speed network. The performances one can get on a cluster heavily depend on the performances of the lowest communication layers. In this paper, we address the special case where the cluster contains multi-processor machines. These shared-memory multi-processors desktop machines (SMPs) with 2 or 4 processors are now becoming very popular and present a high performance/price ratio. We present a software suite for achieving high-performance communications on a Myrinet-based cluster: BIP, BIP-SMP and MPI-BIP. The software suite supports single-processor (Intel PC and Digital Alpha) and multi-processor machines, as well as any combination of the two architectures.     

Flavonoids differentially inhibit guinea pig epidermal cytosolic phospholipase A2

Cytosolic phospholipase A₂ (cPLA₂) is believed to involve the regulation of essential cellular processes. Like other cell types, epidermal cPLA₂ may participate in various metabolic processes including eicosanoid generation. In this investigation, we demonstrated the presence of cPLA₂ in guinea pig epidermis. The epidermal cPLA₂ is Ca²⁺-dependent, active at micromolar concentration of Ca²⁺ and resistant to disulfide-reducing agents. Furthermore, it is inhibited by methyl arachidonyl fluorophosphonate (MAFP), a selective inhibitor of cPLA₂, while 12-epi-scalardial (a sPLA₂ inhibitor) did not cause inhibition. A test of several flavonoids revealed that quercetin (flavonol) weakly inhibited cPLA₂, while flavanone had negligible inhibitory activity. In contrast, amentoflavone and ginkgetin (biflavones) markedly inhibited cPLA₂ activity in the epidermis. These results underscore that different flavonoids do vary in their capability to exert differential effects on arachidonate metabolism in the skin via modulation of epidermal cPLA₂ activity.     

Characterization of Biofilm Formation by Borrelia burgdorferi In Vitro

Borrelia burgdorferi, the causative agent of Lyme disease, has long been known to be capable of forming aggregates and colonies. It was recently demonstrated that Borrelia burgdorferi aggregate formation dramatically changes the in vitro response to hostile environments by this pathogen. In this study, we investigated the hypothesis that these aggregates are indeed biofilms, structures whose resistance to unfavorable conditions are well documented. We studied Borrelia burgdorferi for several known hallmark features of biofilm, including structural rearrangements in the aggregates, variations in development on various substrate matrices and secretion of a protective extracellular polymeric substance (EPS) matrix using several modes of microscopic, cell and molecular biology techniques. The atomic force microscopic results provided evidence that multilevel rearrangements take place at different stages of aggregate development, producing a complex, continuously rearranging structure. Our results also demonstrated that Borrelia burgdorferi is capable of developing aggregates on different abiotic and biotic substrates, and is also capable of forming floating aggregates. Analyzing the extracellular substance of the aggregates for potential exopolysaccharides revealed the existence of both sulfated and non-sulfated/carboxylated substrates, predominately composed of an alginate with calcium and extracellular DNA present. In summary, we have found substantial evidence that Borrelia burgdorferi is capable of forming biofilm in vitro. Biofilm formation by Borrelia species might play an important role in their survival in diverse environmental conditions by providing refuge to individual cells.     

A cluster of Ankyrin and Ankyrin-TPR repeat genes is associated with panicle branching diversity in rice

The number of grains per panicle is an important yield-related trait in cereals which depends in part on panicle branching complexity. One component of this complexity is the number of secondary branches per panicle. Previously, a GWAS site associated with secondary branch and spikelet numbers per panicle in rice was identified. Here we combined gene capture, bi-parental genetic population analysis, expression profiling and transgenic approaches in order to investigate the functional significance of a cluster of 6 ANK and ANK-TPR genes within the QTL. Four of the ANK and ANK-TPR genes present a differential expression associated with panicle secondary branch number in contrasted accessions. These differential expression patterns correlate in the different alleles of these genes with specific deletions of potential cis-regulatory sequences in their promoters. Two of these genes were confirmed through functional analysis as playing a role in the control of panicle architecture. Our findings indicate that secondary branching diversity in the rice panicle is governed in part by differentially expressed genes within this cluster encoding ANK and ANK-TPR domain proteins that may act as positive or negative regulators of panicle meristem’s identity transition from indeterminate to determinate state.