Production of chiral R-3-hydroxyalkanoic acids and R-3-hydroxyalkanoic acid methylesters via hydrolytic degradation of polyhydroxyalkanoate synthesized by pseudomonads.

A novel and efficient method for the production of enantiomericaly pure R-3-hydroxyalkanoic acids and R-3-hydroxyalkanoic acid methylesters was developed. The described method is based on hydrolysis of poly(hydroxyalkanoate) copolymers synthesized by Pseudomonas putida. The polymer was isolated via solvent recovery and hydrolyzed by acid methanolysis. The obtained 3-hydroxyalkanoic acid methylester mixture was distilled into several fractions with an overall yield of 96.6% (w/w). Gas chromatography-mass spectrometry analysis of the fractions showed that 3-hydroxyhexanoic-, 3-hydroxyoctanoic-, 3 hydroxydecanoic-, and 3-hydroxydodecanoic acid methylesters were enriched to purities exceeding 96 mol%, with distillation yields of 99.9, 99.8, 88.4, and 56.8% (w/w), respectively. Subsequent saponification of the purified methylester fractions yielded the corresponding 3-hydroxyalkanoic acids, which were recovered up to 92.8% (w/w). Chiral gas chromatography analysis confirmed that both 3-hydroxyoctanoic acid and 3-hydroxyoctanoic acid methylester are present in the R-form at a very high enantiomeric excess (>99.9%).     

An inhibitor-resistant histone deacetylase in the plant pathogenic fungus Cochliobolus carbonum.

We have partially purified and characterized histone deacetylases of the plant pathogenic fungus Cochliobolus carbonum. Depending on growth conditions, this fungus produces HC-toxin, a specific histone deacetylase inhibitor. Purified enzymes were analyzed by immunoblotting, by immunoprecipitation, and for toxin sensitivity. The results demonstrate the existence of at least two distinct histone deacetylase activities. A high molecular weight complex (430,000) is sensitive to HC-toxin and trichostatin A and shows immunoreactivity with an antibody against Cochliobolus HDC2, an enzyme homologous to yeast RPD3. The second activity, a 60,000 molecular weight protein, which is resistant even to high concentrations of well-known deacetylase inhibitors, such as HC-toxin and trichostatin A, is not recognized by antibodies against Cochliobolus HDC1 (homologous to yeast HOS2) or HDC2 and represents a different and/or modified histone deacetylase which is enzymatically active in its monomeric form. This enzyme activity is not present in the related filamentous fungus Aspergillus nidulans. Furthermore, in vivo treatment of Cochliobolus mycelia with trichostatin A and analysis of HDACs during the transition from non-toxin-producing to toxin-producing stages support an HC-toxin-dependent enzyme activity profile.     

Molecular Analysis of the β-Globin Gene Cluster in the Niokholo Mandenka Population Reveals a Recent Origin of the βS Senegal Mutation

A large and ethnically well-defined Mandenka sample from eastern Senegal was analyzed for the polymorphism of the beta-globin gene cluster on chromosome 11. Five RFLP sites of the 5' region were investigated in 193 individuals revealing the presence of 10 different haplotypes. The frequency of the sickle-cell anemia causing mutation (beta(S)) in the Mandenka estimated from this sample is 11.7%. This mutation was found strictly associated with the single Senegal haplotype. Approximately 600 bp of the upstream region of the beta-globin gene were sequenced for a subset of 94 chromosomes, showing the presence of four transversions, five transitions, and a composite microsatellite polymorphism. The sequence of 22 beta(S) chromosomes was also identical to the previously defined Senegal haplotype, suggesting that this mutation is very recent. Monte Carlo simulations (allowing for a specific balancing selection model, a logistic growth of the population, and variable initial frequencies of the Senegal haplotype) were used to estimate the age of the beta(S) mutation. Resulting maximum-likelihood estimates are 45-70 generations (1,350-2,100 years) for very different demographic scenarios. Smallest confidence intervals (25-690 generations) are obtained under the hypothesis that the Mandenka population is large (N(e) >5,000) and stationary or that it has undergone a rapid demographic expansion to a current size of >5,000 reproducing individuals, which is quite likely in view of the great diversity found on beta(A) chromosomes.     

Stimulation of the NADPH oxidase in activated rat microglia removes nitric oxide but induces peroxynitrite production

Cultured rat microglia produced extracellular superoxide at a rate of 814 +/- 52 pmol/min/million cells when stimulated with phorbol 12-myristate 13-acetate (PMA) as measured by extracellular cytochrome c reduction. This superoxide production resulted in a rapid rate of superoxide dismutase-sensitive nitric oxide (NO) breakdown (155 +/- 30 pmol of NO/min/million cells) when NO was added to PMA stimulated microglia. Lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-activated microglia produce NO at the rate of 145 +/- 42 pmol/min/million cells and activated astrocytes at the rate of 51 +/- 9 pmol/min/million cells as estimated by NO electrode. Both types of cells maintained a steady-state level of 0.5-0.7 microm NO, only in the presence of L-arginine. Addition of PMA to activated microglia (but not activated astrocytes) caused the rapid and complete disappearance of all extracellular NO (but was restored in the presence of superoxide dismutase) followed by the production of peroxynitrite (as measured by urate-sensitive oxidation of dihydrorhodamine). Co-incubation of activated microglia with cerebellar granule neurones resulted in NO inhibition of neuronal respiration, but this was rapidly removed by PMA-induced breakdown of the NO. Thus, microglial NADPH oxidase can regulate the bioavailability of NO and the production of peroxynitrite.     

Evaluation of tissue and cellular biomarkers to assess 2,4,6-trinitrotoluene (TNT) exposure in earthworms: effects-based assessment in laboratory studies using Eisenia andrei

The lysosomal neutral red retention time (NRRT) assay, a biomarker for lysosomal membrane stability, and the total immune activity (TIA) assay, a measure of non-specific immune system activity, were used in laboratory studies to assess the toxic effects of 2,4,6-trinitrotoluene (TNT) on earthworms (Eisenia andrei) in vivo. The results were compared with the concentration of TNT and its metabolites in earthworm tissue, as well as standard sublethal toxicity endpoints including growth (i.e. weight change) and reproduction effects from previously published studies. Filter paper experiments indicated a significant decrease in NRRT at ≥1.8 µg TNT cm^-2, whereas sublethal (weight loss) and lethal effects to earthworms were detected at ≥3.5 and 7.1 µg TNT cm^-2, respectively. Experiments in artificial soil showed that NRRT effects could be detected at lower TNT concentrations ( ≥55 mg TNT kg^-1 soil dry weight) compared with other sublethal endpoints (effects on growth and reproduction). The TIA biomarker did not significantly respond to TNT. Copper (as CuSO₄, filter paper contact tests) and 2-chloroacetamide (soil tests), which were used as reference toxicants, also decreased the NRRT. The use of the NRRT assay linked with tissue concentrations of TNT metabolites in earthworms was identified as a potentially appropriate biomarker approach for TNT exposure assessment under laboratory conditions and a novel tool for effects-based risk assessment.     

Mutation spectrum and splicing variants in the OPA1 gene

Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy that features low visual acuity leading in many cases to legal blindness. We have recently shown, with others, that mutations in the OPA1 gene encoding a dynamin-related mitochondrial protein, underlie the dominant form of optic atrophy. Here we report that OPA1 has eight mRNA isoforms as a result of the alternative splicing of exon 4 and two novel exons named 4b and 5b. In addition, we screened a cohort of 19 unrelated patients with dominant optic atrophy by direct sequencing of the 30 OPA1 exons (including exons 4b and 5b) and found mutations in 17 (89%) of them of which 8 were novel. A majority of these mutations were truncative (65%) and located in exons 8 to 28, but a number of them were amino acid changes predominantly found in the GTPase domain (exons 8 to 15). We hypothesize that at least two modifications of OPA1 may lead to dominant optic atrophy, that is alteration in GTPase activity and loss of the last seven C-terminal amino acids that putatively interact with other proteins.     

Modification of a rapid method for the identification of gene-specific polymorphisms in Cryptosporidium parvum and its application to clinical and epidemiological investigations.

The application of genotyping to clinical isolates of Cryptosporidium has increased significantly our knowledge and understanding of the distribution and epidemiology of this parasite. However, some methods can be laborious and demand specialist technical expertise. PCR-restriction fragment length polymorphism (RFLP) techniques represent a more rapid and simple method of genotyping to support epidemiological and clinical investigations than conventional DNA analytical techniques. We describe a nested PCR-RFLP technique that identifies polymorphisms in the C. parvum thrombospondin-related adhesive protein gene locus; this method offers a sensitive and specific tool for the confirmation and investigation of disease associated with C. parvum. The potential of this enhanced method is demonstrated by its application to the confirmation and epidemiological investigation of an outbreak of cryptosporidiosis associated with a school visit to an open farm.     

Cortical heterogeneity: Implications for visual processing and polysensory integration

Recent studies have revealed substantial variation in pyramidal cell structure in different cortical areas. Moreover, cell morphology has been shown to vary in a systematic fashion such that cells in visual association areas are larger and more spinous than those in the primary visual area. Various aspects of these structural differences appear to be important in influencing neuronal function. At the cellular level, differences in the branching patterns in the dendritic arbour may allow for varying degrees of non-linear compartmentalisation. Differences in total dendritic length and spine number may determine the number of inputs integrated by individual cells. Variations in spine density and geometry may affect cooperativity of inputs and shunting inhibition, and the tangential dimension of the dendritic arbours may determine sampling strategies within cortex. At the systems level, regional variation in pyramidal cell structure may determine thedegree of recurrent excitation through reentrant circuits influencing the discharge properties of individual neurones and the functional signature of the circuits they compose. The ability of pyramidal neurones in visual areas of the parietal and temporal lobes to integrate large numbers of excitatory inputs may also facilitate cortical binding. Here I summarise what I consider to be among the most salient, and testable, aspects of an inter-relationship between morphological and functional heterogeneity in visual cortex.     

Nitric oxide stimulates PC12 cell proliferation via cGMP and inhibits at higher concentrations mainly via energy depletion.

We investigated the mechanisms by which nitric oxide (NO) from an NO donor (DETA/NO) regulates proliferation of pheochromocytoma PC12 cells. The NO donor stimulated proliferation at low concentrations, but reversibly and completely inhibited proliferation at higher concentrations. The stimulation (but not the inhibition) of proliferation was apparently due to NO stimulation of soluble guanylate cyclase to produce cGMP, as it was prevented by a specific cyclase inhibitor (ODQ), and replicated by a cell-permeable form of cGMP. The NO-induced cytostasis was not reversed by inhibitors of MEK kinase or poly(ADP-ribose)polymerase, or by treatments that bypass inhibition of ribonucleotide reductase or ornithine decarboxylase. Cytostatic concentrations of DETA/NO strongly inhibited respiration of PC12 cells, and specific respiratory inhibitors (rotenone, myxothiazol, or azide) caused complete cytostasis. Uridine and pyruvate reversed the cytostasis induced by the specific respiratory inhibitors, but not that induced by DETA/NO. However, the combination of uridine, pyruvate, and N-acetyl-cysteine did reverse DETA/NO-induced cytostasis. DETA/NO strongly and progressively inhibited glycolysis measured by glucose consumption, lactate production, and ATP level, and a specific glycolytic inhibitor (5 mM 2-deoxy-d-glucose) caused complete cytostasis. Our results indicate that NO at low concentrations increases cell proliferation via cGMP, while high concentrations of NO block proliferation via inhibition of both glycolysis and respiration, causing energy depletion.