Regulation of Ca2+-dependent desensitization in the vanilloid receptor TRPV1 by calcineurin and cAMP-dependent protein kinase

The vanilloid receptor TRPV1 is a polymodal nonselective cation channel of nociceptive sensory neurons involved in the perception of inflammatory pain. TRPV1 exhibits desensitization in a Ca2+-dependent manner upon repeated activation by capsaicin or protons. The cAMP-dependent protein kinase (PKA) decreases desensitization of TRPV1 by directly phosphorylating the channel presumably at sites Ser116 and Thr370. In the present study we investigated the influence of protein phosphatase 2B (calcineurin) on Ca2+-dependent desensitization of capsaicin- and proton-activated currents. By using site-directed mutagenesis, we generated point mutations at PKA and protein kinase C consensus sites and studied wild type (WT) and mutant channels transiently expressed in HEK293t or HeLa cells under whole cell voltage clamp. We found that intracellular application of the cyclosporin A.cyclophilin A complex (CsA.CyP), a specific inhibitor of calcineurin, significantly decreased desensitization of capsaicin- or proton-activated TRPV1-WT currents. This effect was similar to that obtained by extracellular application of forskolin (FSK), an indirect activator of PKA. Simultaneous applications of CsA.CyP and FSK in varying concentrations suggested that these substances acted independently from each other. In mutation T370A, application of CsA.CyP did not reduce desensitization of capsaicin-activated currents as compared with WT and to mutant channels S116A and T144A. In a double mutation at candidate protein kinase C phosphorylation sites, application of CsA.CyP or FSK decreased desensitization of capsaicin-activated currents similar to WT channels. We conclude that Ca2+-dependent desensitization of TRPV1 might be in part regulated through channel dephosphorylation by calcineurin and channel phosphorylation by PKA possibly involving Thr370 as a key amino acid residue.     

Cloning, expression, and characterization of an oligoxyloglucan reducing end-specific xyloglucanobiohydrolase from Aspergillus nidulans

An oligoxyloglucan reducing end-specific xyloglucanobiohydrolase from the filamentous fungus Aspergillus nidulans was cloned and expressed in Pichia pastoris as a secreted histidine-tagged protein and purified by affinity chromatography. The enzyme acts on xyloglucan oligomers and releases the first two glycosyl residue segments from the reducing end, provided that neither the first glucose nor the xylose attached to the third glucose residue from the reducing end is not further substituted. The enzyme has a specific activity of 7 U/mg at the pH optimum of 3 and at the temperature optimum of 42 degrees C.     

Bioprospecting microalgae as potential sources of "green energy"--challenges and perspectives (review)

Microalgae and cyanobacteria are potential foods, feeds, sources of high-value bioactive molecules and biofuels, and find tremendous applications in bioremediation and agriculture. Although few efforts have been undertaken to index the microalgal germplasm available in terms of lipid content, information on suitability of strains for mass multiplication and advances in development of methods for extraction and generating biofuel are scarce. Our review summarizes the potential of microalgae, latest developments in the field and analyzes the "pitfalls" in oversimplification of their promise in the years to come. Microalgae represent "green gold mines" for generating energy; however, the path to success is long and winding and needs tremendous and concerted efforts from science and industry, besides political will and social acceptance for overcoming the limitations. The major advantages of second generation biofuels based on microalgal systems, include their higher photon conversion efficiency, growth all around the year, even in wastewaters, and production of environment friendly biodegradable biofuels.     

Differential distribution of simple sequence repeats in eukaryotic genome sequences.

Complete chromosome/genome sequences available from humans, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, and Saccharomyces cerevisiae were analyzed for the occurrence of mono-, di-, tri-, and tetranucleotide repeats. In all of the genomes studied, dinucleotide repeat stretches tended to be longer than other repeats. Additionally, tetranucleotide repeats in humans and trinucleotide repeats in Drosophila also seemed to be longer. Although the trends for different repeats are similar between different chromosomes within a genome, the density of repeats may vary between different chromosomes of the same species. The abundance or rarity of various di- and trinucleotide repeats in different genomes cannot be explained by nucleotide composition of a sequence or potential of repeated motifs to form alternative DNA structures. This suggests that in addition to nucleotide composition of repeat motifs, characteristic DNA replication/repair/recombination machinery might play an important role in the genesis of repeats. Moreover, analysis of complete genome coding DNA sequences of Drosophila, C. elegans, and yeast indicated that expansions of codon repeats corresponding to small hydrophilic amino acids are tolerated more, while strong selection pressures probably eliminate codon repeats encoding hydrophobic and basic amino acids. The locations and sequences of all of the repeat loci detected in genome sequences and coding DNA sequences are available at http://www.ncl-india.org/ssr and could be useful for further studies.     

Protein degradation in aged nematodes (Turbatrix aceti).

The rates of degradation of total soluble proteins in the free-living nematode, $\text{Turbatrix aceti}$, have been estimated by following the loss of acid-insoluble insoluble radioactivity from protein during a nonradioactive chase period after initial labeling with [³⁵S]methionine. These proteins appear to lose label kinetically as a homogeneous class in age-synchronized nematode populations. However, proteins are degraded more slowly in senescent cultures than in young cultures. Protein degradation rates decline progressively during nematode aging. These findings suggest that the protein degradative system in T. aceti may become partially defective with advancing age which may result in the accumulation of aberrant protein molecules in senescent organisms.     

Synthesis of novel nitric oxide (NO)-releasing esters of timolol

A novel class of timolol derivatives with nitric oxide (NO)-donating moieties achieved chemical stability yet under physiologically relevant conditions released timolol and NO. Hindered esters A were designed and synthesized, whose ‘triggered’ release relied on enzymatic hydrolysis of the nitrate ester in A to B, that in turn cyclized to liberate timolol.     

Endophytic bacterial flora in the stem tissue of a tropical maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) genotype: isolation,identification and enumeration

The genotypic diversity of indigenous bacterial endophytes within stem of tropical maize (Zea mays L.) was determined in field and greenhouse experiments. Strains were isolated from stem tissues of a tropical maize cultivar (PEHM-1) by trituration and surface disinfestation and their population dynamics was determined. Endophytes were found in most of the growing season at populations ranging from 1.36–6.12 × 10⁵ colony-forming units per gram fresh weight (c.f.u./gm fw) of stem. Analysis of these bacterial endophytes using Gas Chromatography—Fatty Acid Methyl Ester (GC-FAME) led to the identification of Bacillus pumilus, B. subtilis, Pseudomonas aeruginosa and P. fluorescens as the relatively more predominant group of bacterial species residing in maize stem. When the maize seedlings grown in a greenhouse were inoculated with these four isolates individually, their population densities decreased (1.6–3.1 × 10⁵ c.f.u./gm fw of stem) as compared to the field-grown maize (1.8–3.8 × 10⁵ c.f.u./gm fw of stem). The highest persistence, however, was recovered in the case of B. subtilis with a population density of 3.1 × 10⁵ c.f.u./gm fw of stem tissue on 28 days after emergence (DAE). This is the first report on population dynamics of bacterial endophytes from tropical maize and the results establish that symptomless populations of bacteria exist in the maize stem.