Redox modification of Akt mediated by the dopaminergic neurotoxin MPTP, in mouse midbrain, leads to down-regulation of pAkt

Impairment of Akt phosphorylation, a critical survival signal, has been implicated in the degeneration of dopaminergic neurons in Parkinson's disease. However, the mechanism underlying pAkt loss is unclear. In the current study, we demonstrate pAkt loss in ventral midbrain of mice treated with dopaminergic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), when compared to ventral midbrain of control mice treated with vehicle alone. Thiol residues of the critical cysteines in Akt are oxidized to a greater degree in mice treated with MPTP, which is reflected as a 40% loss of reduced Akt. Association of oxidatively modified Akt with the phosphatase PP2A, which can lead to enhanced dephosphorylation of pAkt, was significantly stronger after MPTP treatment. Maintaining the protein thiol homeostasis by thiol antioxidants prevented loss of reduced Akt, decreased association with PP2A, and maintained pAkt levels. Overexpression of glutaredoxin, a protein disulfide oxidoreductase, in human primary neurons helped sustain reduced state of Akt and abolished MPP(+)-mediated pAkt loss. We demonstrate for the first time the selective loss of Akt activity, in vivo, due to oxidative modification of Akt and provide mechanistic insight into oxidative stress-induced down-regulation of cell survival pathway in mouse midbrain following exposure to MPTP.     

Atherogenic effect of Arecoline: A computational study

There are over 600 million people worldwide covering Asian and Oceanic countries including India have the habit of chewing areca nut as masticator in different forms. Arecoline (C(8)H(13)NO(2)) has been reported as one of the abundant constituents of areca nut. A good number of scientific publications have made Arecoline responsible for oral cancer. Based on observation from clinical situation in North East India, one of the most betel quid chewing region of the country, we suspected a link between consumption of areca nut and Cerebro Vascular Disease like stroke. Therefore, we considered Low Density Lipoprotein (LDL) receptor as target and Arecoline as ligand and studied ligand -target interaction using computational tools. Also we considered High Density Lipoprotein (HDL) receptor as another target to see if Arecoline has any binding potential with it over and above LDL receptor. Docking result indicated that Arecoline and Cholesterol both, have affinity towards extracellular domain of Human LDL receptor but affinity of Arecoline is much higher (-12.3560.) than that of Cholesterol(-0.1810). Docking of Arecoline and 1, 2-Hexyl-1- cyclopentanone thiosemicarbazone (thiosemicarbazone) with Bovine HDL receptor showed that Arecoline also has the potential (Score, -6.2690Kcal/Mol) to block HDL receptor though its potential is less than that (score, -10.0509 Kcal/Mol) of control (thiosemicarbazone). We, therefore, suggest that by inhibiting endocytosis of LDL cholesterol because of blocking LDL receptor function and also by preventing LDL cholesterol uptake by liver from blood because of interference with HDL receptor, Arecoline may contribute to atherosclerosis. The study therefore, indicates a positive correlation between chewing of betel quid and Cerebro Vascular Disease.     

Production and characterization of pharmacologically active recombinant human phosphodiesterase 4B in Dictyostelium discoideum

Phosphodiesterase 4B (PDE4B) is an important therapeutic target for asthma and chronic obstructive pulmonary disease. To identify PDE4 subtype-specific compounds using high-throughput assays, full-length recombinant PDE4 proteins are needed in bulk quantity. In the present study, full-length human PDE4B2 was expressed in the cellular slime mould Dictyostelium discoideum (Dd). A cell density of 2 x 10(7) cells/mL was obtained and up to 1 mg/L recombinant PDE4B2 was purified through Ni-NTA affinity chromatography. The expressed protein was soluble and its activity was comparable to PDE4B2 protein expressed in mammalian cells (K(m)=1.7 microM). The functional significance of the Dd expression system is supported by the demonstration that, in concert with proteins expressed in mammalian systems, there are no major changes in the affinity for PDE4B2 inhibitors and substrates. These findings thus provide the first evidence that Dd can be utilized for the expression and purification of functionally active full-length human PDE4B2 in large amounts required for high-throughput screening of pharmacologically active compounds against this therapeutic target.     

Assessment of fungal diversity in deep-sea sediments by multiple primer approach

Increasing evidence of the fungal diversity in deep-sea sediments has come from amplification of environmental DNA with fungal specific or eukaryote primer sets. In order to assess the fungal diversity in deep-sea sediments of the Central Indian Basin (CIB) at ~5,000 m depth, we amplified sediment DNA with four different primer sets. These were fungal-specific primer pair ITS1F/ITS4 (internal transcribed spacers), universal 18S rDNA primers NS1/NS2, Euk18S-42F/Euk18S-1492R and Euk18S-555F/Euk18S-1269R. One environmental library was constructed with each of the primer pairs, and 48 clones were sequenced per library. These sequences resulted in 8 fungal Operational Taxonomic Units (OTUs) with ITS and 19 OTUs with 18S rDNA primer sets respectively by taking into account the 2% sequence divergence cut-off for species delineation. These OTUs belonged to 20 distinct fungal genera of the phyla Ascomycota and Basidiomycota. Seven sequences were found to be divergent by 79–97% from the known sequences of the existing database and may be novel. A majority of the sequences clustered with known sequences of the existing taxa. The phylogenetic affiliation of a few fungal sequences with known environmental sequences from marine and hypersaline habitat suggests their autochthonous nature or adaptation to marine habitat. The amplification of sequences belonging to Exobasidiomycetes and Cystobasidiomycetes from deep-sea is being reported for the first time in this study. Amplification of fungal sequences with eukaryotic as well as fungal specific primers indicates that among eukaryotes, fungi appear to be a dominant group in the sampling site of the CIB.     

Distal Lateral Subungual Onychomycosis Owing to <Emphasis Type="Italic">Tritirachium oryzae</Emphasis>: A Bystander or Invader?

The genus Tritirachium is a mitosporic fungus which inhabits in soil and decaying plant material and also a notable insect pathogen. Human infections with Tritirachium species though rare were previously reported to cause corneal ulcers, otomycosis, onychomycosis, and dermatomycosis of the scalp and hence may be considered as a potential pathogen. Here we report a case of distal lateral subungual onychomycosis involving right great toenail in a 22-year-old female, wherein direct potassium hydroxide preparations, fungal cultures, and molecular sequencing of the isolate established Tritirachium oryzae as the etiological agent. Antifungal susceptibility performed by the microbroth technique of CLSI revealed increased MICs to amphotericin B and low MICs to azoles and echinocandins. The case was managed with surgical nail avulsion followed by topical application of 2% ketoconazole cream resulting regrowth of normal nail. To the best of our knowledge, this is the first report of non-dermatophytic mold T. oryzae causing onychomycosis in India.     

Retrotransposon based genetic status of North-West Himalayan Zingiber officinale revealed high heterogeneity

Inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) techniques were successfully applied, for the first time, to analyze genetic diversity among 92 ginger landraces collected from north-western Himalayan region of India. Six IRAP primer/combinations generated 75 loci with an average of 12 loci/primer displaying an overall polymorphism of 95.95 %. On the other hand, twenty five REMAP primer combinations produced 414 loci with 96.5 % polymorphism. IRAP showed maximum Rp (5.39) and PIC (0.28) values, while the same in REMAP was observed to be 10.92 and 0.34. Cluster analysis using Jaccard’s similarity coefficient for IRAP and REMAP data ranged between 0.21 to 1.0 and 0.21 to 0.85, respectively distinguishing all the genotypes with diverse genetic makup. The results also confirmed the presence of sukkula retrotransposon (RT6) in the ginger genome which effectively acted as genetic marker revealing high regional genetic diversity in the ginger gene pool. The study will help in giving insight to the genetic constitution of vegetatively grown ginger crop and for its further utilization in improvement, conservation and management programmes.     

Arthrobacter sp. lipase immobilization for preparation of enantiopure masked β-amino alcohols

Recent reports on immobilization of lipase from Arthrobacter sp. (ABL, MTCC 5125; IIIM isolate) on insoluble polymers have shown altered properties including stability and enantioselectivity. Present work demonstrates a facile method for the preparation of enantiopure β-amino alcohols by modulation of ABL enzyme properties via immobilization on insoluble as well as soluble supports using entrapment/covalent binding techniques. Efficacies of immobilized ABL on insoluble supports prepared from tetraethylorthosilicate/aminopropyltriethoxy silane and soluble supports derived from copolymerization of N-vinyl pyrrolidone-allylglycidyl ether (ANP type)/N-vinyl pyrrolidone-glycidyl methacrylate (GNP type) for kinetic resolution of masked β-amino alcohols have been studied vis-à-vis free ABL enzyme/wet cell biomass. The immobilized lipase on different insoluble/soluble supports has shown 21–110 mg/g protein binding and 30–700 U/g activity for hydrolyzing tributyrin substrate. The findings have shown a significant enhancement in enantioselectivity (ee 99%) vis-à-vis wet cell biomass providing ee 70–90% for resolution of β-amino alcohols.