A glutathione-specific aldose reductase of Leishmania donovani and its potential implications for methylglyoxal detoxification pathway

Methylglyoxal is mainly catabolized by two major enzymatic pathways. The first is the ubiquitous detoxification pathway, the glyoxalase pathway. In addition to the glyoxalase pathway, aldose reductase pathway also plays a crucial role in lowering the levels of methylglyoxal. The gene encoding aldose reductase (ALR) has been cloned from Leishmania donovani, a protozoan parasite causing visceral leishmaniasis. DNA sequence analysis revealed an open reading frame (ORF) of approximately 855 bp encoding a putative protein of 284 amino acids with a calculated molecular mass of 31.7 kDa and a predicted isoelectric point of 5.85. The sequence identity between L. donovani ALR (LdALR) and mammals and plants is only 36-44%. The ORF is a single copy gene. A protein with a molecular mass that matched the estimated approximately 74 kDa according to the amino acid composition of LdALR with a maltose binding tag present at its N-terminal end was induced by heterologous expression of LdALR in Escherichia coli. In the presence of glutathione, recombinant LdALR reduced methylglyoxal with a K(m) of approximately 112 microM. Comparative structural analysis of the human ALR structure with LdALR model suggests that the active site anchoring the N-terminal end of the glutathione is highly conserved. However, the C-terminal end of the glutathione backbone is expected to be exposed in LdALR, as the residues anchoring the C-terminal end of the glutathione backbone come from the three loop regions in human, which are apparently shortened in the LdALR structure. Thus, the computational analysis provides clues about the expected mode of glutathione binding and its interactions with the protein. This is the first report of the role of an ALR in the metabolic disposal of methylglyoxal in L. donovani and of thiol binding to a kinetoplastid aldose reductase.     

Exome Sequencing Identifies WDR35 Variants Involved in Sensenbrenner Syndrome

Sensenbrenner syndrome/cranioectodermal dysplasia (CED) is an autosomal-recessive disease that is characterized by craniosynostosis and ectodermal and skeletal abnormalities. We sequenced the exomes of two unrelated CED patients and identified compound heterozygous mutations in WDR35 as the cause of the disease in each of the two patients independently, showing that it is possible to find the causative gene by sequencing the exome of a single sporadic patient. With RT-PCR, we demonstrate that a splice-site mutation in exon 2 of WDR35 alters splicing of RNA on the affected allele, introducing a premature stop codon. WDR35 is homologous to TULP4 (from the Tubby superfamily) and has previously been characterized as an intraflagellar transport component, confirming that Sensenbrenner syndrome is a ciliary disorder.     

Phage display: Concept,innovations, applications and future

Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field.     

Biochemical characterization and potential for textile dye degradation of blue laccase from Aspergillus ochraceus NCIM-1146

In our study, we produced intracellular blue laccase by growing the filamentous fungus Aspergillus ochraceus NCIM-1146 in potato dextrose broth. The enzyme was then purified 22-fold to a specific activity of 4.81 U/mg using anion-exchange and size exclusion chromatography. The molecular weight of purified laccase was estimated as 68 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme showed maximum substrate specificity toward 2,2′-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid than any other substrate. The optimum pH and temperature for laccase activity were 4.0 and 60°C, respectively. The purified enzyme was stable up to 50°C, and high laccase activity was maintained at pH 5.0 ∼ 7.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol, and L-cysteine. Purified laccase decolorized various textile dyes within 4 h in the absence of redox mediators. HPLC and FTIR analysis confirmed degradation of methyl orange. The metabolite formed after decolorization of methyl orange was characterized as p-N,N′-dimethylamine phenyldiazine using GCMS.     

Graft copolymerization of acrylic acid onto guar gum initiated by vanadium (V)–mercaptosuccinic acid redox pair

Guar gum has been modified by graft copolymerization with acrylic acid in aqueous medium using vanadium (V)–mercaptosuccinic acid redox system. The optimum reaction conditions affording maximum grafting ratio, efficiency, add on and conversion have been determined. The grafting parameters have been found to increase with increase in vanadium (V) concentration upto 1.0 × 10⁻² mol dm⁻³, but these parameters decrease on further increasing the vanadium (V) concentration. On increasing the mercaptosuccinic acid concentration from 1.0 × 10⁻² to 4.0 × 10⁻² mol dm⁻³ grafting ratio, efficiency and add on increase up to 2.0 × 10⁻² mol dm⁻³ but decrease with further increase in mercaptosuccinic acid concentration. On varying the acrylic acid concentration from 5.0 × 10⁻² to 30.0 × 10⁻² mol dm⁻³, maximum grafting ratio, efficiency and add on have been obtained at 20.0 × 10⁻² mol dm⁻³. The grafting ratio, add on and conversion increase, on increasing the H⁺ ion concentration from 1.5 × 10⁻¹ to 6.0 × 10⁻¹ mol dm⁻³. On increasing the guar gum concentration the grafting parameters increase. The grafting ratio, add on and conversion have been found to increase with time period while efficiency started decreasing after 120 min. It has been observed that %G increases on increasing the temperature up to 35 °C. The graft copolymer has been characterized by IR spectroscopy and thermogravimetric analysis.     

Adrenalectomy abolishes the stress-induced increase in pituitary cyclic AMP

We have previously demonstrated that various stressors increase pituitary cyclic AMP in vivo in the rat. In the course of studying the mechanisms mediating this response, we examined the effect of bilateral adrenalectomy on footshock-induced increases in pituitary cyclic AMP. In unoperated rats, intermittent footshock markedly increased pituitary levels of cyclic AMP and plasma levels of corticosterone and prolactin. Adrenalectomy completely abolished the stress-induced increase in pituitary cyclic AMP. The marked increase in plasma prolactin following footshock was not affected by adrenalectomy. Our results indicate that adrenal factors are involved in the stress-induced increase in pituitary cyclic AMP.     

Subsensitive pituitary cyclic AMP response to stress following adrenalectomy is not caused by loss of adrenal epinephrine

We have previously reported that various stressors acutely elevate levels of pituitary cyclic AMP in vivo and that this stress response is not seen in animals tested 7 or 30 days post-adrenalectomy. In this report we present data that demonstrate that the loss of the pituitary cyclic AMP stress response following adrenalectomy is not the result of the loss of stress-induced adrenal epinephrine release. These data show that (1) although administration of epinephrine to intact rats does not elevate levels of pituitary cyclic AMP, administration of epinephrine to adrenalectomized animals does not elevate pituitary cyclic AMP levels in vivo; (2) splanchnic denervation prevents stress-induced adrenal epinephrine release but does not abolish stress-induced increases in pituitary cyclic AMP; and (3) the time course of the developing subsensitive pituitary cyclic AMP response to stress following adrenalectomy is much slower (2 to 3 days) than the loss of circulating epinephrine.