Design of a New Peptide Substrate Probe of the Putative Biomarker Legumain with Potential Application in Prostate Cancer Diagnosis Ex Vivo

International Journal of Peptide Research and Therapeutics - The lysosomal endoprotease legumain (asparaginyl endoprotease) has been proposed as a putative biomarker in prostate tumours, in which...     

Covalent modification of cysteine 193 impairs ATPase function of nucleotide-binding domain of a Candida drug efflux pump

N-ethylmaleimide (NEM) impairs the ATPase function of N-terminal NBD of Candida drug resistance gene product Cdr1p. To identify the reactive cysteine(s) for such a contribution, we adopted a three-arm approach that included covalent modification, cysteine mutagenesis, and structure homology modeling. The covalent modification results clearly indicate the ability of NEM and iodoacetic acid (IAA) to potently inhibit the ATPase activity of N-terminal NBD. Since this domain contains five cysteine residues in its sequence, we mutated each and found four of these (C325A, C363A, C402A, and C462A) to stay sensitive to NEM/IAA modification and influence ATPase activity, while C193A mutation completely abrogated the catalytic function. The structural homology modeling data further validate these biochemical findings by ruling out any plausible interactions within the cysteine residues, and deriving the importance of Cys-193 in lying at a bond length clearly feasible to interact with ATP and divalent cation to critically influence ATP hydrolysis.     

Host plant nodule parameters associated with nitrogen fixation efficiency in French bean (Phaseolus vulgaris L) cultivars

Two cultivars of French bean (Phaseolus vulgaris L.) viz. contender and arka komal were planted in polythene bags containing sand and grown under glasshouse conditions. The nodulation status, shoot/root biomass, activities of several nodule enzymes, total soluble protein and leghaemoglobin contents were monitored over the entire growth period. Allantoinase activity in leaves was measured to monitor the ureide degrading capacity. Significant genotype difference was observed in both the cultivars. All the parameters showed a decline after flowering except uricase, which declined before flowering. Malate dehydrogenase and isocitrate dehydrogenase showed a constant decline throughout the growth period. Degree of decline varied with the genotype for all the parameters. Leghaemoglobin content, PEP carboxylase activity and ureide degrading capacity of leaves did not show an appreciable decline in contender and were significantly higher than in arka komal. These factors can be used to increase nitrogen fixation in French bean.     

Murine dendritic cell type I IFN production induced by human IgG-RNA immune complexes is IFN regulatory factor (IRF)5 and IRF7 dependent and is required for IL-6 production

Dendritic cell (DC) activation by nucleic acid-containing IgG complexes is implicated in systemic lupus erythematosus (SLE) pathogenesis. However, it has been difficult to definitively examine the receptors and signaling pathways by which this activation is mediated. Because mouse FcgammaRs recognize human IgG, we hypothesized that IgG from lupus patients might stimulate mouse DCs, thereby facilitating this analysis. In this study, we show that sera and purified IgG from lupus patients activate mouse DCs to produce IFN-alpha, IFN-beta, and IL-6 and up-regulate costimulatory molecules in a FcgammaR-dependent manner. This activation is only seen in sera with reactivity against ribonucleoproteins and is completely dependent on TLR7 and the presence of RNA. As anticipated, IFN regulatory factor (IRF)7 is required for IFN-alpha and IFN-beta production. Unexpectedly, however, IRF5 plays a critical role in IFN-alpha and IFN-beta production induced not only by RNA-containing immune complexes but also by conventional TLR7 and TLR9 ligands. Moreover, DC production of IL-6 induced by these stimuli is dependent on a functional type I IFNR, indicating the need for a type I IFN-dependent feedback loop in the production of inflammatory cytokines. This system may also prove useful for the study of receptors and signaling pathways used by immune complexes in other human diseases.     

Aptamers as a model for functional evaluation of LNA and 2′-amino LNA

The affinity change upon incorporation of LNA and 2′-amino-LNA monomers into an avidin binding DNA aptamer is described. The kinetic profile of selected modified-aptamer was obtained by surface plasmon resonance experiments and compared with the profile of the parent unmodified DNA aptamer. We report significant improvement of avidin binding affinity by the incorporation of single LNA modifications into the aptamer, and successful incorporation of 2′-amino LNA as a novel monomer in aptamers with potential function as carrier unit for additional molecular entities.     

Iridoid glycosides from Gmelina arborea

Three iridoid glycosides 6-O-(3'-O-benzoyl)-alpha-l-rhamnopyranosylcatalpol (1a), 6-O-(3'-O-trans-cinnamoyl)-alpha-l-rhamnopyranosylcatalpol (2a) and 6-O-(3'-O-cis-cinnamoyl)-alpha-l-rhamnopyranosylcatalpol (3a) were isolated from aerial parts of Gmelina arborea and structures were elucidated by spectral analysis. Additionally a known iridoid 6-O-(3', 4'-O-dibenzoyl)-alpha-l-rhamnopyranosylcatalpol (4) was also isolated and identified.     

Batten disease: biochemical and molecular characterization revealing novel <Emphasis Type="Italic">PPT1</Emphasis> and <Emphasis Type="Italic">TPP1</Emphasis> gene mutations in Indian patients

Background

Neuronal ceroid lipofuscinoses type I and type II (NCL1 and NCL2) also known as Batten disease are the commonly observed neurodegenerative lysosomal storage disorder caused by mutations in the PPT1 and TPP1 genes respectively. Till date, nearly 76 mutations in PPT1 and approximately 140 mutations, including large deletion/duplications, in TPP1 genes have been reported in the literature. The present study includes 34 unrelated Indian patients (12 females and 22 males) having epilepsy, visual impairment, cerebral atrophy, and cerebellar atrophy.

Methods

The biochemical investigation involved measuring the palmitoyl protein thioesterase 1 and tripeptidy peptidase l enzyme activity from the leukocytes. Based on the biochemical analysis all patients were screened for variations in either PPT1 gene or TPP1 gene using bidirectional Sanger sequencing. In cases where Sanger sequencing results was uninformative Multiplex Ligation-dependent Probe Amplification technique was employed. The online tools performed the protein homology modeling and orthologous conservation of the novel variants.

Results

Out of 34 patients analyzed, the biochemical assay confirmed 12 patients with NCL1 and 22 patients with NCL2. Molecular analysis of PPT1 gene in NCL1 patients revealed three known mutations (p.Val181Met, p.Asn110Ser, and p.Trp186Ter) and four novel variants (p.Glu178Asnfs*13, p.Pro238Leu, p.Cys45Arg, and p.Val236Gly). In the case of NCL2 patients, the TPP1 gene analysis identified seven known mutations and eight novel variants. Overall these 15 variants comprised seven missense variants (p.Met345Leu, p.Arg339Trp, p.Arg339Gln, p.Arg206Cys, p.Asn286Ser, p.Arg152Ser, p.Tyr459Ser), four frameshift variants (p.Ser62Argfs*19, p.Ser153Profs*19, p.Phe230Serfs*28, p.Ile484Aspfs*7), three nonsense variants (p.Phe516*, p.Arg208*, p.Tyr157*) and one intronic variant (g.2023_2024insT). No large deletion/duplication was identified in three NCL1 patients where Sanger sequencing study was normal.

Conclusion

The given study reports 34 patients with Batten disease. In addition, the study contributes four novel variants to the spectrum of PPT1 gene mutations and eight novel variants to the TPP1 gene mutation data. The novel pathogenic variant p.Pro238Leu occurred most commonly in the NCL1 cohort while the occurrence of a known pathogenic mutation p.Arg206Cys dominated in the NCL2 cohort. This study provides an insight into the molecular pathology of NCL1 and NCL2 disease for Indian origin patients.

     

Novel selective biocatalytic deacylation studies on key precursors for bicyclonucleosides

Immobilized Candida antarctica lipase and Thermomyces lanuginosus lipase catalyze the deacylation of precursors of LNA analogs, 4'-C-acyloxymethyl-2',3',5'-tri-O-acyl-beta-L-threo-pentofuranosylthymine and 4-C-acyloxymethyl-3,5-di-O-acyl-1,2-O-(1-methylethylidene)-beta-L-threo-pentofuranose, respectively in a highly selective and efficient manner.