Selective inhibition of carbonic anhydrase IX by sulphonylated 1,2,3-triazole incorporated benzenesulphonamides capable of inducing apoptosis

In search of selective carbonic anhydrase (CA) IX inhibitors endowed with apoptotic inducing properties, we designed and synthesised two subsets of 4- and 3-(5-aryl-(4-phenylsulphonyl)-1H-1,2,3-triazol-1-yl)benzenesulphonamides. All compounds were assayed for human carbonic anhydrase (hCA) isoforms I, II, IV, and IX inhibition. Isoforms hCA I and hCA IV were weakly inhibited by most of the synthesised compounds. Many four-substituted benzenesulphonamides displayed low nanomolar inhibition against isoform hCA II, unlike the three-substituted analogues. All target compounds exhibited good inhibition profile with K_I values ranging from 16.4 to 66.0 nM against tumour-associated isoform hCA IX. Some selective and potent inhibitors of hCA IX were assayed for in vitro apoptotic induction in goat testicular cells. Compounds 10d and 10h showed interesting apoptotic induction potential. The present study may provide insights into a strategy for the design of novel anticancer agents based on hCA inhibitors endowed with apoptotic interference.     

TDP-43 triggers immune response via mitochondrial DNA release

The molecular mechanisms of toxicity associated with cytoplasmic accumulation of TAR DNA binding protein-43(TDP-43),a pathological feature of many neurodegenera...     

Withania somnifera (L.) Dunal whole-plant extracts exhibited anti-sporotrichotic effects by destabilizing peripheral integrity of Sporothrix globosa yeast cells

Chronic topical cases of Sporotrichosis, a chronic fungal infection caused by the ubiquitously present cryptic members of the Sporothrix species complex, are treated with oral administrations of itraconazole. However, severe pulmonary or disseminated cases require repeated intra-venous doses of amphotericin B or even surgical debridement of the infected tissue. The unavoidable adverse side-effects of the current treatments, besides the growing drug resistance among Sporothrix genus, demands exploration of alternative therapeutic options. Medicinal herbs, due to their multi-targeting capacity, are gaining popularity amidst the rising antimicrobial recalcitrance. Withania somnifera is a well-known medicinal herb with reported antifungal activities against several pathogenic fungal genera. In this study, the antifungal effect of the whole plant extract of W. somnifera (WSWE) has been explored for the first time, against an itraconazole resistant strain of S. globosa. WSWE treatment inhibited S. globosa yeast form growth in a dose-dependent manner, with IC₅₀ of 1.40 mg/ml. Minimum fungicidal concentration (MFC) was found to be 50 mg/ml. Sorbitol protection and ergosterol binding assays, revealed that anti-sporotrichotic effects of WSWE correlated well with the destabilization of the fungal cell wall and cell membrane. This observation was validated through dose-dependent decrease in overall ergosterol contents in WSWE-treated S. globosa cells. Compositional analysis of WSWE through high performance liquid chromatography (HPLC) exhibited the presence of several anti-microbial phytochemicals like withanone, withaferin A, withanolides A and B, and withanoside IV and V. Withanone and withaferin A, purified from WSWE, were 10–20 folds more potent against S. globosa than WSWE, thus, suggesting to be the major phytocompounds responsible for the observed anti-sporotrichotic activity. In conclusion, this study has demonstrated the anti-sporotrichotic property of the whole plant extract of W. somnifera against S. globosa that could be further explored for the development of a natural antifungal agent against chronic Sporotrichosis.     

Proteomic analysis reveals USP7 as a novel regulator of palmitic acid-induced hepatocellular carcinoma cell death

Nutrient surplus and consequent free fatty acid accumulation in the liver cause hepatosteatosis. The exposure of free fatty acids to cultured hepatocyte and hepatocellular carcinoma cell lines induces cellular stress, organelle adaptation, and subsequent cell death. Despite many studies, the mechanism associated with lipotoxicity and subsequent cell death still remains poorly understood. Here, we have used the proteomics approach to circumvent the mechanism for lipotoxicity using hepatocellular carcinoma cells as a model. Our quantitative proteomics data revealed that ectopic lipids accumulation in cells severely affects the ubiquitin-proteasomal system. The palmitic acid (PA) partially lowered the expression of deubiquitinating enzyme USP7 which subsequently destabilizes p53 and promotes mitotic entry of cells. Our global phosphoproteomics analysis also provides strong evidence of an altered cell cycle checkpoint proteins’ expression that abrogates early G2/M checkpoints recovery with damaged DNA and induced mitotic catastrophe leading to hepatocyte death. We observe that palmitic acid prefers apoptosis-inducing factor (AIF) mediated cell death by depolarizing mitochondria and translocating AIF to the nucleus. In summary, the present study provides evidence of PA-induced hepatocellular death mediated by deubiquitinase USP7 downregulation and subsequent mitotic catastrophe.Subject terms: Apoptosis, Protein-protein interaction networks     

Generation of a cytotoxic T-lymphocyte response using a Salmonella antigen-delivery system

We have constructed a general-use vector for the cloning and stable expression of foreign genes in the chromosome of attenuated Salmonella typhimurium. Using this chromosomal expression vector (CEV), we expressed the circumsporozoite (CS) gene of the mouse malaria Plasmodium yoelii in an aroA S. typhimurium strain. Mice immunized with CS-expressing Salmonella recombinants mount a CS-specific cytotoxic T-lymphocyte (CTL) response. This is the first demonstration that attenuated Salmonella can elicit a specific CTL response to a foreign protein in mice. The ability to easily and stably express foreign genes from the Salmonella chromosome and the generation of specific CTL greatly expands the potential of Salmonella as an antigen-delivery system.     

Agrobacterium-mediated transient GUS gene expression in buffel grass (Cenchrus ciliaris L.)

The study was conducted to standardize a protocol for Agrobacterium-mediated genetic transformation of buffel grass (Cenchrus ciliaris L.). Embryogenic calli, produced from one-year-old mature seeds of buffel grass, were used as target cells for Agrobacterium-mediated transformation. A. tumefaciens strain LBA4404, harbouring pCAMBIA-1301 or pCAMBIA-2301, was used for co-cultivation with embryogenic calli from three genotypes (IG-3108, IG-9757 and IG-97101). Co-culturing of calli with Agrobacterium for 30 minutes, followed by co-cultivation with 0.1 mM acetosyringone for 3 days was found to be optimum for maximum transformation efficiency. Presence of acetosyringone during co-cultivation was found to be necessary for transformation. Transient GUS (beta-glucuronidase) gene expression was used to monitor T-DNA delivery into the target cells. Significant genotypic variations in response to transformation were observed among the tested genotypes. A very high frequency (63.3%) of GUS gene expression was obtained following Agrobacterium-mediated gene transfer into embryogenic calli. The standardized protocol would be useful for Agrobacterium-mediated genetic transformation of buffel grass with genes of agronomic importance.     

Immobilization of oligodeoxyribonucleotides with multiple anchors to microchips

Facile modification of oligodeoxyribonucleotides is required for efficient immobilization to a pre-activated glass surface. This report presents an oligodeoxyribonucleotide which contains a hairpin stem–loop structure with multiple phosphorothioate moieties in the loop. These moieties are used to anchor the oligo to glass slides that are pre-activated with bromoacetamidopropylsilane. The efficiency of the attachment reaction was improved by increasing the number of phosphorothioates in the loop, as shown in the remarkable enhancement of template hybridization and single base extension through catalysis by DNA polymerase. The loop and stem presumably serve as lateral spacers between neighboring oligodeoxyribonucleotides and as a linker arm between the glass surface and the single-stranded sequence of interest. The oligodeoxyribonucleotides of this hairpin stem–loop architecture with multiple phosphorothioate moieties have broad application in DNA chip-based gene analysis.     

Nematophagous Fungi Associated with Root Galls of Rice Caused by Meloidogyne graminicola and its Control by Arthrobotrys dactyloides and Dactylaria brochopaga

Root galls of rice caused by Meloidogyne graminicola were examined for natural colonization by nematophagous fungi from four fields with different nematode infestations. Old galls from severely infested fields had a higher frequency of Monacrosporium eudermatum and Stylopaga hadra than young galls. The frequency of Arthrobotrys oligospora, Arthrobotrys dactyloides, Dactylaria brochopaga and Monacrosporium gephyropagum was lower. A greater proportion (%) of root galls were colonized by nematophagous fungi in those fields in which rice roots had a greater root gall index. This indicated that disease severity supported the colonization of galls by nematophagous fungi. In vitro predacity tests of four fungi showed that A. dactyloides was most effective in capturing and killing J₂ of Mel. graminicola followed by D. brochopaga and Mon. eudermatum. Application of inocula of A. dactyloides and D. brochopaga in soil infested with Mel. graminicola, respectively, reduced the number of root galls by 86% and of females by 94%, and eggs and juveniles by 94%. The application of these fungi to soil increased plant growth: shoot length by 42.7% and 39.8%, root length by 45.5% and 48.9%, fresh weight of shoot by 59.9% and 56.7% and fresh weight of root by 20.3% and 25.1%, respectively, compared to these parameters for plants grown in nematode‐infested soil.