Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.     

SD-8, a novel therapeutic agent active against multidrug-resistant Gram positive cocci

Anti-bacterial drug resistance is one of the most critical concerns among the scientist worldwide. The novel antimicrobial decapeptide SD-8 is designed and its minimal inhibitory concentration and therapeutic index (TI) was found in the range of 1–8 μg/ml and 45–360, respectively, against major group of Gram positive pathogens (GPP). The peptide was also found to be least hemolytic at a concentration of 180 μg/ml, i.e., nearly 77 times higher than its average effective concentration. The kinetics assay showed that the killing time is 120 min for methicillin-sensitive Staphylococcus aureus (MSSA) and 90 min for methicillin-resistant S. aureus (MRSA). Membrane permeabilization is the cause of peptide antimicrobial activity as shown by the transmission electron microscopy studies. The peptide showed the anti-inflammatory property by inhibiting COX-2 with a K _D and K _i values of 2.36 × 10⁻⁹ and 4.8 × 10⁻⁸ M, respectively. The peptide was also found to be effective in vivo as derived from histopathological observations in a Staphylococcal skin infection rat model with MRSA as causative organism.     

Nitric oxide induces apoptosis in cutaneous T cell lymphoma (HuT-78) by downregulating constitutive NF-κB

Constitutive active NF-κB have been shown to protect cutaneous T cell lymphoma (CTCL) cells from apoptosis. In the present study, we have studied the cytotoxic potential of nitric oxide generating compound, sodium nitroprusside (SNP) on CTCL cell line, HuT-78. Treatment of cells with SNP resulted in decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and poly (ADP ribose) polymerase cleavage. SNP treatment inhibited activation of NF-κB in a concentration dependent manner. SNP increased the expression of IκBα without affecting the phosphorylation of IκBα. Downregulation of NF-κB by SNP decreased p65 nuclear translocation as evident by confocal fluorescence microscopy. Further it was found that SNP treatment caused downregulation of Bcl-2 family member (Bcl-xl) in HuT-78 cells. Thus, we have provided evidence that SNP induces apoptosis in CTCL cell line, HuT-78 by downregulating constitutive NF-κB and thereby Bcl-xl expression.     

Production of thermostable pectinase and xylanase for their potential application in bleaching of kraft pulp

A very high level of alkalophilic and thermostable pectinase and xylanase has been produced from newly isolated strains of Bacillus subtilis and Bacillus pumilus respectively. Enzyme production for pectinase was carried out under SSF using combinations of cheap agricultural residues while xylanase was produced under submerged fermentation using wheat bran as substrate to minimize the cost of production of these enzymes Among the various substrates tested, the highest yield of pectinase production was observed by using combination of WB + CW (6592 U/g of dry substrate) supplemented with 4% yeast extract when incubated at 37 °C for 72 h using deionized water of pH 7.0 as moistening agent. The biobleaching effect of these cellulase free enzymes on kraft pulp was determined. Both xylanase and pectinase showed stability over a broad range of pH from 6 to 10 and temperature from 55 to 70 °C. The bleaching efficiency of the pectinase and xylanase on kraft pulp was maximum after 150 min at 60 °C using enzyme dosage of 5 IU/ml of each enzyme at 10% pulp consistency with about 16% reduction in kappa number and 84% reduction in permanganate number. Enzyme treated pulp when subjected to CDED₁D₂ steps, 25% reduction in chlorine consumption and upto 19% reduction in consumption of chlorine dioxide was observed for obtaining the same %ISO brightness. Also an increase of 22 and 84% in whiteness and fluorescence respectively and a decrease of approximately 19% in the yellowness of the biotreated pulp were observed by pretreatment of the pulp with our enzymatic mixture.     

Efficient shoots regeneration and genetic transformation of Bacopa monniera

Bacopa monniera is an important source of metabolites with pharmaceutical value. It has been regarded as a valuable medicinal plant and its entire commercial requirement is met from wild natural population. Recently, metabolic engineering has emerged as an important solution for sustained supply of assured and quality raw material for the production of active principles. Present report describes efficient in vitro multiplication and transformation method for genetic manipulation of this species. MS medium supplemented with 2 mgl⁻¹ BA and 0.2 mgl⁻¹ IAA was found optimum for maximum shoot regeneration (98.33 %) from in vitro leaves with 2–3 longitudinal cuts. Agrobacterium tumefaciens-mediated transformation method was used for generating transgenic B. monniera plants. Putative transformants were confirmed by GUS assay and PCR based confirmation of hptII gene. DNA blot analysis showed single copy insertion of transgene cassette. An average of 87.5 % of the regenerated shoots were found PCR positive for hptII gene and GUS activity was detected in leaves of transgenic shoots at a frequency of 82.5 % The efficient multiple shoots regeneration system described herein may help in mass production of B. monniera plant. Also, the high frequency transformation protocol described here can be used for genetic engineering of B. monniera for enhancement of its pharmaceutically important metabolites.     

An external validation study reporting poor correlation between the claims-based index for rheumatoid arthritis severity and the disease activity score

IntroductionWe conducted an external validation study to examine the correlation of a previously published claims-based index for rheumatoid arthritis severity (CIRAS) with disease activity score in 28 joints calculated by using C-reactive protein (DAS28-CRP) and the multi-dimensional health assessment questionnaire (MD-HAQ) physical function score.MethodsPatients enrolled in the Brigham and Women’s Hospital Rheumatoid Arthritis Sequential Study (BRASS) and Medicare were identified and their data from these two sources were linked. For each patient, DAS28-CRP measurement and MD-HAQ physical function scores were extracted from BRASS, and CIRAS was calculated from Medicare claims for the period of 365 days prior to the DAS28-CRP measurement. Pearson correlation coefficient between CIRAS and DAS28-CRP as well as MD-HAQ physical function scores were calculated. Furthermore, we considered several additional pharmacy and medical claims-derived variables as predictors for DAS28-CRP in a multivariable linear regression model in order to assess improvement in the performance of the original CIRAS algorithm.ResultsIn total, 315 patients with enrollment in both BRASS and Medicare were included in this study. The majority (81%) of the cohort was female, and the mean age was 70 years. The correlation between CIRAS and DAS28-CRP was low (Pearson correlation coefficient = 0.07, P = 0.24). The correlation between the calculated CIRAS and MD-HAQ physical function scores was also found to be low (Pearson correlation coefficient = 0.08, P = 0.17). The linear regression model containing additional claims-derived variables yielded model R² of 0.23, suggesting limited ability of this model to explain variation in DAS28-CRP.ConclusionsIn a cohort of Medicare-enrolled patients with established RA, CIRAS showed low correlation with DAS28-CRP as well as MD-HAQ physical function scores. Claims-based algorithms for disease activity should be rigorously tested in distinct populations in order to establish their generalizability before widespread adoption.     

Role of Salmonella surface components in immunomodulation of inflammatory mediators

Salmonella enterica serovar Typhimurium and its surface components were assessed for their inflammatory potential by footpad oedema test using plethysmometer. Inflammation was found to be the highest when outer membrane proteins (OMPs) were used as inflammagen followed by lipid associated protein-lipopolysaccharide complex (LAP-LPS) and lipopolysaccharides (LPS). Inflammation produced by OMPs was found to be comparable to that by carrageenan (a known positive inflammagen). However, injection of L-histidine (an antioxidant) prior to administration of carrageenan or Salmonella enterica serovar Typhimurium inhibited the inflammation, which indicated the involvement of oxidants during inflammatory response. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nitric oxide (NO) production by peritoneal macrophages from infected mice exhibited a significant increase as compared to those of the immunized mice. In contrast, glutathione production was found to be the maximum in the macrophages taken from OMPs-immunized mice followed by LAP-LPS and LPS alone. The biochemical studies correlated well with histopathological studies of intestinal tissue of animals from various groups. Based upon these parameters, inflammation seems to be modulated by OMPs and LAP-LPS, which may be because of the protein moieties present in the components. Hence, immunization with protein moieties having L-histidine or L-histidine-like structures may suggest an alternative to the potential therapeutic values of anti-inflammatory drugs. Thus the results of this study form the basis for evaluating these antigens (either alone or in combination with polysaccharides) for preventive intervention rather than therapeutic. (Mol Cell Biochem 270: 167–175, 2005)     

Diastereoselective annulation of 4-hydroxypyran-2H-ones with enantiopure 2,3-dideoxy-alpha,beta-unsaturated sugar aldehydes derived from respective glycals

One-pot condensations of 4-hydroxypyran-2H-ones 1 and 2, respectively, with various enantiopure 2,3-dideoxy-alpha,beta-unsaturated carbohydrate enals in the presence of l-proline in EtOAc at room temperature generated pyrano-pyrones. It was observed that, while benzyl-protected carbohydrate enals on condensation with 1 or 2 under the above conditions produced an inseparable diastereomeric mixture in a ratio of 1:1, the acyl-protected carbohydrate enals on treatment with 1 or 2 under identical conditions yielded products with moderate to very high diastereoselectivity. A remarkable asymmetric induction was noticed from the C-4 stereogenic center of the acyl-protected carbohydrate enals. An almost complete diastereoselectivity was observed in those reactions that involved condensation of 1 with acetyl-protected enals 5 and 7. The reaction of 2 with 5 also proceeded diastereoselectively to furnish the corresponding annulated product. The reaction presumably took place by C-1,2-addition of the pyrone onto the iminium salt of the alpha,beta-unsaturated carbohydrate enal generated in situ, followed by beta-elimination and cyclization of the 1-oxatriene involving a 6pi-electron electrocyclic process to yield a 2H,5H-pyrano[3,2-c]pyran-5-one derivative.