Isolation of novel lipolytic genes from uncultured bacteria of pond water

Metagenomic libraries give access to gene pool of bacteria present in environmental samples avoiding the culture bias. A metagenomic library of pond water microbial assemblage in plasmid vector containing about 532 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 11 unique lipolytic clones with an ability to hydrolyze tributyrin. DNA sequence of the lipolytic genes varied in G+C composition from 57% to 75%. Twelve lipolytic genes encoding proteins with 25-70% amino acid identity with proteins in the databases were identified. Ten of the encoded proteins belonged to seven known lipolytic protein families. One of the proteins was similar to recently identified esterase BioH. A lipolytic protein with high similarity to yet uncharacterized alpha/beta hydrolase protein family abh_upf0017 was identified from one of the clones. Conserved motif for lipolytic enzymes GXSXG, conserved aspartic and histidine residues were identified in this encoded protein.     

Phytochemical Profiling,Antimicrobial, Antiproliferative and Apoptotic Effects of Stemodia viscosa Roxb. of Western Ghats Region,India

The present study shows the chemical profile, antimicrobial, antiproliferative, and apoptotic effects of Stemodia viscosa extracts. Thirteen bioactive compounds were identified in the 80 % ethanolic extract by GC/MS analysis. The acetone extract exhibited a higher content of flavonoids and phenols of 805.10 μg QE/mg DW and 89.31 μg GAE/mg DW extracts, respectively. Furthermore, the acetone extract possessed the highest antioxidant activity (IC₅₀=9.96 μg/mL). The 80 % ethanolic extract exhibited significant antimicrobial activity; the highest activity was observed against Staphylococcus aureus with a zone of inhibition of 25±0.51 mm, MIC value of 4 mg/mL, and MBC value of 8 mg/mL. The antiproliferative results revealed the presence of anticancer activity with an IC₅₀=91.562 and 74.362 μg/mL against the B16F10 skin and COLO205 colon cancer cells, respectively. The flow cytometric analysis shows that the plant extracts cause cancer cell death through the induction of apoptosis. Our findings confirmed that Stemodia viscosa is a potential source of biologically active compounds.     

A spirostanol glycoside from Yucca aloifolia.

From an ethanolic extract of the influorescence of Yucca aloifolia a new spirostanol glycoside has been isolated and characterized as 3-O[(alpha-L-rhamnopyranosyl(1----3)-beta-D-xylopyranosyl(1----2))(beta- D-glucopyranosyl(1----3)-beta-D-glucopyranosyl(1----3)-beta-D-glucopy ranosyl]-25R,5 alpha-spirostan-2 alpha, 3 beta-diol.     

Structural conservation of residues in BH1 and BH2 domains of Bcl-2 family proteins

The sequence of Bcl-2 homology domains, BH1 and BH2, is known to be conserved among anti- and pro-apoptotic members of Bcl-2 family proteins. But structural conservation of these domains with respect to functionally active residues playing role in heterodimerization-mediated regulation of apoptosis has never been elucidated. Here, we have suggested the formation of an active site by structurally conserved residues in BH1 (glycine, arginine) and BH2 (tryptophan) domains of Bcl-2 family members, which also accounts for the functional effect of known mutations in BH1 (G145A, G145E) and BH2 (W188A) domains of Bcl-2.     

Alteration of sphingolipid metabolism as a putative mechanism underlying LPS‐induced BBB disruption

Septic encephalopathy with confusion and agitation occurs early during sepsis and contributes to the severity of the disease. A decrease in the sphingosine‐1‐phosphate (S1P) blood levels has been shown in patients and in animal models of sepsis. The lipid mediator S1P is known to be involved in endothelial barrier function in a context‐dependent manner. We utilized lipopolysaccharide (LPS )‐injected mice as a model for septic encephalopathy and first performed tracer permeability assays to assess the blood–brain barrier (BBB ) breakdown in vivo. At time points corresponding to the BBB breakdown post LPS injection, we aimed to characterize the regulation of the sphingolipid signaling pathway at the BBB during sepsis. We measured sphingolipid concentrations in blood, in mouse brain microvessels (MBMV s), and brain tissue. We also analyzed the expression of S1P receptors, transporters, and metabolizing enzymes in MBMV s and brain tissue. Primary mouse brain microvascular endothelial cells (MBMEC s) were isolated to evaluate the effects of LPS on transendothelial electrical resistance (TEER ) as a measure of permeability in vitro . We observed a relevant decrease in S1P levels after LPS injection in all three compartments (blood, MBMV s, brain tissue) that was accompanied by an increased expression of the S1P receptor type 1 and of sphingosine kinase 1 on one hand and of the S1P degrading enzymes lipid phosphate phosphatase 1 (LPP 1) and S1P phosphatase 1 on the other hand, as well as a down‐regulation of sphingosine kinase 2. Application of LPS to a monolayer of primary MBMEC s did not alter TEER , but serum from LPS ‐treated mice lead to a breakdown of the barrier compared to serum from vehicle‐treated mice. We observed profound alterations of the sphingolipid metabolism at the BBB after LPS injection that point toward a therapeutic potential of drugs interfering with this pathway as novel approach for the detrimental overwhelming immune response in sepsis.

Read the Editorial Highlight for this article on page 115 . Cover Image for this Issue: doi. 10.1111/jnc.14161 .