A pregnane ester triglycoside from Sarcostemma brevistigma

A new pregnane ester glycoside, brevine, was isolated from the dried twigs of Sarcostemma brevistigma. Its chemical and spectroscopic properties were consistent with the structure 11-O-benzoyl-sarcogenin-3-O-α-L-diginopyranosyl (1 → 4)-O-α-L-diginopyranosyl(1 → 4)-O-α-L-diginopyranoside.     

Processing of Rat Preprocortistatin in Mouse AtT-20 Cells

Preprocortistatin (PPCST) has been recently identified as a novel somatostatin (SST)-related gene expressed only in brain. PPCST shares 11 of 14 residues with SST-14 at its C-terminal segment, where it features Lys-Lys and Lys-Arg basic sites for cleavage to putative cortistatin (CST)-14 and CST-29 peptides, respectively. Although synthetic replicates of the two putative CST peptides interact with SST receptors, they also display novel effects suggesting independent biological functions. Nothing is currently known about the naturally occurring mature cleavage products of PPCST posttranslational processing. Here we have cloned rat PPCST cDNA, stably expressed it in AtT-20 pituitary cells, and characterized the cellular and releasable products of PPCST processing by HPLC and radioimmunoassay using a SST-14 antibody that recognizes synthetic CST-14 and CST-29. Transfected cells released 120 +/- 21 pg of total CST-LI per plate basally, with an increase to 204 +/- 33 pg per plate with forskolin stimulation (p < 0.05). HPLC chromatograms of cell extracts revealed three peaks corresponding to CST-14, CST-29, and unprocessed PPCST (ratio, 41:55:4.5). CST was released preferentially as CST-14 (63-70%) compared with CST-29 (30-37%) under basal and forskolin-stimulated conditions. These studies demonstrate efficient processing of PPCST to both CST-14 and CST-29 through putative cleavage at both C-terminal dibasic sites of PPCST. Although the two peptides are synthesized approximately equally, CST-14 is released preferentially via the regulated secretory pathway.     

Rapid effects of 1,25(OH)2 vitamin D3 on signal transduction systems in colonic cells.

Previous work from our laboratory demonstrated that 1,25(OH)2D3 rapidly stimulated hydrolysis of membrane polyphosphoinositides (PI) in rat colonocytes and in Caco-2 cells, generating the second messengers DAG and IP3. [Ca2+]i subsequently increased due to IP3-mediated release of intracellular Ca2+ stores, and to Ca2+ influx through a receptor-mediated Ca channel. Studies examining purified antipodal plasma membranes and experiments using Caco-2 cell monolayers found that 1,25(OH)2D3 influenced PI turnover only in the basolateral (BLM) and not brush border (BBM) membranes. Vitamin D analogues with poor affinity for the vitamin D receptor were found to effectively stimulate PI turnover, suggesting the presence of a unique vitamin D receptor in the BLM. Studies from our laboratory have demonstrated saturable, reversible binding of 1,25(OH)2 D3 to colonocyte BLM. Recently, we found that 1,25(OH)2D3 activated the tyrosine kinase c-src in colonocyte BLM by a heterotrimeric guanine nucleotide binding protein (G-protein)-dependent mechanism, with subsequent phosphorylation, translocation to the BLM, and activation of PI-specific phospholipase C gamma. Due to the rise in [Ca2+]i and DAG, two isoforms of protein kinase C (PKCalpha and PKCbeta2), but not other isoforms were activated by 1,25(OH)2D3 in rat colonocytes. Recent studies demonstrated that the seco-steroid translocated the beta2 isoform to the BLM, but not the BBM. In contrast, the alpha isoform did not translocate to either antipodal plasma membrane, but modulated IP3-mediated Ca2+ release from the endoplasmic reticulum. Preliminary studies have shown that 1,25(OH)2D3 also activated phosphatidylcholine phospholipase D (PLD) in Caco-2 cells, generating phosphatidic acid and contributing to the sustained rise in DAG. PLD stimulation occurred by both PKC-dependent and -independent mechanisms. Inhibitors of G-proteins, c-src, and PKC blunted the seco-steroid-mediated activation of PLD. Cells stably transfected with sense PKCalpha showed increased 1,25(OH)2D3-stimulated PLD activation, whereas transfectants with antisense PKCalpha had an attenuated response. In addition, 1,25(OH)2D3 also regulated PLD by activating the monomeric G-protein rho A by a mechanism independent of the G-protein/ c-src/PKC pathway.     

Studies on ontogeny and reproductive behaviour of Lepisorus nudus (Hook.) Ching (Polypodiaceae)

Journal of Plant Research - In-vitro studies of the ontogeny and mating system of the gametophytes of Lepisorus nudus were carried out through multispore and isolate cultures lasting...     

Downstream processing,characterization, and structure–function relationship of solvent‐, detergent‐, psychro‐, thermo‐, alkalistable metalloprotease from metal‐, solvent‐tolerant psychrotrophic Pseudomonas putida SKG‐1 isolate

The purification and characterization of psychro‐thermoalkalistable protease from psychrotrophic Pseudomonas putida isolate is being reported for the first time. A ～53 kDa protease was purified 21.4‐folds with 57.2% recovery by ultrafiltration and hydrophobic interaction chromatography. Kinetic analyses revealed the K_m and V_max to be 1.169 mg mL^?1 and 0.833 mg mL^?1 min^?1, respectively. The k_cat value of 3.05 × 10² s^?1 indicated high affinity and catalytic efficiency toward casein. The protease was most active at pH 9.5 and 40°C, with 100% stability in pH and temperature range of 6.0–11.0 and 10–40°C, respectively. Presence of Zn²⁺ increased the thermostability of protease (at 70°C) by 433%. Ethylene diamine tetra acetic acid (EDTA) and 1,10‐phenanthroline were inhibitory, whereas phenyl methyl sulfonyl fluoride (PMSF), p‐chloro mercuric benzoate (PCMB), and β‐mercaptoethanol were ineffective, revealing the enzyme to be a metalloprotease. Zinc, calcium, iron, nickel, and copper at 1 mM increased the enzyme activity (102–134%). Complete reversion of enzyme inhibition (caused by Ethylene diamine tetra acetic acid [EDTA]) by Zn²⁺ affirmed this enzyme as zinc‐dependent metalloprotease. At 0.1% concentration, Triton X‐100 and Tween 80 slightly increased, while SDS and H₂O₂ reduced the protease activity. In the presence of 0.1% commercial detergents, the enzyme was fairly stable (54–81%). In the presence of organic solvent, the protease was remarkably stable exhibiting 72–191% activities. In contrast, savinase exhibited good stability in the presence of hydrophilic solvents, while chymotrypsin showed elevated activities with benzene, toluene, and xylene only. Circular dichroism analysis revealed the protease as a β‐rich protein, having large fraction (～40%) of β‐sheets. Presence of different environmental conditions altered the β‐content, which accordingly affected the protease activity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Structure of brevobiose

A new disaccharide, brevobiose (1), has been isolated from the dried twigs of Sarcostemma brevistigma. The structure of 1 has been established as 4-O-(6-deoxy-2-O-methyl-β-d-allopyranosyl)-d-boivinose on the basis of chemical and spectroscopic evidence, and identification of its hydrolysis products.