Regulation of β-catenin stabilization in human platelets

The Wnt/β-catenin pathway controls developmental processes and homeostasis; however, abnormal activation of this pathway has been linked to several human diseases. Recent reports have demonstrated regulation of platelet function by canonical and non-canonical Wnt signalling. Platelet aggregation plays a crucial role in haemostasis and thrombosis. Here we report for the first time that, induction of sustained aggregation of platelets by a strong agonist in the presence of calcium was associated with nearly complete proteolysis of β-catenin, which was abrogated upon depletion of calcium from platelet suspension. β-catenin cleavage was disallowed in absence of aggregation, thus implicating integrin α_IIbβ₃ engagement in β-catenin proteolysis. Degradation of β-catenin was blocked partially by inhibitors of either proteasome or calpain and completely when cells were exposed to both the inhibitors. Protein kinase C inhibition, too, abolished β-catenin degradation. Thus activities of proteasome, calpain and protein kinase C regulate stabilization of β-catenin in aggregated human platelets.     

Impaired male meiosis, morphology and distribution pattern of different cytotypes of Bupleurum lanceolatum Wall. (Apiaceae) from the Western Himalayas

Detailed male meiosis, critical morphological observations and distribution pattern of diploid as well as tetraploid cytotypes of the Western Himalayan species, Bupleurum lanceolatum have been evaluated at present. A diploid (n = 8) cytotype is reported from Kashmir, whereas, both diploid (n = 8) and tetraploid (n = 16) cytotypes are available from two districts Kangra and Sirmaur of Himachal Pradesh. Out of these, the tetraploid cytotype makes new addition for the species on a worldwide basis. As per behavior, a tetraploid cytotype is characterized by abnormal meiosis leading to high pollen sterility and size variation of the pollen grains. Morphologically, tetraploids are noted to be luxuriant in comparison to the diploids.     

Isolation and physico-chemical characterisation of extracellular polymeric substances produced by the marine bacterium Vibrio parahaemolyticus

A marine bacterial strain identified as Vibrio parahaemolyticus by 16S rRNA gene (HM355955) sequencing and gas chromatography (GC) coupled with MIDI was selected from a natural biofilm by its capability to produce extracellular polymeric substances (EPS). The EPS had an average molecule size of 15.278 μm and exhibited characteristic diffraction peaks at 5.985°, 9.150° and 22.823°, with d-spacings of 14.76661, 9.29989 and 3.89650 Å, respectively. The Fourier-transform infrared spectroscopy (FTIR) spectrum revealed aliphatic methyl, primary amine, halide groups, uronic acid and saccharides. Gas chromatography mass spectrometry (GCMS) confirmed the presence of arabinose, galactose, glucose and mannose. ¹HNMR (nuclear magnetic resonance) revealed functional groups characteristic of polysaccharides. The EPS were amorphous in nature (CI_xrd 0.092), with a 67.37% emulsifying activity, thermostable up to 250°C and displayed pseudoplastic rheology. MALDI-TOF–TOF analysis revealed a series of masses, exhibiting low-mass peaks (m/z) corresponding to oligosaccharides and higher-mass peaks for polysaccharides consisting of different ratios of pentose and hexose moieties. This is the first report of a detailed characterisation of the EPS produced by V. parahaemolyticus, which could be further explored for biotechnological and industrial use.     

Understanding the specificity of serpin–protease complexes through interface analysis

Serpins such as antithrombin, heparin cofactor II, plasminogen activator inhibitor, antitrypsin, antichymotrypsin, and neuroserpin are involved in important biological processes by inhibiting specific serine proteases. Initially, the protease recognizes the mobile reactive loop of the serpin eliciting conformational changes, where the cleaved loop together with the protease inserts into β-sheet A, translocating the protease to the opposite side of inhibitor leading to its inactivation. Serpin interaction with proteases is governed mainly by the reactive center loop residues (RCL). However, in some inhibitory serpins, exosite residues apart from RCL have been shown to confer protease specificity. Further, this forms the basis of multi-specificity of some serpins, but the residues and their dimension at interface in serpin-protease complexes remain elusive. Here, we present a comprehensive structural analysis of the serpin-protease interfaces using bio COmplexes COntact MAPS (COCOMAPS), PRotein Interface Conservation and Energetics (PRICE), and ProFace programs. We have carried out interface, burial, and evolutionary analysis of different serpin-protease complexes. Among the studied complexes, non-inhibitory serpins exhibit larger interface region with greater number of residue involvement as compared to the inhibitory serpins. On comparing the multi-specific serpins (antithrombin and antitrypsin), a difference in the interface area and residue number was observed, suggestive of a differential mechanism of action of these serpins in regulating their different target proteases. Further, detailed study of these multi-specific serpins listed few essential residues (common in all the complexes) and certain specificity (unique to each complex) determining residues at their interfaces. Structural mapping of interface residues suggested that individual patches with evolutionary conserved residues in specific serpins determine their specificity towards a particular protease.     

Cardiac-specific overexpression of perilipin 5 provokes severe cardiac steatosis via the formation of a lipolytic barrier

Cardiac triacylglycerol (TG) catabolism critically depends on the TG hydrolytic activity of adipose triglyceride lipase (ATGL). Perilipin 5 (Plin5) is expressed in cardiac muscle (CM) and has been shown to interact with ATGL and its coactivator comparative gene identification-58 (CGI-58). Furthermore, ectopic Plin5 expression increases cellular TG content and Plin5-deficient mice exhibit reduced cardiac TG levels. In this study we show that mice with cardiac muscle-specific overexpression of perilipin 5 (CM-Plin5) massively accumulate TG in CM, which is accompanied by moderately reduced fatty acid (FA) oxidizing gene expression levels. Cardiac lipid droplet (LD) preparations from CM of CM-Plin5 mice showed reduced ATGL- and hormone-sensitive lipase-mediated TG mobilization implying that Plin5 overexpression restricts cardiac lipolysis via the formation of a lipolytic barrier. To test this hypothesis, we analyzed TG hydrolytic activities in preparations of Plin5-, ATGL-, and CGI-58-transfected cells. In vitro ATGL-mediated TG hydrolysis of an artificial micellar TG substrate was not inhibited by the presence of Plin5, whereas Plin5-coated LDs were resistant toward ATGL-mediated TG catabolism. These findings strongly suggest that Plin5 functions as a lipolytic barrier to protect the cardiac TG pool from uncontrolled TG mobilization and the excessive release of free FAs.     

Alcohol Consumption Negates Estrogen-mediated Myocardial Repair in Ovariectomized Mice by Inhibiting Endothelial Progenitor Cell Mobilization and Function

We have shown previously that estrogen (estradiol, E2) supplementation enhances voluntary alcohol consumption in ovariectomized female rodents and that increased alcohol consumption impairs ischemic hind limb vascular repair. However, the effect of E2-induced alcohol consumption on post-infarct myocardial repair and on the phenotypic/functional properties of endothelial progenitor cells (EPCs) is not known. Additionally, the molecular signaling of alcohol-estrogen interactions remains to be elucidated. This study examined the effect of E2-induced increases in ethanol consumption on post-infarct myocardial function/repair. Ovariectomized female mice, implanted with 17β-E2 or placebo pellets were given access to alcohol for 6 weeks and subjected to acute myocardial infarction. Left ventricular functions were consistently depressed in mice consuming ethanol compared with those receiving only E2. Alcohol-consuming mice also displayed significantly increased infarct size and reduced capillary density. Ethanol consumption also reduced E2-induced mobilization and homing of EPCs to injured myocardium compared with the E2-alone group. In vitro, exposure of EPCs to ethanol suppressed E2-induced proliferation, survival, and migration and markedly altered E2-induced estrogen receptor-dependent cell survival signaling and gene expression. Furthermore, ethanol-mediated suppression of EPC biology was endothelial nitric oxide synthase-dependent because endothelial nitric oxide synthase-null mice displayed an exaggerated response to post-acute myocardial infarction left ventricular functions. These data suggest that E2 modulation of alcohol consumption, and the ensuing EPC dysfunction, may negatively compete with the beneficial effects of estrogen on post-infarct myocardial repair.     

Cytological studies of Brassicaceae Burn. (Cruciferae Juss.) from Western Himalayas

Cytological studies have been carried out on 12 species of Brassicaceae Burn. on population basis from different geographical areas of Kashmir and Himachal Pradesh in the Western Himalayas. Variable chromosome reports for Barbaraea intermedia (n = 16), Cardamine loxostemonoides (n = 8), Nasturtium officinale (n = 8), Sisymbrium orientale (n = 14) on world-wide basis have been added to the previous reports of these species. The chromosome numbers in seven species as Barbaraea intermedia (n = 8), B. vulgaris (n = 8), Capsella bursa-pastoris (n = 8), Descuriania Sophia (n = 10), Rorippa islandica (n = 8), Sisymbrium strictum (n = 7) and Thlaspi alpestre (n = 7) have been worked out for the first time from India. The meiotic course in the populations of seven species such as Barbaraea intermedia, Capsella bursa-pastoris, Coronopus didymus, Descuriania sophia, Nasturtium officinale, Sisymbrium orientale and S. strictum varies from normal to abnormal while all the populations of two species Barbaraea vulgaris and Sisymbrium irio show abnormal meiotic course. Meiotic abnormalities are in the form of cytomixis, chromosomal stickiness, unoriented bivalents, inter-bivalent connections, formation of laggards and bridges, all resulting into abnormal microsporogenesis. Heterogenous sized fertile pollen grains and reduced reproductive potentialities have invariably been observed in all the meiotically abnormal populations. However, the meiotic course in all the populations of Cardamine loxostemonoides, Rorippa islandica and Thalspi alpestre is found to be normal with high pollen fertility.     

Differential responses of antioxidant enzymes to aluminum toxicity in two rice (Oryza sativa L.) cultivars with marked presence and elevated activity of Fe SOD and enhanced activities of Mn SOD and catalase in aluminum tolerant cultivar

Seedlings of two Indica rice (Oryza sativa L.) cvs. HUR-105 and Vandana, differing in Al-tolerance were used to identify the key mechanisms involved in their differential behaviour towards Al toxicity. Cv. HUR-105 appeared to be Al sensitive by showing significant reduction (p ≤ 0.01) in root/shoot length, fresh weight, dry weight and water content in presence of 421 μM Al³⁺ in growth medium whereas cv. Vandana appeared to be fairly Al³⁺ tolerant. A conspicuous and significant reduction in dry weight of root and shoot was observed in Al sensitive cv. HUR-105 with 178 μM Al³⁺ treatment for 3 days. Al was readily taken up by the roots and transported to shoots in both the rice cultivars. Localization of absorbed Al was always greater in roots than in shoots. Our results of the production of reactive oxygen species (ROS) H₂O₂ and O₂ ^.? and activities of major antioxidant enzymes such as total superoxide dismutase (SOD), Cu/Zn SOD, Mn SOD, Fe SOD, catalase (CAT) and guaiacol peroxidase revealed Al induced higher oxidative stress, greater production of ROS and lesser capacity to scavenge ROS in cv. HUR-105 than Vandana. With Al treatment, higher oxidative stress was noted in shoots than in roots. Greatly enhanced activities of SOD (especially Fe and Mn SOD) and CAT in Al treated seedlings of cv. Vandana suggest the role of these enzymes in Al tolerance. Furthermore, a marked presence of Fe SOD in roots and shoots of the seedlings of Al tolerant cv. Vandana and its significant (p ≤ 0.01) increase in activity due to Al-treatment, appears to be the unique feature of this cultivar and indicates a vital role of Fe SOD in Al-tolerance in rice.     

A novel strain of Brevibacillus laterosporus produces chitinases that contribute to its biocontrol potential

A novel strain exhibiting entomopathogenic and chitinolytic activity was isolated from mangrove marsh soil in India. The isolate was identified as Brevibacillus laterosporus by phenotypic characterization and 16S rRNA sequencing and designated Lak1210. When grown in the presence of colloidal chitin as the sole carbon source, the isolate produced extracellular chitinases. Chitinase activity was inhibited by allosamidin indicating that the enzymes belong to the family 18 chitinases. The chitinases were purified by ammonium sulfate precipitation followed by chitin affinity chromatography yielding chitinases and chitinase fragments with 90, 75, 70, 55, 45, and 25 kDa masses. Mass spectrometric analyses of tryptic fragments showed that these fragments belong to two distinct chitinases that are almost identical to two putative chitinases, a 89.6-kDa four-domain chitodextrinase and a 69.4-kDa two-domain enzyme called ChiA1, that are encoded on the recently sequenced genome of B. laterosporus LMG15441. The chitinase mixture showed two pH optima, at 6.0 and 8.0, and an optimum temperature of 70 °C. The enzymes exhibited antifungal activity against the phytopathogenic fungus Fusarium equiseti. Insect toxicity bioassays with larvae of diamondback moths (Plutella xylostella), showed that addition of chitinases reduced the time to reach 50 % mortality upon infection with non-induced B. laterosporus from 3.3 to 2.1 days. This study provides evidence for the presence of inducible, extracellular chitinolytic enzymes in B. laterosporus that contribute to the strain’s antifungal activity and insecticidal activity.     

Design and synthesis of (4E)-4-(4-substitutedbenzylideneamino)-3-substituted-2,3-dihydro-2-thioxothiazole-5-carbonitrile as novel A2A receptor antagonists

Novel 2-thioxothiazole derivatives (6–19) as potential adenosine A_2A receptor (A_2AR) antagonists were synthesized. The strong interaction of the compounds (6–19) with A_2AR in docking study was confirmed by high binding affinity with human A_2AR expressed in HEK293T cells using radioligand-binding assay. The compound 19 demonstrated very high selectivity for A_2AR as compared to standard A_2AR antagonist SCH58261. Decrease in A_2AR-coupled release of endogenous cAMP in treated HEK293T cells demonstrated in vitro A_2AR antagonist potential of the compound 19. Attenuation in haloperidol-induced impairment (catalepsy) in Swiss albino male mice pre-treated with compound 19 is evocative to explore its prospective in therapy of PD.