Malabarase,a serine protease with anticoagulant activity from Trimeresurus malabaricus venom

In the present study we describe the purification and characterization of Malabarase, a serine protease from Trimeresurus malabaricus venom. Purification was achieved by gel-permeation chromatography on Sephadex G-75 followed by ion-exchange chromatography on CM Sephadex C-25. Homogeneity of Malabarase was confirmed by RP-HPLC. Malabarase is a monomer that migrated as a single protein band on SDS-PAGE under both reducing and non-reducing conditions. The molecular mass of Malabarase was determined to be 23.4 kDa using MALDI-TOF mass spectrometry. Malabarase is the first serine protease purified from T. malabaricus venom and is selective for fibrinogen. Malabarase hydrolyzes Aα and Bβ but not γ-chains of fibrinogen similar to the metalloproteases, Malabarin and Trimarin, isolated from the same venom. However, the action of Malabarase on plasma coagulation is opposite than those of Malabarin, Trimarin and the whole venom. Malabarase significantly prolonged plasma coagulation time from 152–341 s; whereas Malabarin, Trimarin, and whole venom, greatly reduce plasma clotting time from 152 to 12, 48, and 14 s, respectively. Malabarase did not show hemorrhagic or myotoxic activity. In contrast, Malabarin, Trimarin and whole venom are highly hemorrhagic and myotoxic. These observations support the specificity of Malabarase towards fibrinogen and its non-toxic nature. In conclusion, Malabarase is a fibrinogen-specific, anti-coagulant, and non-toxic serine protease. Its selective action and non-toxic nature might make it useful for treating thrombotic disorders.     

Saccharomyces cerevisiae DNA Ligase IV Supports Imprecise End Joining Independently of Its Catalytic Activity

DNA ligase IV (Dnl4 in budding yeast) is a specialized ligase used in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation in vitro and NHEJ activity in vivo, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that dnl4-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast) at DSBs was observed in wild-type as well as dnl4 point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5′ resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9.     

Analysis of the Territorial, Courtship and Coupling Behavior of the Hemipterophagous Butterfly, Spalgis epius (Westwood) (Lepidoptera: Lycaenidae)

In this study we examined the territorial, courtship and coupling behavior, mating interruptions and influence of sunlight on mating behavior in Spalgis epius (Westwood). We constructed an ethogram of territorial and courtship behavior, and catalogued the sequence of behavioral acts associated with mating behavior. Mating behavioral acts were divided into four repertoires i.e. pre-courtship, pre-coupling, coupling and post-coupling behaviors. Totally 22 behavioral acts were recorded from four repertoires. Tree canopy, canopy height and sunlight conditions are the important factors that influence copulation in S. epius. The courtship activity led to successful copulation in 71.9 % pairs. Incidence of different types of courtship and copula interruptions in S. epius was also recorded.     

Ambient Light Promotes Selective Subcellular Proteotoxicity after Endogenous and Exogenous Porphyrinogenic Stress

Hepatic accumulation of protoporphyrin-IX (PP-IX) in erythropoietic protoporphyria (EPP) or X-linked-dominant protoporphyria (XLP) cause liver damage. Hepatocyte nuclear lamin aggregation is a sensitive marker for PP-IX-mediated liver injury. We tested the hypothesis that extracellular or intracellular protoporphyria cause damage to different subcellular compartments, in a light-triggered manner. Three hepatoma cell lines (HepG2, Hepa-1, and Huh-7) were treated with exogenous PP-IX (mimicking XLP extrahepatic protoporphyria) or with the iron chelator deferoxamine and the porphyrin precursor 5-aminolevulinic acid (ALA) (mimicking intracellular protoporphyrin accumulation in EPP). Exogenous PP-IX accumulated predominantly in the nuclear fraction and caused nuclear shape deformation and cytoplasmic vacuoles containing electron-dense particles, whereas ALA+deferoxamine treatment resulted in higher PP-IX in the cytoplasmic fraction. Protein aggregation in the nuclear and cytoplasmic fractions paralleled PP-IX levels and, in cell culture, the effects were exclusively ambient light-mediated. PP-IX and ALA caused proteasomal inhibition, whereas endoplasmic reticulum protein aggregation was more prominent in ALA-treated cells. The enhanced ALA-related toxicity is likely due to generation of additional porphyrin intermediates including uroporphyrin and coproporphyrin, based on HPLC analysis of cell lysates and the culture medium, as well as cell-free experiments with uroporphyrin/coproporphyrin. Mouse livers from drug-induced porphyria phenocopied the in vitro findings, and mass spectrometry of liver proteins isolated in light/dark conditions showed diminished (as compared with light-harvested) but detectable aggregation under dark-harvested conditions. Therefore, PP-IX leads to endoplasmic reticulum stress and proteasome inhibition in a manner that depends on the source of porphyrin buildup and light exposure. Porphyrin-mediated selective protein aggregation provides a potential mechanism for porphyria-associated tissue injury.     

Mass rearing of Spalgis epius (Lepidoptera: Lycaenidae), a potential predator of mealybugs (Hemiptera: Pseudococcidae)

Spalgis epius (Lepidoptera: Lycaenidae) has been recorded as a potential predator of various species of mealybug crop pests worldwide. We describe the mass rearing of S. epius, as no information on this topic is available. Outdoor nylon tent cages of different dimensions were provided to achieve mating and oviposition as S. epius adults did not mate in the laboratory cages. Adults mated only in the tent cage (6×6×10 m) placed over a native tree (9 m height). The presence of a tree canopy inside the cage is essential to achieve courtship and mating. Gravid females of S. epius deposited eggs on the mealybug-infested pumpkins inside the different sized nylon cages with or without a bush/tree. Spalgis epius eggs were maintained on mealybug-infested pumpkins in the laboratory and developmental stages of the predator were reared. Adults fed on various diets laid significantly higher number of eggs than those of starved individuals. Spalgis epius with a life cycle completed in 21.2 days and 55.7 larvae, could be reared on a single mealybug-infested pumpkin.     

Virus isolation and molecular epidemiology of highly pathogenic avian influenza A(H5N8) from an outbreak in free-ranging wild birds,India 2016

We report the molecular epidemiology of highly pathogenic avian influenza (HPAI) virus involved in an outbreak causing death in free-ranging wild birds at Mysore, Karnataka state of India. The virus was typed as HPAI A(H5N8) by conventional and TaqMan probe based real-time PCR assays. Six isolates of HPAI virus were recovered in 9-day-old embryonated chicken eggs. Haemagglutinin gene-based phylogeny of virus isolates showed >?99.9% nucleotide sequence identity with HPAI A(H5N8) isolates from migratory birds and domestic poultry from China and Korea indicating either these wild birds have routed their migration through Korea and/or eastern China or these dead birds must have directly or indirectly contacted with wild birds migrating from Eastern China and/or Korean regions. The study emphasises the role of migratory wild birds in spread of HPAI across the globe.     

Isolation and characterization of BetaM protein encoded by ATP1B4--a unique member of the Na,K-ATPase β-subunit gene family

ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a β-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase β-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS–PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was also characterized by SELDI-TOF mass spectrometry before and after deglycosylation. This allowed us to determine that the carbohydrate moiety of BetaM has molecular mass 5.9 kDa and consists of short high-mannose type N-glycans. The results of direct analysis of the purified native eutherian BetaM protein provide first insights into structural properties underlying its entirely new evolutionarily acquired functions.     

Mass-Spectrometric Identification of T-Kininogen I/Thiostatin as an Acute-Phase Inflammatory Protein Suppressed by Curcumin and Capsaicin

Curcumin and capsaicin are dietary xenobiotics with well-documented anti-inflammatory properties. Previously, the beneficial effect of these spice principles in lowering chronic inflammation was demonstrated using a rat experimental model for arthritis. The extent of lowering of arthritic index by the spice principles was associated with a significant shift in macrophage function favoring the reduction of pro-inflammatory molecules such as reactive oxygen species and production and release of anti-inflammatory metabolites of arachidonic acid. Beyond the cellular effects on macrophage function, oral administration of curcumin and capsaicin caused alterations in serum protein profiles of rats injected with adjuvant to develop arthritis. Specifically, a 72 kDa acidic glycoprotein, GpA72, which was elevated in pre-arthritic rats, was significantly lowered by feeding either curcumin or capsaicin to the rats. Employing the tandem mass spectrometric approach for direct sequencing of peptides, here we report the identification of GpA72 as T-kininogen I also known as Thiostatin. Since T-kininogen I is an early acute-phase protein, we additionally tested the efficiency of curcumin and capsaicin to mediate the inflammatory response in an acute phase model. The results demonstrate that curcumin and capsaicin lower the acute-phase inflammatory response, the molecular mechanism for which is, in part, mediated by pathways associated with the lowering of T-kininogen I.     

Synthesis and biological evaluation of a novel structural type of serotonin 5-HT3 receptor antagonists

A series of novel 3-substituted quinoxalin-2-carboxamides were designed as per the pharmacophoric requirement for 5-HT(3) receptor antagonists and prepared by microwave irradiation and also by conventional method. The compounds were characterized by spectral data (IR, (1)H NMR, and MS) and the purity was ascertained by microanalysis. The synthesized compounds were evaluated for 5-HT(3) antagonisms in longitudinal muscle-myenteric plexus preparation from guinea pig ileum against 5-HT(3) agonist, 2-methyl-5-HT. Among the test compounds, N-{3-[(4-methylpiperazin-1-yl)methyl]-4-hydroxyphenyl}-3-methoxyquinoxalin-2-carboxamide 4e showed most favorable 5-HT(3) receptor antagonism.     

The interaction profile of homologous recombination repair proteins RAD51C,RAD51D and XRCC2 as determined by proteomic analysis

The RAD51 family of proteins is involved in homologous recombination (HR) DNA repair and maintaining chromosome integrity. To identify candidates that interact with HR proteins, the mouse RAD51C, RAD51D and XRCC2 proteins were purified using bacterial expression systems and each of them used to co‐precipitate interacting partners from mouse embryonic fibroblast cellular extracts. Mass spectroscopic analysis was performed on protein bands obtained after 1‐D SDS‐PAGE of co‐precipitation eluates from cell extracts of mitomycin C treated and untreated mouse embryonic fibroblasts. Profiling of the interacting proteins showed a clear bias toward nucleic acid binding and modification proteins. Interactions of four candidate proteins (SFPQ, NONO, MSH2 and mini chromosome maintenance protein 2) were confirmed by Western blot analysis of co‐precipitation eluates and were also verified to form ex vivo complexes with RAD51D. Additional interacting proteins were associated with cell division, embryo development, protein and carbohydrate metabolism, cellular trafficking, protein synthesis, modification or folding, and cell structure or motility functions. Results from this study are an important step toward identifying interacting partners of the RAD51 paralogs and understanding the functional diversity of proteins that assist or regulate HR repair mechanisms.