Genome size and restriction fragment length polymorphism analysis of Vibrio cholerae strains belonging to different serovars and biotypes

Abstract The genome size of Vibrio cholerae has been determined by pulsed field gel electrophoresis following digestion of chromosomal DNA with endonucleases. The genome size of all the classical strains examined was about 3000 kb and that of El Tor biotype was 2500 kb. The Not I and S fi I digestion patterns of the genomes of several V. cholerae straimns belonging to different serovars and biotypes showed distinct restriction fragment length polymorphism (RFLP). RFLP analysis together with the genome size can be used to differentiate strains of different serovars and biotypes of V. cholerae .     

Endogenous protease mediated manifestation of a Ca2+-stimulated ATPase in purified dog gastric microsomes

An endogenous soluble protease has been demonstrated to unmask a Ca²⁺-stimulated ATPase activity in purified dog gastric microsomes. The presence of ATP during protease treatment appears essential for the manifestation of the gastric Ca²⁺-stimulated ATPase activity. The endogenous protease appears to have trypsin-like activity, since soybean trypsin inhibitor completely blocks the protease effect. Manifestation of the Ca²⁺-stimulated ATPase occurs without affecting the microsomal (H⁺ +K⁺)-ATPase activity and associated H⁺ uptake ability. The unmasked Ca²⁺-stimulated ATPase appears insensitive to calmodulin. Possible roles of the enzyme in the regulation of gastric H⁺ transport have been discussed.     

EST-SSRs reveal genetic distinction between lac and grain yielding genotypes of pigeonpea

Journal of Plant Biochemistry and Biotechnology - Pigeonpea, an important legume crop is a good host plant for lac cultivation in North East India. In the present study, sixty-three polymorphic EST...     

Pathophysiological relationship between hypoxia associated oxidative stress,Epithelial-mesenchymal transition,stemness acquisition and alteration of Shh/ Gli-1 axis during oral sub-mucous fibrosis and oral squamous cell carcinoma

Oral sub-mucous fibrosis (OSF) is a pathophysiological state of oral cavity or oropharynx having a high chance of conversion to oral squamous cell carcinoma (OSCC). It involves fibrotic transformation of sub-epithelial matrix along with epithelial abnormalities. The present work aims to unveil the mechanistic domain regarding OSF to OSCC conversion exploring the scenario of hypoxia associated oxidative stress, epithelial-mesenchymal transition (EMT), metastasis and stemness acquisition. The study involves histopathological analysis of the diseased condition along with the exploration of oxidative stress status, assessment of mitochondrial condition, immunohistochemical analysis of HIF-1α, E-cadherin, vimentin, ERK, ALDH-1, CD133, Shh, Gli-1 and survivin expressions in the oral epithelial region together with the quantitative approach towards collagen deposition in the sub-epithelial matrix. Oxidative stress was found to be associated with type-II EMT in case of OSF attributing the development of sub-epithelial fibrosis and type-III EMT in case of OSCC favoring malignancy associated metastasis. Moreover, the acquisition of stemness during OSCC can also be correlated with EMT. Alteration of Shh and Gli-1 expression pattern revealed the mechanistic association of hypoxia with the phenotypic plasticity and disease manifestation in case of OSF as well as OSCC. Shh/ Gli-1 signaling can also be correlated with survivin mediated cytoprotective phenomenon under oxidative stress. Overall, the study established the correlative network of hypoxia associated oxidative stress, EMT and manifestation of oral pre-cancerous and cancerous condition in a holistic approach that may throw rays of hope in the therapeutic domain of the concerned diseases.     

Ubiquitin-Like Protein from Human Placental Extract Exhibits Collagenase Activity

An aqueous extract of human placenta exhibits strong gelatinase/collagenase activity in zymography. 2-D gel electrophoresis of the extract with gelatin zymography in the second dimension displayed a single spot, identified as ubiquitin-like component upon MALDI/TOF MS/MS analysis. Immunoblot indicated presence of ubiquitin and absence of collagenase in the extract. Collagenase activity of the ubiquitin-like component was confirmed from the change in solubility of collagen in aqueous buffer, degradation of collagen by size-exclusion HPLC and atomic force microscopy. Quantification with DQ-gelatin showed that the extract contains 0.04 U/ml of collagenase activity that was inhibited up to 95% by ubiquitin antibody. Ubiquitin from bovine erythrocytes demonstrated mild collagenase activity. Bioinformatics studies suggest that placental ubiquitin and collagenase follow structurally divergent evolution. This thermostable intrinsic collagenase activity of placental extract might have wide physiological relevance in degrading and remodeling collagen as it is used as a drug for wound healing and pelvic inflammatory diseases.     

Ulltrastructural and trace metal studies on radiographers’ hair and nails

Scalp hair and fingernail samples of 42 medical radiographers and 42 nonradiographers (control) with matching age groups and food habits were collected for this study. Trace metal estimation by atomic absorption spectrometry (AAS) has indicated a significant increase (P < 0.001) in Zn, Cu, and Cd contents in the radiographers’ hair and nails. Scanning electron microscopy (SEM) reveled structural changes in the hair and nails of radiographers. Significant alterations in the Zn and Cd contents along with extensive structural damage in the hair and nails probably indicate that low-dose Χ-radiation imposes stress on these radiation workers.     

Insulin regulates MAP kinase phosphatase-1 induction in Hirc B cells via activation of both extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK)

Previously, we have reported that insulin induces the expression of the dual-specificity tyrosine phosphatase Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) and that this may represent a negative feedback mechanism to regulate insulin-stimulated MAP kinase activity. In this work, the mechanism of regulation of MKP-1 expression by insulin was examined, particularly the role of the MAP kinase superfamily. Inhibition of the ERK pathway attenuated insulin-stimulated MKP-1 mRNA expression. Expression of dominant negative molecules of the JNK pathway also abolished insulin-stimulated MKP-1 expression. However, inhibition of p38^MAPK activity by SB202190 had no effect on insulin-stimulated MKP-1 induction. Simultaneous inhibition of the ERK and JNK pathways abolished the ability of insulin to stimulate MKP-1 expression, however, this combined inhibition was neither additive nor synergistic, suggesting these pathways converge to act on a common final effector. In conclusion, induction of MKP-1 mRNA expression in Hirc B cells by insulin requires activation of both the ERK and JNK pathways, but not p38^MAPK.     

Phosphorylation and activation of protein tyrosine phosphatase (PTP) 1B by insulin receptor

We have previously reported a direct in vivo interaction between the activated insulin receptor and protein-tyrosine phosphatase-1B (PTP1B), which leads to an increase in PTP1B tyrosine phosphorylation. In order to determine if PTP1B is a substrate for the insulin receptor tyrosine kinase, the phosphorylation of the Cys 215 Ser, catalytically inactive mutant PTP1B (CS-PTP1B) was measured in the presence of partially purified and activated insulin receptor. In vitro, the insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1B. 53% of the total cellular PTP1B became tyrosine phosphorylated in response to insulin in vivo. Tyrosine phosphorylation of PTP1B by the insulin receptor was absolutely dependent upon insulin-stimulated receptor autophosphorylation and required an intact kinase domain, containing insulin receptor tyrosines 1146, 1150 and 1151. Tyrosine phosphorylation of wild type PTP1B by the insulin receptor kinase increased phosphatase activity of the protein. Intermolecular transdephosphorylation was demonstrated both in vitro and in vivo, by dephosphorylation of phosphorylated CS-PTP1B by the active wild type enzyme either in a cell-free system or via expression of the wild type PTP1B into Hirc-M cell line, which constitutively overexpress the human insulin receptor and CS-PTP1B. These results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP1B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated (the active form) and dephosphorylated (the inactive form) state.     

Functional identification of sll1383 from<Emphasis Type="Italic"> Synechocystis</Emphasis> sp PCC 6803 as L-<Emphasis Type="Italic">myo</Emphasis>-inositol 1-phosphate phosphatase (EC 3.1.3.25): molecular cloning,expression and characterization

The genome sequence of the cyanobacterium Synechocystis sp. PCC6803 revealed four Open reading frame (ORF) encoding putative inositol monophosphatase or inositol monophosphatase-like proteins. One of the ORFs, sll1383, is ∼870 base pair long and has been assigned as a probable myo-inositol 1 (or 4) monophosphatase (IMPase; EC 3.1.3.25). IMPase is the second enzyme in the inositol biosynthesis pathway and catalyses the conversion of L-myo-inositol 1-phosphate to free myo-inositol. The present work describes the functional assignment of ORF sll1383 as myo-inositol 1-phosphate phosphatase (IMPase) through molecular cloning, bacterial overexpression, purification and biochemical characterization of the gene product. Affinity (K _m) of the recombinant protein for the substrate DL-myo-inositol 1-phosphate was found to be much higher (0.0034 ± 0.0003 mM) compared to IMPase(s) from other sources but in comparison V _max (∼0.033 μmol Pi/min/mg protein) was low. Li⁺ was found to be an inhibitor (IC₅₀ 6.0 mM) of this enzyme, other monovalent metal ions (e.g. Na⁺, K⁺ NH₄⁺) having no significant effect on the enzyme activity. Like other IMPase(s), the activity of this enzyme was found to be totally Mg²⁺ dependent, which can be substituted partially by Mn²⁺. However, unlike other IMPase(s), the enzyme is optimally active at ∼42°C. To the best of our knowledge, sll1383 encoded IMPase has the highest substrate affinity and specificity amongst the known examples from other prokaryotic sources. A possible application of this recombinant protein in the enzymatic coupled assay of L-myo-inositol 1-phosphate synthase (MIPS) is discussed.     

Prophylactic role of D-Saccharic acid-1,4-lactone in tertiary butyl hydroperoxide induced cytotoxicity and cell death of murine hepatocytes via mitochondria-dependent pathways

D-Saccharic acid 1,4-lactone (DSL) is a derivative of D-glucaric acid. It is a beta-glucuronidase inhibitor and possesses anticarcinogenic, detoxifying, and antioxidant properties. In the present study, the protective effects of DSL were investigated against tertiary butyl hydroperoxide (TBHP) induced cytotoxicity and cell death in vitro using murine hepatocytes. Exposure of TBHP caused a reduction in cell viability, enhanced the membrane leakage, and disturbed the intracellular antioxidant machineries in murine hepatocytes. Investigating the signaling mechanism of TBHP-induced cellular pathophysiology and protective action of DSL, we found that TBHP exposure disrupted mitochondrial membrane potential, facilitated cytochrome c release in the cytosol, and led to apoptotic cell death via mitochondria-dependent pathways. DSL counteracted these changes and maintained normalcy in hepatocytes. Combining, results suggest that DSL possesses the ability to ameliorate TBHP-induced oxidative insult, cytotoxicity, and apoptotic cell death probably due to its antioxidant activity and functioning via mitochondria-dependent pathways.