Cloning and partial characterization of a novel hemolysin gene of Vibrio tubiashii and the development of a PCR-based detection assay

Vibrio tubiashii expresses virulence factors, such as a vulnificolysin-like hemolysin or cytolysin and a zinc metalloprotease, similar to those of other pathogenic vibrios. In this study, we report the cloning of a novel hemolysin gene of V.?tubiashii in Escherichia coli . A V.?tubiashii gene library was screened for hemolytic activity on sheep blood agar. Three hemolytic clones pGem:hly1, pGem:hly2, and pGem:hly3 were sequenced, and the sequences showed a strong homology to the ribA gene coding for guanosine triphosphate cyclohydrolase II (GCH II), required for riboflavin biosynthesis and reported to be responsible for hemolytic activity in Helicobacter pylori . The plasmids pGem:hly1 and pGem:hly3 when introduced into E. coli BSV18 (ribA18::Tn5) were able to restore growth of strain BSV18 in a medium without riboflavin and also produced hemolytic activity on blood agar. PCR primers based on the cloned hly-ribA sequence were tested using 23 different Vibrio strains representing 10 different species. Amplification of ribA gene locus only occurred with V.?tubiashii strains. In summary, our results indicate that we have cloned a ribA homolog of V.?tubiashii that imparts hemolytic activity to E.?coli clones, and primers based on this gene locus might be useful as a species-specific identification tool for V.?tubiashii.     

Genotyping of Toxoplasma gondii Isolates from Wild Boars in Peninsular Malaysia

Toxoplasma gondii is a parasitic protozoan that infects nearly one-third of the world population. The present study was done to isolate and genotype T. gondii from wild boar from forests of Pahang, Malaysia. A total of 30 wild boars'' blood, heads and hearts were obtained for this study and 30 (100.0%) were found to be seropositive when assayed with modified agglutination test (MAT≥6). The positive samples were inoculated into mice and T. gondii was only isolated from samples that had strong seropositivity (MAT≥1∶24).The isolates were subjected to PCR-RFLP analysis and all the Peninsular Malaysia isolates of T. gondii are of clonal type I.     

Salmonella Typhimurium methionine sulfoxide reductase A (MsrA) prefers TrxA in repairing methionine sulfoxide

Intraphagocytic survival of Salmonella Typhimurium (ST) depends (at least in part) upon its ability to repair oxidant-damaged macromolecules. Met residues either free or in protein bound form are highly susceptible to phagocyte-generated oxidants. Oxidation of Mets leads to Met-SO formation, consequently loss of protein functions that results in cell death. Methionine sulfoxide reductase (Msr) reductively repairs Met-SO to Met in the presence of thioredoxin (trx) and thioredoxin reductase (trxR). Earlier we reported that methionine sulfoxide reductase A (msrA) gene deletion strain of ST suffered oxidative stress.^[1 Trivedi, R.N.; Agarwal, P.; Kumawat, M.; Pesingi, P.K.; Gupta, V.K.; Goswami, T.K.; Mahawar, M. Methionine Sulfoxide Reductase A (MsrA) Contributes to Salmonella Typhimurium Survival Against Oxidative Attack of Neutrophils. Immunobiology 2015, 220(12), 1322–1327.[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]^] Thioredoxin system of ST comprises of two thioredoxins (trxA and trxC) and one thioredoxin reductase (trxB). Preferred trx utilized in MsrA-mediated repair of Met-SO is not known. In current study, we cloned, expressed, and purified ST TrxA, TrxB, TrxC, and MsrA in recombinant forms. The migration of TrxA, TrxB, TrxC, and MsrA proteins was approximately 10, 36, 16, and 26?kDa on SDS-gels. The nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-linked reductase assays interpreted that MsrA utilized two times more NADPH for the reduction of S-methyl p-tolyl sulfoxide when TrxA was included in the assays as compared to TrxC.     

Rad50 adenylate kinase activity regulates DNA tethering by Mre11/Rad50 complexes

Mre11 and Rad50 are the catalytic components of a highly conserved DNA repair complex that functions in many aspects of DNA metabolism involving double-strand breaks. The ATPase domains in Rad50 are related to the ABC transporter family of ATPases, previously shown to share structural similarities with adenylate kinases. Here we demonstrate that Mre11/Rad50 complexes from three organisms catalyze the reversible adenylate kinase reaction in vitro. Mutation of the conserved signature motif reduces the adenylate kinase activity of Rad50 but does not reduce ATP hydrolysis. This mutant resembles a rad50 null strain with respect to meiosis and telomere maintenance in S. cerevisiae, correlating adenylate kinase activity with in vivo functions. An adenylate kinase inhibitor blocks Mre11/Rad50-dependent DNA tethering in vitro and in cell-free extracts, indicating that adenylate kinase activity by Mre11/Rad50 promotes DNA-DNA associations. We propose a model for Rad50 that incorporates both ATPase and adenylate kinase reactions as critical activities that regulate Rad50 functions.     

ImitateDB: A database for domain and motif mimicry incorporating host and pathogen protein interactions

Amino Acids - Molecular mimicry of host proteins by pathogens constitutes a strategy to hijack the host pathways. At present, there is no dedicated resource for mimicked domains and motifs in the...     

Gene therapy in cardiac arrhythmias

Gene therapy has progressed from a dream to a bedside reality in quite a few human diseases. From its first application in adenosine deaminase deficiency, through the years, its application has evolved to vascular angiogenesis and cardiac arrhythmias. Gene based biological pacemakers using viral vectors or mesenchymal cells tested in animal models hold much promise. Induction of pacemaker activity within the left bundle branch can provide stable heart rates. Genetic modification of the AV node mimicking beta blockade can be therapeutic in the management of atrial fibrillation. G protein overexpression to modify the AV node also is experimental. Modification and expression of potassium channel genes altering the delayed rectifier potassium currents may permit better management of congenital long QT syndromes. Arrhythmias in a failing heart are due to abnormal calcium cycling. Potential targets for genetic modulation include the sarcoplasmic reticulum calcium pump, calsequestrin and sodium calcium exchanger. Lastly the ethical concerns need to be addressed.     

Curcumin loaded-PLGA nanoparticles conjugated with Tet-1 peptide for potential use in Alzheimer's disease

Alzheimer's disease is a growing concern in the modern world. As the currently available medications are not very promising, there is an increased need for the fabrication of newer drugs. Curcumin is a plant derived compound which has potential activities beneficial for the treatment of Alzheimer's disease. Anti-amyloid activity and anti-oxidant activity of curcumin is highly beneficial for the treatment of Alzheimer's disease. The insolubility of curcumin in water restricts its use to a great extend, which can be overcome by the synthesis of curcumin nanoparticles. In our work, we have successfully synthesized water-soluble PLGA coated- curcumin nanoparticles and characterized it using different techniques. As drug targeting to diseases of cerebral origin are difficult due to the stringency of blood-brain barrier, we have coupled the nanoparticle with Tet-1 peptide, which has the affinity to neurons and possess retrograde transportation properties. Our results suggest that curcumin encapsulated-PLGA nanoparticles are able to destroy amyloid aggregates, exhibit anti-oxidative property and are non-cytotoxic. The encapsulation of the curcumin in PLGA does not destroy its inherent properties and so, the PLGA-curcumin nanoparticles can be used as a drug with multiple functions in treating Alzheimer's disease proving it to be a potential therapeutic tool against this dreaded disease.     

Structure of E. coli ketopantoate hydroxymethyl transferase complexed with ketopantoate and Mg2+, solved by locating 160 selenomethionine sites

We report the crystal structure of E. coli ketopantoate hydroxymethyltransferase (KPHMT) at 1.9 A resolution, in complex with its product, ketopantoate. KPHMT catalyzes the first step in the biosynthesis of pantothenate (vitamin B(5)), the precursor of coenzyme A and the acyl carrier protein cofactor. The structure of the decameric enzyme was solved by multiwavelength anomalous dispersion to locate 160 selenomethionine sites and phase 560 kDa of protein, making it the largest structure solved by this approach. KPHMT adopts the (betaalpha)(8) barrel fold and is a member of the phosphoenolpyruvate/pyruvate superfamily. The active site contains a ketopantoate bidentately coordinated to Mg(2+). Similar binding is likely for the substrate, alpha-ketoisovalerate, orienting the C3 for deprotonation.     

Protective effect of ferulic acid on gamma-radiation-induced micronuclei, dicentric aberration and lipid peroxidation in human lymphocytes

In this study we examined radioprotective effect of ferulic acid (FA) on gamma radiation-induced dicentric aberration and lipid peroxidation with reference to alterations in cellular antioxidant status in cultured lymphocytes. To establish most effective protective support we used three different concentrations of FA (1, 5 and 10 microg/ml) and three different doses of gamma-radiation (1, 2 and 4 Gy). Treatment of lymphocytes with FA alone (at 10 microg/ml) gave no significant change in micronuclei (MN), dicentric aberration (DC), thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) or glutathione peroxidase (GPx) activities when compared with normal lymphocytes; irradiation at 1, 2 and 4 Gy increased the MN and DC frequencies in a dose-dependent manner. Treatment with FA for 30 min before radiation exposure resulted in a significant decline of MN and DC yields as FA concentration increased. Compared to 1 Gy exposure alone, the extent to which FA (1 microg/ml) reduced the MN and DC yields was 75% and 50%, respectively. With 4 Gy irradiation, FA (10 microg/ml) decreased 45% MN and 25% DC frequencies. FA-pretreated lymphocytes (1, 5 and 10 microg/ml) showed progressively decreased TBARS levels after irradiation. Irradiation (1, 2 and 4 Gy) significantly decreased GSH levels, SOD, CAT and GPx activities in a dose-dependent manner. Pretreatment with 10 microg/ml of FA significantly (p<0.05) prevented the decreases in the radiation-induced GSH, SOD, CAT and GPx activities. These findings suggest potential use and benefit of FA as a radioprotector.