Virulence variation of Puccinia striiformis Westend. f. sp. tritici in Pakistan

Yellow rust populations of Pakistan were characterised for their virulence pathotypes/races and pathogenetic variation using seedling evaluation of differential genotypes under glasshouse conditions in Murree (6000 feet above sea level). Differential genotypes comprised a world set, an European set, near isogenic lines and the universally susceptible bread wheat cultivar “Morocco”. Over the two-year study a total of 18 race groups were identified. Out of these 18 race groups, several (68E0, 64E0, 66E0, 70E0, 6E0, 71E0, 6E0, 2E0, 67E0, and 68E16) were found previously. The new race group 70E32 found probably evolved because of mutation from the previously existing 70E16. Virulence frequencies of yellow rust (Yr) resistance genes were also determined on near isogenic lines. The highest virulence frequencies (%) were found for Yr7 (88%), Yr9 (57%), Yr18 (70%), and Yr24 (69%). Virulence frequencies were low for Yr 1 (4%), Yr5 (7%), Yr10 (10%) and Yr15 (4%). Our studies indicated that virulence existed for almost all yr genes, necessitating regular monitoring of the yellow rust populations and intensifying efforts to identify new sources of resistance to this pathogen.     

Inhibition and eradication of Salmonella Typhimurium biofilm using P22 bacteriophage,EDTA and nisin

Abstract

P22 phage >10⁵ PFU ml^?1 could be used to inhibit Salmonella Typhimurium biofilm formation by 55–80%. Concentrations of EDTA >1.25?mM and concentrations of nisin >1,200?µg ml^?1 were also highly effective in reducing S. Typhimurium biofilm formation (≥96% and ≥95% reductions were observed, respectively). A synergistic effect was observed when EDTA and nisin were combined whereas P22 phage in combination with nisin had no synergistic impact on biofilm formation. Triple combination of P22 phage, EDTA and nisin could be also used to inhibit biofilm formation (≥93.2%) at a low phage titer (10² PFU ml^?1), and low EDTA (1.25?mM) and nisin (9.375?µg ml^?1) concentrations. A reduction of 70% in the mature biofilm was possible when 10⁷ PFU ml^?1 of P22 phage, 20?mM of EDTA and 150?μg ml^?1 of nisin were used in combination. This study revealed that it could be possible to reduce biofilm formation by S. Typhimurium by the use of P22 phage, EDTA and nisin, either alone or in combination. Although, removal of the mature biofilm was more difficult, the triple combination could be successfully used for mature biofilm of S. Typhimurium.     

Methylation of the PTPRO gene in human hepatocellular carcinoma and identification of VCP as its substrate

We have previously reported that the gene encoding protein tyrosine phosphatase receptor type‐O (PTPRO) is suppressed by promoter methylation in a rat model of hepatocellular carcinoma (HCC) and it functions as tumor suppressor in leukemia and lung cancer. Here, we explored the methylation and expression of PTPRO as well as its function in human HCC. MassARRAY analysis of primary human HCC and matching liver samples (n = 24) revealed significantly higher (P = 0.004) methylation density at the promoter CGI in tumors. Combined bisulfite restriction analysis (COBRA) of another set of human HCC samples (n = 17) demonstrated that the CGI was methylated in 29% of tumors where expression of PTPRO was lower than that in corresponding matching livers. A substrate‐trapping mutant of PTPRO that stabilizes the bound substrates was used to identify its novel substrate(s). VCP/p97 was found to be a PTPRO substrate by mass spectrometry of the peptides pulled down by the substrate‐trapping mutant of PTPRO. Tyrosyl dephosphorylation of VCP following ectopic expression of wild‐type PTPRO in H293T and HepG2 cells confirmed that it is a bona fide substrate of PTPRO. Treatment of PTPRO overexpressing HepG2 cells with Doxorubicin, a DNA damaging drug commonly used in therapy of primary HCC, sensitized these cells to this potent anticancer drug that correlated with dephosphorylation of VCP. Taken together, these results demonstrate methylation and downregulation of PTPRO in a subset of primary human HCC and establish VCP as a novel functionally important substrate of this tyrosine phosphatase that could be a potential molecular target for HCC therapy. J. Cell. Biochem. 114: 1810–1818, 2013. © 2013 Wiley Periodicals, Inc.     

Discovery of 7-azaindole based anaplastic lymphoma kinase (ALK) inhibitors: Wild type and mutant (L1196M) active compounds with unique binding mode

We have identified a novel 7-azaindole series of anaplastic lymphoma kinase (ALK) inhibitors. Compounds 7b, 7m and 7n demonstrate excellent potencies in biochemical and cellular assays. X-ray crystal structure of one of the compounds (7k) revealed a unique binding mode with the benzyl group occupying the back pocket, explaining its potency towards ALK and selectivity over tested kinases particularly Aurora-A. This binding mode is in contrast to that of known ALK inhibitors such as Crizotinib and NVP-TAE684 which occupy the ribose binding pocket, close to DFG motif.     

Predicted binding of certain antifilarial compounds with glutathione-S-transferase of human Filariids

Glutathione-S-transferase is a major phase-II detoxification enzyme in parasitic helminthes. Previous research highlights the importance of GSTs in the establishment of chronic infections in cytotoxic microenvironments. Filarial nematodes depend on these detoxification enzymes for their survival in the host. GST plays an important role in filariasis and other diseases. GST from W.bancrofti and B.malayi are very much different from human GST. This structural difference makes GST potential chemotherapeutic targets for antifilarial treatment. In this study we have checked the efficacy of some well known antifilarial compounds against GST from B.malayi and W.bancrofti. The structure of BmGST was modeled using modeller9v10 and was submitted to PMDB. Molecular docking study reveals arbindazole to be the most potent compounds against GST from both the filarial parasites. Role of some residues playing important role in the binding of compounds within the active site of GST has also been revealed in the present study. The BmGST and WbGST structural information and docking studies could aid in screening new antifilarials or selective inhibitors for chemotherapy against filariasis.

Abbreviations

GST - Glutathione-S-transferase, Bm - Brugia malayi, Wb - Wuchereria bancrofti.     

Improving of Catalase Stability Properties by Encapsulation in Alginate/Fe3O4 Magnetic Composite Beads for Enzymatic Removal of H2O2

The aim of this study was enhancing of stability properties of catalase enzyme by encapsulation in alginate/nanomagnetic beads. Amounts of carrier (10–100 mg) and enzyme concentrations (0.25–1.5 mg/mL) were analyzed to optimize immobilization conditions. Also, the optimum temperature (25–50°C), optimum pH (3.0–8.0), kinetic parameters, thermal stability (20–70°C), pH stability (4.0–9.0) operational stability (0–390 min), and reusability were investigated for characterization of the immobilized catalase system. The optimum pH levels of both free and immobilized catalase were 7.0. At the thermal stability studies, the magnetic catalase beads protected 90% activity, while free catalase maintained only 10% activity at 70°C. The thermal profile of magnetic catalase beads was spread over a large area. Similarly, this system indicated the improving of the pH stability. The reusability, which is especially important for industrial applications, was also determined. Thus, the activity analysis was done 50 times in succession. Catalase encapsulated magnetic alginate beads protected 83% activity after 50 cycles.     

Enzymatic characterization of a serralysin-like metalloprotease from the entomopathogen bacterium, Xenorhabdus

We investigated the enzymatic properties of a serralysin-type metalloenzyme, provisionally named as protease B, which is secreted by Xenorhabdus bacterium, and probably is the ortholog of PrA peptidase of Photorhabdus bacterium. Testing the activity on twenty-two oligopeptide substrates we found that protease B requires at least three amino acids N-terminal to the scissile bond for detectable hydrolysis. On such substrate protease B was clearly specific for positively charged residues (Arg and Lys) at the P1 substrate position and was rather permissive in the others. Interestingly however, it preferred Ser at P1 in the oligopeptide substrate which contained amino acids also C-terminal to the scissile bond, and was cleaved with the highest k(cat)/K(M) value. The pH profile of activity, similarly to other serralysins, has a wide peak with high values between pH 6.5 and 8.0. The activity was slightly increased by Cu(2+) and Co(2+) ions, it was not sensitive for serine protease inhibitors, but it was inhibited by 1,10-phenanthroline, features shared by many Zn-metalloproteases. At the same time, EDTA inhibited the activity only partially even either after long incubation or in excess amount, and Zn(2+) was inhibitory (both are unusual among serralysins). The 1,10-phenanthroline inhibited activity could be restored with the addition of Mn(2+), Cu(2+) and Co(2+) up to 90-200% of its original value, while Zn(2+) was inefficient. We propose that both the Zn inhibition of protease B activity and its resistance to EDTA inhibition might be caused by an Asp in position 191 where most of the serralysins contain Asn.     

Phenotypic and genetic characterization of multidrug-resistant <Emphasis Type="Italic">Salmonella</Emphasis> Infantis strains isolated from broiler chicken meats in Turkey

Twenty Salmonella Infantis strains resistant against kanamycin, tetracycline, neomycin, spectinomycin, sulphonamide, nalidixic acid and trimethoprim were selected for this study out of 103 Salmonella strains isolated from broiler samples collected from several markets in the Bolu and Ankara regions of Turkey. The resistance genes aadA1, aphA1, sul1, tet(A), dfrA5/dfrA14 and gyrA were determined for these multidrug-resistant S. Infantis strains. S. Infantis strains contained a mega plasmid with the molecular size of 206 kb. The strains were divided into three groups according to the pulsed field gel electrophoresis patterns of XbaI digested chromosomal DNA. A Ser83→Tyr83 point mutation was found in the gyrA gene of all quinolone-resistant isolates. Filter mating experiments showed that 206 kb plasmid transferred nalidixic acid resistance associated with class I integrons.     

Zinc,Parity, Infection,and Severe Anemia Among Pregnant Women in Kassla,Eastern Sudan

The study was conducted to investigate determinants (clinical, nutritional, and nonnutritional factors) of anemia among pregnant women in Kassala, eastern Sudan. Sociodemographic characteristics were gathered; serum ferritin, zinc, albumin, and C-reactive protein were measured using different laboratory methods in a cross-sectional study of 250 pregnant women. Of the 250 women, 58.4% had anemia (hemoglobin (HB) <11 g/dl), 6.8% had severe anemia (HB < 7 g/dl), 19.6% had iron deficiency (S-ferritin <15 μg/l), 14.8% had iron deficiency anemia (<11 g/dl and S-ferritin <15 μg/l), and 38% had zinc deficiency (<80 μg/ml). S-albumin, zinc, and ferritin were significantly lower in patients with severe anemia. While age, gestational age, ferritin, and C-reactive protein were not predictors for anemia, primigravidae (OR = 2.7, 95% CI = 1.1–6.7, P = 0.02), low S-albumin (OR = 5.9, 95% CI = 1.4–25.2, P = 0.01), and low S-zinc (OR = 2.6, 95% CI = 1.0–6.6, P = 0.03) were the predictors for anemia. While there was no significant correlation between hemoglobin, S-zinc, and S-ferritin, there was a significant positive correlation between hemoglobin and S-albumin (r = 0.308, P = 0.001) and significant inverse correlation between hemoglobin and C-reactive protein (r = 0.169, P = 0.007). Thus, the role of chronic inflammation and zinc as possible contributing factors to anemia in pregnancy has important implications for the clinical evaluation and treatment of these women.