Effect of vitamin E and menadione supplementation on riboflavin production and stress parameters in Ashbya gossypii

Ashbya gossypii is a filamentous fungus which overproduces riboflavin as a pseudo-secondary metabolite. Vitamin E supplemented at 1, 2.5 and 5 μM levels in the growth medium of A. gossypii increased the extracellular secretion of riboflavin and at 50, 100 and 240 μM levels reduced the biomass and riboflavin yield. With 2.5 μM vitamin E total riboflavin production and extracellular riboflavin secretion on day 2 was higher than non-supplemented control. By day 3 the production in supplemented was nearly the same as in non-supplemented, but the intracellular riboflavin levels were lower and extracellular levels higher. Supplemented cells showed increased levels of catalase, glutathione peroxidase, lipid peroxides and membrane lipid peroxides, and decreased glutathione indicating that vitamin E, a well-known antioxidant, had acted as a pro-oxidant at low levels of 2.5 μM and had increased the oxidative stress. Menadione, a well known oxidant also increased riboflavin production and secretion at 1.0, 2.5 and 5.0 μM level. This is the first report were vitamin E and menadione effects support the concept that overproduction of riboflavin is a stress induced phenomenon. These findings are not only of scientific interest but also useful for improving the industrial production of riboflavin.     

Fast inactivation in potassium channels: An interplay of cytoplasmic domains

Fast inactivation in voltage-gated potassium channels has traditionally been associated exclusively with the N-terminus. Here, we explore the role of the T1 domain using a series of chimeric channels. A chimeric channel, 4N/2, (N-terminus from the rapidly inactivating hKv1.4, and the channel body from the non-inactivating hKv1.2), exhibited slower and incomplete inactivation as compared to the wild-type hKv1.4. Replacing the T1 domain of 4N2 with that from hKv1.2 (4N/2T1/2), restored inactivation, while that from hKv1.1 (4N/1T1/2) completely abolished inactivation. Based on these observations, we hypothesize a correlation between the tetramerization domain and the putative inactivation domain receptor in the process of rapid inactivation of hKv1 channels.     

Antimicrobial susceptibility patterns,emm type distribution and genetic diversity of Streptococcus pyogenes recovered in Brazil

Streptococcus pyogenes is responsible for a variety of infectious diseases and immunological complications. In this study, 91 isolates of S. pyogenes recovered from oropharynx secretions were submitted to antimicrobial susceptibility testing, emm typing and pulsed-field gel electrophoresis (PFGE) analysis. All isolates were susceptible to ceftriaxone, levofloxacin, penicillin G and vancomycin. Resistance to erythromycin and clindamycin was 15.4%, which is higher than previous reports from this area, while 20.9% of the isolates were not susceptible to tetracycline. The macrolide resistance phenotypes were cMLSB (10) and iMLSB (4). The ermB gene was predominant, followed by the ermA gene. Thirty-two emm types and subtypes were found, but five (emm1, emm4, emm12, emm22, emm81) were detected in 48% of the isolates. Three new emm subtypes were identified (emm1.74, emm58.14, emm76.7). There was a strong association between emm type and PFGE clustering. A variety of PFGE profiles as well as emm types were found among tetracycline and erythromycin-resistant isolates, demonstrating that antimicrobial resistant strains do not result from the expansion of one or a few clones. This study provides epidemiological data that contribute to the development of suitable strategies for the prevention and treatment of such infections in a poorly studied area.     

Association of genetic and non-genetic risk factors with the development of prostate cancer in Malaysian men

There is growing global interest to stratify men into different levels of risk to developing prostate cancer, thus it is important to identify common genetic variants that confer the risk. Although many studies have identified more than a dozen common genetic variants which are highly associated with prostate cancer, none have been done in Malaysian population. To determine the association of such variants in Malaysian men with prostate cancer, we evaluated a panel of 768 SNPs found previously associated with various cancers which also included the prostate specific SNPs in a population based case control study (51 case subjects with prostate cancer and 51 control subjects) in Malaysian men of Malay, Chinese and Indian ethnicity. We identified 21 SNPs significantly associated with prostate cancer. Among these, 12 SNPs were strongly associated with increased risk of prostate cancer while remaining nine SNPs were associated with reduced risk. However, data analysis based on ethnic stratification led to only five SNPs in Malays and 3 SNPs in Chinese which remained significant. This could be due to small sample size in each ethnic group. Significant non-genetic risk factors were also identified for their association with prostate cancer. Our study is the first to investigate the involvement of multiple variants towards susceptibility for PC in Malaysian men using genotyping approach. Identified SNPs and non-genetic risk factors have a significant association with prostate cancer.     

Fragments of Bacterial Endoglycosidase S and Immunoglobulin G Reveal Subdomains of Each That Contribute to Deglycosylation

Endoglycosidase S (EndoS) is a glycoside-hydrolase secreted by the bacterium Streptococcus pyogenes. EndoS preferentially hydrolyzes the N-linked glycans from the Fc region of IgG during infection. This hydrolysis impedes Fc functionality and contributes to the immune evasion strategy of S. pyogenes. Here, we investigate the mechanism of human serum IgG deactivation by EndoS. We expressed fragments of IgG1 and demonstrated that EndoS was catalytically active against all of them including the isolated CH2 domain of the Fc domain. Similarly, we sought to investigate which domains within EndoS could contribute to activity. Bioinformatics analysis of the domain organization of EndoS confirmed the previous predictions of a chitinase domain and leucine-rich repeat but also revealed a putative carbohydrate binding module (CBM) followed by a C-terminal region. Using expressed fragments of EndoS, circular dichroism of the isolated CBM, and a CBM-C-terminal region fusion revealed folded domains dominated by β sheet and α helical structure, respectively. Nuclear magnetic resonance analysis of the CBM with monosaccharides was suggestive of carbohydrate binding functionality. Functional analysis of truncations of EndoS revealed that, whereas the C-terminal of EndoS is dispensable for activity, its deletion impedes the hydrolysis of IgG glycans.     

Evidence for the presence of somatic mitochondrial DNA mutations in right atrial appendage tissues of coronary artery disease patients

Coronary artery disease (CAD) is a multifactorial disease with the underlying involvement of environment, life style and nuclear genetics. However, the role of extranuclear genetic material in terms of somatically acquired mutations in mitochondrial tRNA and protein coding genes in the initiation or progression of CAD is not well defined. Hence, in the present study, right atrial appendage tissues and matched blood samples of 150 CAD patients were screened for mutations in nucleotide regions encompassing the Cytochrome c oxidase subunit II (MT-CO2), tRNA lysine (MT-TK), ATP synthase F0 subunit 8 (MT-ATP8) and Cytochrome b (MT-CYB) genes of mitochondrial DNA. We have found 9 different somatic mutations in 6 % of the CAD patients. Out of these mutations, 4 each were localized in MT-TK gene (T8324A, A8326G, A8331G and A8344G) and MT-CYB genes (T15062C, C15238A, T15378G and C15491G) in addition to one mutation in non-coding region 7 (A8270T) of mitochondrial genome. In addition, we noticed that majority (85.3 %) of CAD patients showed double repeats of germ-line “CCCCCTCTA” intergenic sequence between MT-CO2 and MT-TK genes. Our in-silico investigations of missense mutations revealed that they may alter the free energy and stability of polypeptide chains of MT-CYB protein of complex III of mitochondrial respiratory chain. Based on our study findings, we hypothesize that the somatically acquired variations in MT-TK and MT-CYB genes may negatively impact the energy metabolism of cardiomyocytes in right atrial appendage tissues and contribute in the cardiac dysfunction among CAD patients. In conclusion, our findings may be likely to have potential implications in understanding the disease pathophysiology, diagnosis as well as for the better therapeutic management of CAD patients.     

Genetic characterization of the polycotyledon locus in tomato

Developmental mutants serve as a useful material to unravel the mechanisms necessary for organ development. The polycotyledon (poc) mutant of tomato, with multiple cotyledons in the seedling and varied phenotypic effects in the adult plant is one such mutant. Studies using physiological and anatomical methods in our lab suggest that POC is involved in the negative regulation of polar auxin transport, which is likely the reason for the pleiotropic phenotype in the mutant. Because of the physiological significance of the polycotyledon mutant described in this paper and also being first of its kind in tomato and also other plant species, we are using a map-based cloning approach to map the polycotyledon gene. Molecular mapping of this locus using segregating interspecific F₂ mapping population localized polycotyledon gene close to TG424 marker on the long arm of chromosome 9. The closest marker mapped was a PCR marker identified in this study, E8A2 at a distance of 7.4 cM from the poc locus. The absence of tightly linked RAPD markers and the non-availability of more mapped markers in this region led us to initiate chromosome walk to polycotyledon gene. Both the flanking markers TG248 and E8A2 were used to screen the BAC library and a contig was developed for TG248 marker. The BAC-end sequences were analyzed for their use as RFLP markers to enrich this region for markers. Analysis of the BAC-end sequences revealed that poc is localized in the region surrounded by copia-like retrotransposon elements explaining the absence of markers in the euchromatin region on long arm of chromosome 9. Further studies identified two BAC-end sequences which mapped around the poc locus and also indicated very low physical versus genetic distance ratio in this region. The double mutant analyses of poc with the other two known polycotyledon mutants of tomato, pct and dem revealed allelism with pct; therefore, the poc mutant was named as pct1-2, and also the original pct mutant was renamed as pct1-1.     

Identification of catalytic residues in the As(III) S-adenosylmethionine methyltransferase

The enzyme As(III) S-adenosylmethionine methyltransferase (EC 2.1.1.137) (ArsM or AS3MT) is found in members of every kingdom, from bacteria to humans. In these enzymes, there are three conserved cysteine residues at positions 72, 174, and 224 in the CmArsM orthologue from the thermophilic eukaryotic alga Cyanidioschyzon sp. 5508. Substitution of any of the three led to loss of As(III) methylation. In contrast, a C72A mutant still methylated trivalent methylarsenite [MAs(III)]. Protein fluorescence of a single-tryptophan mutant reported binding of As(III) or MAs(III). As(GS)(3) and MAs(GS)(2) bound significantly faster than As(III), suggesting that the glutathionylated arsenicals are preferred substrates for the enzyme. Protein fluorescence also reported binding of Sb(III), and the purified enzyme methylated and volatilized Sb(III). The results suggest that all three cysteine residues are necessary for the first step in the reaction, As(III) methylation, but that only Cys174 and Cys224 are required for the second step, methylation of MAs(III) to dimethylarsenite [DMAs(III)]. The rate-limiting step was identified as the conversion of DMAs(III) to trimethylarsine, and DMAs(III) accumulates as the principal product.     

Immunomodulatory effects of alcbholic and hydroalcoholic extracts of Moringa olifera Lam leaves

Effects of 50, 100 and 200 mg/kg body weight of the alcoholic and hydro-alcoholic extract of leaves of M. olifera were studied on various immune paradigms like delayed type hypersensitivity reaction using SRBC as an antigen, determination of antibody titer, neutrophil adhesion test as an indicator for neutrophil index, total leucocyte count in cyclophosphamide induced immunosuppressed animals and carbon clearance assay as a measure of phagocytic activity. Hydro-alcoholic extract of M. olifera substantially enhanced cellular immune response, humoral immune response, neutrophil index and phagoctic activity in doses of 100 and 200 mg/kg body weight. The ethanolic extract (200 mg/kg body weight) was efficient in improving immune response. The results suggest that M. olifera has a significant role to play as an immune stimulator.