PURIFICATION AND PROPERTIES OF AN AZO-REDUCTASE FROM BACILLUS SP.

Joint Research on Environmental Science and Technology for the Eart

DECOLORIZATION OF AZO DYES BY PURPLE NON-SULFUR BACTERIA

Joint Research on Environmental Science and Technology for the Eart

GENERATION OF BACILLUS SUBTILIS CLONE DISPLAYING METAL-BINDING POLYHISTIDYL PEPTIDE

Joint Research on Environmental Science and Technology for the Eart

Genetic Interaction Between Two VNTRs in the SLC6A4 Gene Regulates Nicotine Dependence in Vietnamese Men

Nicotine dependence is an addiction to tobacco products and a global public health concern. Association between the SLC6A4 polymorphisms and nicotine dependence is controversial. Two variable number tandem repeat (VNTR) domains, termed HTTLPR and STin2, in the SLC6A4 gene are well characterized transcriptional regulatory elements. Their polymorphism in the copy number of the repeat correlates with the particular personality and psychiatric traits. We analyzed nicotine dependence in 1,804 participants from Central Vietnam. The Fagerström Test (FTND) was used to evaluate the nicotine dependence and PCR was used to determine the SLC6A4 HTTLPR and STin2 VNTRs. The HTTLPR VNTR was associated with difficulties to refrain from smoking in a prohibiting environment. The STIn2 10/10 genotype was associated with (1) years of smoking, (2) difficulties to quit the first cigarette, and (3) higher number of cigarettes smoked per day (CPD). Stratification analysis was used to find the genetic interaction between these two VNTRs in nicotine dependence as they may synergistically regulate the SLC6A4 expression. Smokers with the S/S HTTLPR genotypes showed a much stronger association between STin2 10/10 variant and CPD. This finding is consistent with the molecular evidence for the functional interaction between HTTLPR and STin2 in cell line models, where STin2 has described as a stronger expressional regulator. Similarly, we found that STin2 is a much stronger modifier of smoking with 10/10 genotype related to higher nicotine dependence. The present study supports genetic interaction between HTTLPR and STin2 VNTRs in the regulation of nicotine dependence with the dominance of the STin2 effects. This finding could be explained by their differential effect on the SLC6A4 expression

Pichia pastoris versus Saccharomyces cerevisiae:a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor

BACKGROUND: Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker's yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts. RESULTS: Codon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1. CONCLUSION: Our data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production

Genetic Interaction Between Two VNTRs in the SLC6A4 Gene Regulates Nicotine Dependence in Vietnamese Men

Nicotine dependence is an addiction to tobacco products and a global public health concern. Association between the SLC6A4 polymorphisms and nicotine dependence is controversial. Two variable number tandem repeat (VNTR) domains, termed HTTLPR and STin2, in the SLC6A4 gene are well characterized transcriptional regulatory elements. Their polymorphism in the copy number of the repeat correlates with the particular personality and psychiatric traits. We analyzed nicotine dependence in 1,804 participants from Central Vietnam. The Fagerström Test (FTND) was used to evaluate the nicotine dependence and PCR was used to determine the SLC6A4 HTTLPR and STin2 VNTRs. The HTTLPR VNTR was associated with difficulties to refrain from smoking in a prohibiting environment. The STIn2 10/10 genotype was associated with (1) years of smoking, (2) difficulties to quit the first cigarette, and (3) higher number of cigarettes smoked per day (CPD). Stratification analysis was used to find the genetic interaction between these two VNTRs in nicotine dependence as they may synergistically regulate the SLC6A4 expression. Smokers with the S/S HTTLPR genotypes showed a much stronger association between STin2 10/10 variant and CPD. This finding is consistent with the molecular evidence for the functional interaction between HTTLPR and STin2 in cell line models, where STin2 has described as a stronger expressional regulator. Similarly, we found that STin2 is a much stronger modifier of smoking with 10/10 genotype related to higher nicotine dependence. The present study supports genetic interaction between HTTLPR and STin2 VNTRs in the regulation of nicotine dependence with the dominance of the STin2 effects. This finding could be explained by their differential effect on the SLC6A4 expression

Observation of the foreign protein expression in the yeast cell by using the enhanced cyan fluorescent protein

We have successfully constructed two plasmids pC-Z-Ste and pZ-C-Ste, which express a fusion protein containing the ECFP (Enhanced Cyan Fluorescent Protein) and the protein A encoded by the Z gene on the cytoplasmic side of the plasma membrane of Saccharomyces cerevisiae MT8-1. The oligonucleotide encoding the C-terminal 9 amino acids of the Ste18p protein was cloned downstream of the genes encoding the fusion proteins in order to anchor the fusion proteins on the cytoplasmic side of the plasma membrane. A GS linker was used between the fusion protein and the C-terminal of the Ste18p protein for minimizing the interaction among themselves. The Z gene was cloned upstream or downstream of the ECFP gene in order to examine whether the protein A at C terminal or N terminal of CFP affects the expression of the fusion protein. These two plasmids were sequenced to check the inframe between the gene ECFP, Z and the C terminal Ste. Under the control of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) promoter, the expression of the fusion proteins ECFP-Protein A or Protein A-ECFP in the yeast cell could be confirmed by the fluorescent microscope

Antibiotic Resistance in Food-Borne Bacterial Contaminants in Vietnam▿

This study was conducted to examine the rate of contamination and the molecular characteristics of enteric bacteria isolated from a selection of food sources in Vietnam. One hundred eighty raw food samples were tested; 60.8% of meat samples and 18.0% of shellfish samples were contaminated with Salmonella spp., and more than 90% of all food sources contained Escherichia coli. The isolates were screened for antibiotic resistance against 15 antibiotics, and 50.5% of Salmonella isolates and 83.8% of E. coli isolates were resistant to at least one antibiotic. Isolates were examined for the presence of mobile genetic elements conferring antibiotic resistance. Fifty-seven percent of E. coli and 13% of Salmonella isolates were found to contain integrons, and some isolates contained two integrons. Sequencing results revealed that the integrons harbored various gene cassettes, including aadA1, aadA2, and aadA5 (resistance to streptomycin and spectinomycin), aacA4 (resistance to aminoglycosides), the dihydrofolate reductase gene cassettes dhfrXII, dfrA1, and dhfrA17 (trimethoprim resistance), the beta-lactamase gene blaPSE1 (ampicillin resistance), and catB3 (chloramphenicol resistance). Plasmids were also detected in all 23 antibiotic-resistant Salmonella isolates and in 33 E. coli isolates. Thirty-five percent of the Salmonella isolates and 76% of the E. coli isolates contained plasmids of more than 95 kb, and some of the isolates contained two large plasmids. Conjugation experiments showed the successful transfer of all or part of the antibiotic resistance phenotypes among the Salmonella and E. coli food isolates. Our results show that enteric bacteria in raw food samples from Vietnam contain a pool of mobile genetic elements and that the transfer of antibiotic resistance can readily occur between similar bacteria

A study on using strong promoter Pgrac212 to enhance the secretional expression of reporter alpha-amylase in Bacillus subtilis

Bacillus subtilis has been a model for research on Gram-positive bacteria for more than four decades. It is a non-pathogenic bacterium and is conferred the GRAS (Generally Recognized As Safe) status by FDA. B. subtilis has also been widely used in the production of industrial proteins because of its high secretion capacity into the culture medium. To enhance the utility of B. subtilis in protein expression system, the pHT vectors with strong promoter Pgrac were constructed. This promoter is based on the strong σA-dependent promoter preceding the groELS operon of B. subtilis fused with the lac operator (lacO) of Escherichia coli, which allows a target protein to be expressed up to 16% of total intracellular proteins. In the previous study, amyQ gene encoding reporter α-amylase (AmyQ) from Bacillus amyloliquefaciens fused with Pgrac promoter to construct the secretional expression plasmid pHT43-amyQ (Pgrac-amyQ) and expressed fairly good in B. subtilis. In this study, the promoter-Pgrac212 was selected from a library of 80 promoters to construct pHT1200 (Pgrac212-amyQ) with the aim of investigating the enhancement of the α-amylase expression. The results showed that Pgrac212-amyQ secreted α-amylase more effectively than Pgrac-amyQ in B. subtilis 1012 and B. subtilis WB800N, an extracellular protease-deficient strain. This report demonstrates that Pgrac212 is a strong promoter that can be used to produce other secretional proteins in B. subtilis

Melanogenesis Inhibitory Effects of Ethanol Extract of Perilla frutescens’s Leaves on B16 Melanoma Cells: Perilla frutescens’s Leaves Melanogenesis Inhibitory Effects

Perilla frutescens (L.) Britt. is a Vietnamese herb containing many polyphenol compounds potential on antioxidants and tyrosinase inhibitors for cosmetic applications. Natural compounds and products have got more attention as they have less undesirable side effects and satisfy the diverse needs of consumers that include anti-melanogenesis, antioxidation, etc. This study evaluated the antioxidant capacity, inhibition of melanin synthesis in B16 melanoma cells of perilla leaves ethanol extract (PEE). PEE showed the high antioxidant properties in vitro by 2, 2′-diphenyl-1-picrylhydrazyl radical assay (DPPH) and Ferric reducing antioxidant power (FRAP). In addition, PEE has inhibitory activities for mushroom tyrosinase (TYR). The results showed that concentrations of 100 - 300 μg/mL of PEE were not affected on B16 cell viability. In B16 cells, melanin content and cellular tyrosinase activity were significantly decreased in PEE-treated cells in a dose-dependent manner. These results suggested that Perilla frutescens (L.) Britt. had a great potential as natural sources which could be used as agents in skincare products