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1.
Article in English | WPRIM | ID: wpr-887737

ABSTRACT

Objective@#To evaluate multidrug resistant loop-mediated isothermal amplification (MDR-LAMP) assay for the early diagnosis of multidrug-resistant tuberculosis and to compare the mutation patterns associated with the @*Methods@#MDR-LAMP assay was evaluated using 100 @*Results@#The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MDR-LAMP were 85.5%, 93.6%, 96.7%, and 74.4% for the detection of resistance to isoniazid and rifampicin, respectively, and 80.5%, 92.3%, 98.6%, and 41.4% for the detection of @*Conclusion@#MDR-LAMP is a rapid and accessible assay for the laboratory identification of rifampicin and isoniazid resistance of


Subject(s)
Antitubercular Agents , Bacterial Proteins/genetics , Catalase/genetics , DNA, Bacterial/analysis , DNA-Directed RNA Polymerases/genetics , Drug Resistance, Multiple, Bacterial/genetics , Isoniazid , Molecular Diagnostic Techniques/methods , Mutation , Mycobacterium tuberculosis/isolation & purification , Nucleic Acid Amplification Techniques/methods , Oxidoreductases/genetics , Phenotype , Rifampin , Whole Genome Sequencing
2.
Electron. j. biotechnol ; 25: 1-8, ene. 2017. graf, ilus
Article in English | LILACS | ID: biblio-1008175

ABSTRACT

Background: Ascorbic acid (Asc) is one of the most abundant antioxidants and it serves as a major contributor to protect plants against oxidative damage. Plants use two enzymes that participate in the metabolic recycling of Asc. One of these two enzymes is dehydroascorbate reductase (DHAR). It directly regenerates Asc from its oxidized state and thus prevents Asc from being irreversibly hydrolyzed to 2, 3-diketogulonic acid. This study aimed to examine whether over-expression of DHAR leads to an enhanced oxidative stress tolerance in tobacco plants. Results: In this study, we functionally characterized a novel JcDHAR gene from Jatropha curcas and found via quantitative RT-PCR analysis that JcDHAR can be induced with H2O2, salt and PEG stresses. The DHAR activities of transgenic tobacco plants increased from 2.0 to 5.3 fold compared to wild-type plants. As a result, the transgenic plants displayed enhanced tolerance to oxidative stress. Conclusions: Our results indicate that JcDHAR expression can effectively enhance the tolerance to oxidative stress in plants.


Subject(s)
Oxidoreductases/metabolism , Ascorbic Acid/administration & dosage , Tobacco/enzymology , Plants, Genetically Modified/enzymology , Antioxidants/administration & dosage , Oxidoreductases/isolation & purification , Oxidoreductases/genetics , Ascorbic Acid/metabolism , Stress, Physiological , Tobacco/drug effects , Blotting, Western , Plants, Genetically Modified/drug effects , Reactive Oxygen Species , Oxidative Stress , Reverse Transcriptase Polymerase Chain Reaction , Salt Tolerance , Antioxidants/metabolism
3.
Electron. j. biotechnol ; 18(3): 221-230, May 2015. ilus, graf, tab
Article in English | LILACS | ID: lil-750651

ABSTRACT

Background In this study, the detection of nifH and nifD by a polymerase chain reaction assay was used to screen the potential photosynthetic bacteria capable of producing hydrogen from five different environmental sources. Efficiency of photo-hydrogen production is highly dependent on the culture conditions. Initial pH, temperature and illumination intensity were optimized for maximal hydrogen production using response surface methodology with central composite design. Results Rhodobacter sp. KKU-PS1 (GenBank Accession No. KC478552) was isolated from the methane fermentation broth of an UASB reactor. Malic acid was the favored carbon source while Na-glutamate was the best nitrogen source. The optimum conditions for simultaneously maximizing the cumulative hydrogen production (Hmax) and hydrogen production rate (Rm) from malic acid were an initial of pH 7.0, a temperature of 25.6°C, and an illumination intensity of 2500 lx. Hmax and Rm levels of 1264 ml H2/l and 6.8 ml H2/L-h were obtained, respectively. The optimum initial pH and temperature were further used to optimize the illumination intensity for hydrogen production. An illumination intensity of 7500 lx gave the highest values of Hmax (1339 ml H2/l) and Rm (12.0 ml H2/L-h) with a hydrogen yield and substrate conversion efficiency of 3.88 mol H2/mol malate and 64.7%, respectively. Conclusions KKU-PS1 can produce hydrogen from at least 8 types of organic acids. By optimizing pH and temperature, a maximal hydrogen production by this strain was obtained. Additionally, by optimizing the light intensity, Rm was increased by approximately two fold and the lag phase of hydrogen production was shortened.


Subject(s)
Oxidoreductases/metabolism , Rhodobacter/metabolism , Nitrogenase/metabolism , Oxidoreductases/genetics , Temperature , Polymerase Chain Reaction , Rhodobacter/isolation & purification , Bioreactors , Fermentation , Hydrogen/metabolism , Hydrogen-Ion Concentration , Nitrogenase/genetics
4.
Mem. Inst. Oswaldo Cruz ; 109(3): 307-314, 06/2014. tab
Article in English | LILACS | ID: lil-711730

ABSTRACT

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.


Subject(s)
Bacterial Proteins/genetics , Catalase/genetics , Drug Resistance, Multiple, Bacterial/genetics , Mutation/genetics , Mycobacterium tuberculosis/genetics , Oxidoreductases/genetics , Colorimetry , DNA, Bacterial/genetics , Genotyping Techniques , Isoniazid/pharmacology , Multiplex Polymerase Chain Reaction , Mycobacterium tuberculosis/drug effects , Nucleic Acid Hybridization , Rifampin/pharmacology
5.
Braz. j. microbiol ; 44(4): 1279-1283, Oct.-Dec. 2013. tab
Article in English | LILACS | ID: lil-705268

ABSTRACT

Mercury-resistant Aeromonas strains isolated from diarrhea were studied. Resistance occurs via mercuric ion reduction but merA and merR genes were only detected in some strains using PCR and dot hybridization. Results indicate a high variability in mer operons in Aeromonas. To our knowledge, this is the first report of mercury-resistant clinical Aeromonas strains.


Subject(s)
Humans , Aeromonas/drug effects , Drug Resistance, Bacterial , Diarrhea/microbiology , Gram-Negative Bacterial Infections/microbiology , Mercury/toxicity , Aeromonas/isolation & purification , Bacterial Proteins/genetics , Mercury/metabolism , Nucleic Acid Hybridization , Oxidation-Reduction , Oxidoreductases/genetics , Polymerase Chain Reaction , Transcription Factors/genetics
6.
Mem. Inst. Oswaldo Cruz ; 107(7): 903-908, Nov. 2012. tab
Article in English | LILACS | ID: lil-656047

ABSTRACT

Mycobacterium tuberculosis is the bacterium that causes tuberculosis (TB), a leading cause of death from infectious disease worldwide. Rapid diagnosis of resistant strains is important for the control of TB. Real-time polymerase chain reaction (RT-PCR) assays may detect all of the mutations that occur in the M. tuberculosis 81-bp core region of the rpoB gene, which is responsible for resistance to rifampin (RIF) and codon 315 of the katG gene and the inhA ribosomal binding site, which are responsible for isoniazid (INH). The goal of this study was to assess the performance of RT-PCR compared to traditional culture-based methods for determining the drug susceptibility of M. tuberculosis. BACTEC TM MGIT TM 960 was used as the gold standard method for phenotypic drug susceptibility testing. Susceptibilities to INH and RIF were also determined by genotyping of katG, inhA and rpoB genes. RT-PCR based on molecular beacons probes was used to detect specific point mutations associated with resistance. The sensitivities of RT-PCR in detecting INH resistance using katG and inhA targets individually were 55% and 25%, respectively and 73% when combined. The sensitivity of the RT-PCR assay in detecting RIF resistance was 99%. The median time to complete the RT-PCR assay was three-four hours. The specificities for tests were both 100%. Our results confirm that RT-PCR can detect INH and RIF resistance in less than four hours with high sensitivity.


Subject(s)
Humans , Antitubercular Agents/pharmacology , Isoniazid/pharmacology , Mycobacterium tuberculosis/drug effects , Rifampin/pharmacology , Bacterial Proteins/genetics , Catalase/genetics , DNA, Bacterial/genetics , Drug Resistance, Bacterial/genetics , Mutation , Microbial Sensitivity Tests/methods , Mycobacterium tuberculosis/genetics , Oxidoreductases/genetics , Real-Time Polymerase Chain Reaction
7.
Electron. j. biotechnol ; 15(4): 2-2, July 2012. ilus, tab
Article in English | LILACS | ID: lil-646952

ABSTRACT

Background: For successful in vitro plant regeneration, plant cell lines with multiple transgene integration and low transgene expression levels need to be ruled out. Although real-time polymerase chain reaction (RT-PCR) is a rapid way to accomplish this, it is also expensive and typically limits the size of the target sequence. Quantitative competitive PCR (QC-PCR) is proven to be a safe and accurate method for determination of both copy number and quantification of transcript levels of synthetic transgenes in transformed plants. Results: The glyphosate oxidoreductase genewas chemically synthesized and used to transform Brassica napus L. via Agrobactrium-mediated transformation. A construct containing the mutated form of a synthetic glyphosate oxidoreductase (gox) gene (internal standard) was prepared. Gene copy number was estimated in nine independent transgenic lines using QC-PCR as well as the standard method of Southern blot analysis. By quantitative RT-PCR, transcript levels were also determined in these lines. High (> 3), medium to high (2.2-3), medium to low (1-2.2), and low (< 1) levels of transcript were detected. Conclusions: No direct relationship was found between copy number and transgene expression levels. QC-PCR method could be implemented to screen putative transgenic plants and quickly select single T-DNA inserts. QC-PCR methods and the prepared competitor construct may be useful for future quantification of commercial transgenic food and feed.


Subject(s)
Brassica napus/enzymology , Brassica napus/genetics , Glycine/genetics , Oxidoreductases/genetics , Polymerase Chain Reaction/methods , Glycine/analogs & derivatives , Glycine/physiology , Oxidoreductases/physiology , Transcription, Genetic , Transgenes
8.
Braz. j. infect. dis ; 16(1): 57-62, Jan.-Feb. 2012. ilus, tab
Article in English | LILACS | ID: lil-614551

ABSTRACT

OBJECTIVE: Isoniazid (INH) and rifampin (RIF) are the most effective first line antibiotics against Mycobacterium tuberculosis. Mutations in several genes determine resistance of M. tuberculosis to INH, with the most common gene target of katG, and resistance to RIF is due to mutation in rpoB gene. The aim of present study was to assess the mutations in the regions related to RIF and INH resistance. METHODS: We characterized 80 clinical isolates of confirmed M. tuberculosis to analyze the most commonly observed INH and RIF mutations. PCR analysis and sequencing were used to detect mutations related to RIF and INH resistance. The multiplex allele-specific-PCR (MAS-PCR) was performed as a comparative assay and for evaluation of this method. RESULTS: The sequencing of the 250-bp region of katG codon 315, revealed point mutations at 5 different codons in 13.7 percent of the M. tuberculosis isolates. The sequencing of the 270-bp central region of the rpoB gene revealed point mutations at 7 different codons in 12 (15 percent) of the M. tuberculosis isolates. The results obtained with MAS-PCR are in accordance with PCR-sequencing with high sensitivity and specificity for katG315, inhA15, and rpoB (531, 516, 526). CONCLUSION: The results of this study suggested that molecular techniques can be used as a rapid tool for the identification of drug resistance in clinical isolates of M. tuberculosis. Both DNA sequencing and MAS-PCR yielded high sensitivity for the detection of RIF and INH mutations and detecting multi-drug resistant tuberculosis cases.


Subject(s)
Humans , Bacterial Proteins/genetics , Catalase/genetics , Drug Resistance, Bacterial/genetics , Mycobacterium tuberculosis/genetics , Oxidoreductases/genetics , Point Mutation/genetics , Alleles , Antitubercular Agents/pharmacology , Isoniazid/pharmacology , Multiplex Polymerase Chain Reaction/methods , Mycobacterium tuberculosis/drug effects , Rifampin/pharmacology , Sequence Analysis, DNA
9.
Mem. Inst. Oswaldo Cruz ; 106(6): 655-661, Sept. 2011.
Article in English | LILACS | ID: lil-602047

ABSTRACT

Drug resistance is one of the major concerns regarding tuberculosis (TB) infection worldwide because it hampers control of the disease. Understanding the underlying mechanisms responsible for drug resistance development is of the highest importance. To investigate clinical data from drug-resistant TB patients at the Tropical Diseases Hospital, Goiás (GO), Brazil and to evaluate the molecular basis of rifampin (R) and isoniazid (H) resistance in Mycobacterium tuberculosis. Drug susceptibility testing was performed on 124 isolates from 100 patients and 24 isolates displayed resistance to R and/or H. Molecular analysis of drug resistance was performed by partial sequencing of the rpoB and katGgenes and analysis of the inhA promoter region. Similarity analysis of isolates was performed by 15 loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing. The molecular basis of drug resistance among the 24 isolates from 16 patients was confirmed in 18 isolates. Different susceptibility profiles among the isolates from the same individual were observed in five patients; using MIRU-VNTR, we have shown that those isolates were not genetically identical, with differences in one to three loci within the 15 analysed loci. Drug-resistant TB in GO is caused by M. tuberculosis strains with mutations in previously described sites of known genes and some patients harbour a mixed phenotype infection as a consequence of a single infective event; however, further and broader investigations are needed to support our findings.


Subject(s)
Adult , Female , Humans , Male , Middle Aged , Young Adult , Antitubercular Agents/pharmacology , Drug Resistance, Bacterial/genetics , Isoniazid/pharmacology , Mycobacterium tuberculosis/genetics , Rifampin/pharmacology , Tuberculosis, Multidrug-Resistant/microbiology , Bacterial Proteins/genetics , Catalase/genetics , DNA, Bacterial/genetics , Mutation/genetics , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/isolation & purification , Oxidoreductases/genetics , Phenotype , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length
10.
Clinics ; 66(8): 1361-1366, 2011. tab
Article in English | LILACS | ID: lil-598376

ABSTRACT

INTRODUCTION: 21-hydroxylase deficiency is an autosomal recessive disorder that causes glucocorticoid deficiency and increased androgen production. Treatment is based on glucocorticoid replacement; however, interindividual variability in the glucocorticoid dose required to achieve adequate hormonal control has been observed. OBJECTIVE: The present study aimed to evaluate the association between polymorphic variants involved inglucocorticoid action and/or metabolism and the mean daily glucocorticoid dose in 21-hydroxylase deficiency patients. METHODS: We evaluated 53 patients with classical forms of 21-hydroxylase deficiency who were receiving cortisone acetate. All patients were between four and six years of age and had normal androgen levels. RESULTS: The P450 oxidoreductase A503V, HSD11B1 rs12086634, and CYP3A7*1C variants were found in 19 percent, 11.3 percent and 3.8 percent of the patients, respectively. The mean ± SD glucocorticoid dose in patients with the CYP3A7*1C and wild-type alleles was 13.9 ± 0.8 and 19.5 ± 3.2 mg/m²/d, respectively. We did not identify an association between the P450 oxidoreductase or HSD11B1 allelic variants and the mean glucocorticoid dose. CONCLUSION: Patients carrying the CYP3A7*1C variant required a significantly lower mean glucocorticoid dose. Indeed, the CYP3A7*1C allele accounted for 20 percent of the variability in the cortisone acetate dose. The analysis of genes involved in glucocorticoid metabolism may be useful in the optimization of treatment of 21-hydroxylase deficiency.


Subject(s)
Child , Child, Preschool , Female , Humans , Infant , Male , Adrenal Hyperplasia, Congenital/drug therapy , Adrenal Hyperplasia, Congenital/genetics , Cortisone/analogs & derivatives , Glucocorticoids/administration & dosage , Oxidoreductases/genetics , Polymorphism, Genetic , Adrenal Hyperplasia, Congenital/enzymology , Cortisone/administration & dosage , Hormone Replacement Therapy
11.
Article in English | WPRIM | ID: wpr-170199

ABSTRACT

BACKGROUND: In Korea, tuberculosis is resistant to isoniazid (INH) and/or rifampicin (RIF) in more than 10% of cases. To prevent the spread of resistant Mycobacterium tuberculosis strains, it is crucial to develop more rapid resistance detection methods. METHODS: To determine the feasibility of using direct sequencing for detecting INH- and RIF-resistant strains, the katG gene, the regulatory region of the inhA gene, and the 81-bp hot-spot region of the rpoB gene from 95 culture isolates and 46 respiratory specimens were sequenced. Total 141 culture isolates were classified by conventional drug susceptibility testing (DST) as INH(R)/RIF(R) (N=30), INH(R)/RIF(S) (N=23), INH(S)/RIF(R) (N=15), and INH(S)/RIF(S) (N=73). RESULTS: Compared with phenotypic DST, the overall sensitivity and specificity of sequencing were 83.0% (44/53) and 96.6% (85/88), respectively, for INH resistance, and 93.3% (42/45) and 100% (96/96), respectively, for RIF resistance. The rates were similar between culture isolates and respiratory specimens. Interestingly, three specimens with inhA -15C>T mutation were susceptible to INH by conventional DST. CONCLUSIONS: Detection of mutations in the katG codon 315, the inhA regulatory region, and the hot-spot region of rpoB would be useful for rapid detection of INH and RIF resistance in Korea.


Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/genetics , Catalase/genetics , Drug Resistance, Multiple, Bacterial , Genotype , Humans , Isoniazid/pharmacology , Mutation , Mycobacterium tuberculosis/genetics , Oxidoreductases/genetics , Republic of Korea , Rifampin/pharmacology , Sensitivity and Specificity , Sequence Analysis, DNA/methods
12.
KOOMESH-Journal of Semnan University of Medical Sciences. 2008; 9 (2): 99-104
in Persian | IMEMR | ID: emr-88593

ABSTRACT

Biological desulforezation is a microbial procedure which uses manipulated bacterial strains for increasing of desulforezation from petroleum products. The method is being tested by several big oil companies for making a safe fuel and for preventing air pollution one of the methods for enhancement of desulforezation of microbial oil strains is increasing of copy number of oxidoreductase gene, this gene is able to supply the required FMNH2 for desulforezation genes of Rhodococcus erythropolis IGTS8, which is used as a model micro organism in desulforezation researches, was amplified with PCR and was cloned in to the PTZ57R vector. Then the oxidoreductase gene was extracted by agarose gel DNA extraction kit as a 600 bp fragment and was inserted in to the pkk223-3 expression vector. After induction, the desired protein was weakly detected as a 25 KD band in SDS-PAGE gel. However using another expression vector [PET 21a], a sharp band was detected in the same area. At present, the studies was focused on increasing the production in National Instutute of genetic Engineering and bio technology we are trying to apply genetic Engineering tools to increase gene dosage of oxidoreductase gene to enhancement of desulforezation gene expression


Subject(s)
Air Pollution , Oxidoreductases/genetics , Rhodococcus
13.
An. acad. bras. ciênc ; 79(4): 649-663, Dec. 2007. ilus, graf
Article in English | LILACS | ID: lil-470038

ABSTRACT

Trypanosoma cruzi is highly sensitive to oxidative stress caused by reactive oxygen species. Trypanothione, the parasite's major protection against oxidative stress, is kept reduced by trypanothione reductase, using NADPH; the major source of the reduced coenzyme seems to be the pentose phosphate pathway. Its seven enzymes are present in the four major stages in the parasite's biological cycle; we have cloned and expressed them in Escherichia coli as active proteins. Glucose 6-phosphate dehydrogenase, which controls glucose flux through the pathway by its response to the NADP/NADPH ratio, is encoded by a number of genes per haploid genome, and is induced up to 46-fold by hydrogen peroxide in metacyclic trypomastigotes. The genes encoding 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase and transketolase are present in the CL Brener clone as a single copy per haploid genome. 6-phosphogluconate dehydrogenase is very unstable, but was stabilized introducing two salt bridges by site-directed mutagenesis. Ribose-5-phosphate isomerase belongs to Type B; genes encoding Type A enzymes, present in mammals, are absent. Ribulose-5-phosphate epimerase is encoded by two genes. The enzymes of the pathway have a major cytosolic component, although several of them have a secondary glycosomal localization, and also minor localizations in other organelles.


Trypanosoma cruzi é altamente sensível ao estresse oxidativo causado por espécies reativas do oxigênio. Tripanotiona, o principal protetor do parasita contra o estresse oxidativo, é mantido reduzido pela tripanotiona redutase, pela presença deNADPH; a principal fonte da coenzima reduzida parece ser a via da pentose fosfato. As sete enzimas dessa via estão presentes nos quatro principais estágios do ciclo biológico do parasita; nós clonamos e expressamos as enzimas em Escherichia coli como proteínas ativas. Glucose 6-fosfato desidrogenase, que controla o fluxo da glucose da via em resposta à relação NADP/NADPH, é codificada por um número de genes por genoma haplóide e é induzida até 46-vezes por peróxido de hidrogênio em trypomastigotas metacíclicos. Os genes que codificam 6-fosfogluconolactonase, 6-fosfogluconato desidrogenase, transaldolase e transcetolase estão presentes no clone CL Brener como cópia única por genoma haplóide. 6-fosfogluconato desidrogenase é muito instável, mas foi estabilizada introduzindo duas pontes salinas por mutagênese sítio-dirigida. A Ribose-5-fosfato isomerase pertence ao Tipo B; genes que codificam enzimas Tipo A, presentes em mamíferos estão ausentes. A Ribulose-5-fosfato epimerase é codificada por dois genes. As enzimas da via têm um componente citosólico principal, embora várias delas tenham uma localização glicosomal secundária e também, localizações em menor número em outras organelas.


Subject(s)
Animals , Pentose Phosphate Pathway/genetics , Trypanosoma cruzi/enzymology , Amino Acid Sequence , Aldehyde-Ketone Transferases/genetics , Aldehyde-Ketone Transferases/metabolism , Chagas Disease/drug therapy , Hydrolases/genetics , Hydrolases/metabolism , Isomerases/genetics , Isomerases/metabolism , Molecular Sequence Data , Oxidation-Reduction , Oxidoreductases/genetics , Oxidoreductases/metabolism , Sequence Alignment , Trypanosoma cruzi/genetics
14.
Article in English | IMSEAR | ID: sea-46897

ABSTRACT

Bacterial plasmids encode resistance systems for toxic metal ions including Hg2+ functioning by energy-dependent efflux of toxic ions. The inducible mercury resistance (mer) operon encodes both a mercuric ion uptake and a detoxification enzymes. In Gram-negative bacteria especially in E. coli, a periplasmic protein, MerP, an inner- membrane transport protein, MerT, and a cytoplasmic enzyme, mercuric reductase (the MerA protein), are responsible for the transport of mercuric ions into cell and their reduction to elemental mercury, Hg0. Phytoremediation involves the use of plants to extract, detoxify and/or sequester environmental pollutants from soil and water. Transgenic plants cleave mercury ions from methyl-mercury complexes; reduce mercury ions to the metallic form; take up metallic mercury through their roots; and evolve less toxic elemental mercury. PCR were performed to detect 1695 bp of mercuric reductase gene (merA), which is mainly responsible for the conversion of mercuric (Hg+2) and mercurous (Hg+1) ions into non-toxic elemental mercury. PCR products of putative merA genes from environmental E. coli strains were purified and cloned into a plant expression vector pRT100. The construct will be transformed in calli of Nicotiana plants.


Subject(s)
Bacteriocin Plasmids/genetics , Escherichia coli/drug effects , Gene Amplification , Humans , Mercury Compounds/analysis , Oxidoreductases/genetics , Phytotherapy , Pilot Projects , Soil Microbiology , Tobacco/genetics , Water Microbiology
15.
Article in English | IMSEAR | ID: sea-32239

ABSTRACT

A total of 29 Thai multi-drug-resistant/isoniazid-resistant Mycobacterium tuberculosis isolates were analyzed for mutations in katG from codons 254 to 549, inhA promoter and inhA open reading frame by DNA sequencing and single strand conformation polymorphism. Twenty-five multi-drug resistant isolates exhibited single point mutations (17 isolates at Ser315Thr plus Arg463Leu, 1 at Thr308Pro plus Arg463Leu, 7 at either Ser315Thr or Arg463Leu) while the other 4 isoniazid-resistant isolates had single point mutation only at Arg463Leu. Seven of 25 multi-drug-resistant isolates [4 at C(-15)T, 1 at T(-8)C; 1 at C(-15)T plus Ser94Ala and 1 at Ile21Val] and 2 of 4 isoniazid-resistant isolates [1 at C(-15)T, 1 at C (-15)T plus Ile21Thr] had mutations in inhA promoter and open reading frame, while the other 20 isolates had no mutation at any position. No frame shift mutation was observed in any tested isolates. This is the first report of two mutations, Trp308Pro of katG and T (-8)C of inhA in Mycobacterium tuberculosis isolates.


Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/genetics , Catalase/genetics , DNA Primers , Humans , Isoniazid/pharmacology , Mutation/genetics , Mycobacterium tuberculosis/drug effects , Oxidoreductases/genetics , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Thailand , Tuberculosis, Multidrug-Resistant/drug therapy
16.
J Environ Biol ; 2007 Jan; 28(1): 83-6
Article in English | IMSEAR | ID: sea-113847

ABSTRACT

Distinct morphophysiological variations observed for over 2 years with-in short distances among four perennial plants indicated genetic diversity among the lines growing at three places. The isozyme and SDS polyacrylamide gel banding patterns as genetic markers were used to investigate four perennial species, namely Dalbergia sissoo Roxb., Delonix regia (Boj.) Refin., Cassia fistula L. and Calotropis procera R. Br. Plant materials collected from three locations (Agra, Gwalior and Lucknow) differing in climo-edaphic variables were examined for 4 enzyme systems, viz., esterase, polyphenol oxidase, peroxidase and superoxide dismutase (EST, PPO, PRX and SOD). Among the four isozymes SOD and PRX revealed best discriminating power. Protein banding patterns as well as zymogram revealed that Dalbergia sissoo growing at Gwalior was closer to that of Agra; Delonix regia depicted highest similarity between Lucknow and Agra and Calotropis procera of Lucknow location was more closer to Gwalior than Agra. The results confirm genetic diversity in the species as a means of adaptation to differing climo-edaphic variables.


Subject(s)
Alleles , Apocynaceae/enzymology , Catechol Oxidase/genetics , Ecosystem , Fabaceae/enzymology , Genetic Variation , Genotype , India , Isoenzymes/genetics , Oxidoreductases/genetics , Plant Proteins/genetics
17.
Article in English | WPRIM | ID: wpr-634240

ABSTRACT

To examine the deletion and point mutation of WWOX (WW domain containing oxidoreductase) exons 6-8 in human non-small cell lung cancer and their possible relationship with pathological stages, tumor tissues and the corresponding normal tissues were obtained from 44 Chinese patients who had undergone surgery for non-small cell lung cancer. RNA was extracted from each sample and deletion and mutation of WWOX exons 6-8 were analyzed by RT-PCR and DNA sequencing. Our results showed that 28 of 44 (63.6%) lung cancer samples showed loss of WWOX exons 6-8 transcript and the deletion was detected in only 3 of 44 (6.8%) corresponding adjacent normal tissues (P < 0.05). The transcript sequencing analyses of the 16 lung cancer samples without transcript loss of WWOX exons 6-8 revealed no difference from the sequence of GenBank. Moreover, the deletion of WWOX exons 6-8 was significantly higher in the smokers when compared with the non-smokers. It is also higher in the men and squamous carcinomas than in women and adenocarcinomas (P < 0. 05). The deletion, however, was not found to be associated with pathological stages of the tumors. Our study documented a high incidence of deletion of WWOX exons 6-8 in non-small cell lung cancer in Chinese patients and suggested that the frequent loss of WWOX exons 6-8 might play an important role in the tumorigenesis of non-small cell lung cancer in Chinese. WWOX exons 6-8 may serves as a candidate molecular target of smoking carcinogenesis, and point mutation is not a predominant way of alteration of WWOX exons 6-8.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Exons/genetics , Gene Deletion , Loss of Heterozygosity , Lung Neoplasms/genetics , Oxidoreductases/genetics , Point Mutation , Sequence Analysis, DNA , Tumor Suppressor Proteins
18.
J Biosci ; 2002 Mar; 27(2): 105-12
Article in English | IMSEAR | ID: sea-111031

ABSTRACT

The human gene TM7SF2 encodes a polypeptide (SR-1) with high sequence similarity to sterol C-14 reductase, a key sterol biosynthetic enzyme in fungi, plants and mammals. In Neurospora and yeast this enzyme is encoded by the erg-3 and erg24 genes respectively. In an effort to demonstrate sterol C-14 reductase activity for SR-1 we constructed six recombinant genes coding for chimeras of the Neurospora erg-3 and SR-1 protein sequences and tested them for complementation of the Neurospora erg-3 mutant. To our surprise, all the chimeras failed to complement erg-3. A few of the chimeric proteins were also tested against the yeast erg24 mutant, but again there was no complementation. We discuss some reasons that might account for these unexpected findings.


Subject(s)
Amino Acid Sequence , Animals , Genetic Complementation Test , Humans , Membrane Proteins/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Neurospora crassa/enzymology , Oxidoreductases/genetics , Oxidoreductases Acting on CH-CH Group Donors , Recombinant Fusion Proteins/genetics , Sequence Alignment
19.
Indian J Exp Biol ; 1999 Aug; 37(8): 787-92
Article in English | IMSEAR | ID: sea-55910

ABSTRACT

A partial genomic library was prepared in E. coli JM109 using pBR322 as vector and 2.4 kb Sau 3A I chromosomal fragment, encoding a nitroaryl reductase (nbr A) gene, from Streptomyces aminophilus strain MCMB 411. From the library, 2.4 kb fragment was recloned in E. coli JM109 and S. lividans TK64 using pUC18 and pIJ702 as vectors respectively. The recombinant plasmids pSD103 and pSD105 expressed the reductase gene and exported the enzyme in periplasmic space of E. coli and in cytoplasm of S. lividans TK64. The proteins expressed by E. coli and S. lividans had the same molecular mass (70 kD) as that expressed by parent strain, which suggested that the enzyme was processed similarly by all strains. Activities of the enzymes cloned in E. coli JM109 and S. lividans TK64 containing recombinant plasmids pSD103 and pSD105 respectively were optimum at 30 degrees C and pH 9 and requirement of cofactors was same as that of the parent strain.


Subject(s)
Bacterial Proteins/genetics , Cloning, Molecular , Escherichia coli/genetics , Oxidoreductases/genetics , Species Specificity , Streptomyces/enzymology
20.
Acta bioquím. clín. latinoam ; 32(3): 387-95, sept. 1998. ilus, tab
Article in Spanish | LILACS | ID: lil-235068

ABSTRACT

El surgimiento de resistencia en poblaciones de insectos es uno de los efectos indeseables asociados al uso de insecticidas, y es un buen ejemplo del modo en que ocurren los procesos microevolutivos. En 1908 se documentó por primera vez la existencia de insectos resistentes a insecticidas. Ahora se conocen casos de resistencia en más de 500 especies de artrópodos. Los principales mecanismos que confieren resistencia a insecticidas son penetración cuticular reducida, metabolismo degradativo aumentado y reducción en la susceptibilidad de los sitios de acción. Los métodos de la biología molecular permiten identificar las bases moleculares de esos mecanismos. El propósito de este artículo es reseñar el conocimiento disponible acerca de la biología molecular de la resistencia a insecticidas: mutaciones puntuales en genes de acetilcolinesterasa (Drosophila melanogaster) y del receptor de GABA (varias especies), inserciones en genes de transferasas (D. melanogaster) y del citocromo P450 (D. melanogaster), amplificación de genes de esterasas (Myzus persicae y Culex pipiens / quinquefasciatus complex), cambios que afectan la expresión del gen del citocromo P450 (Musca domestica), y una mutación ligada al gen del canal de sodio dependiente de voltaje (M. domestica)


Subject(s)
Insecticide Resistance/genetics , Insecticides/adverse effects , Insecta/drug effects , Pest Control/standards , Acetylcholinesterase/drug effects , Acetylcholinesterase/genetics , Aedes/drug effects , Culicidae/drug effects , Cytochrome P-450 Enzyme System/drug effects , Cytochrome P-450 Enzyme System/genetics , Drosophila melanogaster/drug effects , Esterases/drug effects , Esterases/genetics , Insect Control/standards , Insect Control/trends , Insecticides/classification , Houseflies , Oxidoreductases/drug effects , Oxidoreductases/genetics , Receptors, GABA/drug effects , Receptors, GABA/genetics , Transferases/drug effects , Transferases/genetics
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