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1.
Arq. neuropsiquiatr ; 78(3): 163-168, Mar. 2020. tab
Article in English | LILACS | ID: biblio-1098075

ABSTRACT

Abstract Herpes simplex virus (HSV) is a cause of a severe disease of the central nervous system (CNS) in humans. The demonstration of specific antibodies in the cerebrospinal fluid (CSF) may contribute to the retrospective neurological diagnosis. However, the commercial immunological tests for HSV infection are for use in serum samples. Objective: The aim of the present study was to adapt a commercial kit anti-HSV IgG used for serum samples to be performed with a CSF sample. Methods: Forty CSF specimens from 38 patients with suspected CNS HSV infection were serially diluted for detecting anti-HSV IgG by enzyme immunoassay (EIA). The same samples were also analyzed with the polymerase chain reaction (PCR). Results: The sensitivity of EIA test for HSV was 5% (dilution 1:40) and 65% (dilution 1:2) in CSF, and HSV DNA PCR was 15%. The combined analysis of EIA (dilution 1:2) and PCR increased the sensitivity up to 72.5%. The inflammatory CSF was associated with positive HSV PCR. Conclusions: We demonstrated the importance to adapt serological anti-HSV IgG EIA test for CSF assays to increase the accuracy of the analysis, considering the low concentration of specific antibodies in CSF.


Resumo O vírus herpes simples (HSV) é um dos agentes causadores de uma doença grave no sistema nervoso central (SNC) em humanos. A detecção de anticorpos específicos no líquido cefalorraquidiano (LCR) pode contribuir para o diagnóstico neurológico retrospectivo. Entretanto, os testes imunológicos comerciais são para uso em amostras de soro. Objetivo: Adaptar um kit comercial sorológico anti-HSV IgG para ser utilizado no de LCR. Metodos: Quarenta amostras de LCR de 38 pacientes com suspeita de infecção por HSV no SNC foram diluídas pesquisa de anticorpos anti-HSV IgG pelo método imunoenzimático (EIA). Além disso, as mesmas amostras também foram analisadas por reação em cadeia da polimerase (PCR). Resultados: A sensibilidade do teste EIA para o HSV consistiu em 5% (diluição 1:40) e 65% (diluição 1:2) no LCR, e o PCR do DNA do HSV, 15%. A análise combinada de EIA (diluição 1:2) e PCR aumentou a sensibilidade para 72,5%. Houve associação entre presença do LCR inflamatório e PCR positiva para HSV. Conclusões: Demonstramos a importância na adaptação previa do teste sorológico anti-HSV IgG EIA para ensaios do no LCR, a fim de aumentar a acuracia da análise, considerando a baixa concentração de anticorpos específicos no LCR.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Cerebrospinal Fluid/virology , Simplexvirus/isolation & purification , Herpes Simplex/diagnosis , Herpes Simplex/virology , Antibodies, Viral/cerebrospinal fluid , Viral Proteins , DNA, Viral/genetics , Polymerase Chain Reaction/methods , Retrospective Studies , Simplexvirus/genetics , DNA-Directed DNA Polymerase/genetics , Exodeoxyribonucleases , Herpes Simplex/cerebrospinal fluid , Nervous System
2.
Mundo saúde (Impr.) ; 42(2): 269-282, jun. 2018. tab, ilus
Article in English, Portuguese | LILACS | ID: biblio-1000102

ABSTRACT

The genus Eugenia sp. (Myrtaceae) comprises plants with reported antioxidant and antidiarrheal capability among other therapeutic potentials. The aim of this study was to evaluate the antimicrobial activity of essential oil; diuretic and hypotensive activities of aqueous extracts from leaves of Eugenia uniflora. The antimicrobial activity was evaluated . The diuretic and hypotensive activities were evaluated in normotensive Wistar rats by measuring blood pressure and urine flow after received four different concentrations of aqueous extracts (10%, 15%, 20% and 25%). Essential oil inhibited the growth of Candida parapsilosis and Candida albicans with MIC values lower than 14.41 mg/mL, equal to 57.75 mg/mL for Candida krusei. Among antibacterial effect, essential oil inhibited growth with a MIC equals to 153.93 mg/mL for all strains tested, except for Escherichia coli (MIC equals to 307.96 mg/mL. Aqueous extracts showed powerful reductions of the arterial pressure (34% and 31% lower than the control), after administration of 10% and 25% of aqueous extract, respectively. However, the animals that received the aqueous extract at the 15% and 20% concentrations presented a discrete hypotensive effect (20% and 21% lower than control group, respectively) concomitantly to powerful diuretic effect (280% and 91% higher than control group, respectively). These data confirmed the potential biological effect of this species, and represents an important step toward a depth study on the therapeutic properties of this species


O gênero Eugenia sp. (Myrtaceae) compreende plantas com capacidade antimicrobiana e antioxidante entre outros potenciais terapêuticos. O objetivo deste estudo foi avaliar a atividade antimicrobiana de óleo essencial; atividade diurética e hipotensora de extrato aquoso de folhas de Eugenia uniflora. A atividade antimicrobiana foi avaliada pela determinação da concentração inibitória mínima (MIC) e concentração mínima bactericida (MBC) de cepas bacterianas e concentração fungicida mínima (MFC) para fungos. A atividade diurética e hipotensora foi avaliada em ratos Wistar normotensos pela mensuração da pressão sanguínea e fluxo urinário após administração de quatro diferentes concentrações de extrato aquoso (10%, 15%, 20% e 25%). Óleo essencial inibiu o crescimento de Candida parapsilosis e Candida albicans com valores de MIC menores que 14,41 mg/mL, igual a 57,75 mg/mL para Candida krusei. A respeito do efeito antimicrobiano, o óleo essencial inibiu o crescimento de todas as cepas testadas, com MIC igual a 153,93 mg/mL, exceto para Escherichia coli (MIC igual a 307.96 mg/mL). O extrato aquoso mostrou redução importante da pressão arterial (34% e 31% quando comparado ao controle), após administração de 10% e 25% do extrato aquoso, respectivamente. Contudo, os animais que receberam o extrato aquoso na concentração de 15% e 20% apresentaram discreto efeito hipotensor (20% e 21% menor que o grupo controle, respectivamente) concomitantemente ao importante efeito diurético (280% e 91% maior quando comparado ao grupo controle, respectivamente). Esses achados confirmam o potencial efeito biológico dessa espécie, e representa um importante embasamento para estudos relacionados as propriedades terapêuticas da Eugenia uniflora


Subject(s)
Humans , Oils , Diuretics , Eugenia , Hyperglycemia , Anti-Infective Agents , Antifungal Agents , Antihypertensive Agents , Brazil , DNA-Directed DNA Polymerase , Antioxidants
3.
Acta bioquím. clín. latinoam ; 52(2): 205-211, jun. 2018. ilus, tab
Article in Spanish | LILACS | ID: biblio-949334

ABSTRACT

La reacción en cadena de la polimerasa (PCR) es la técnica más importante en Biología Molecular. Su principal deficiencia es la susceptibilidad a la contaminación de la muestra, ya sea con productos de amplificación generados en reacciones previas o con material genético proveniente de otras muestras. Se entiende por contaminación a la presencia indeseada de moléculas de ADN o ARN, que pueden actuar como templados en reacciones de amplificación. El objetivo del trabajo fue demostrar la importancia de la implementación de un control de contaminación ambiental periódico monitoreando la integridad del circuito de PCR. Para ello, se hisoparon puntos correspondientes a diferentes áreas de trabajo y se investigó la presencia de amplicones mediante PCR Real Time en Lighcycler 2.0, Roche® y Ampliprep Cobas s201, Roche®. En puntos críticos del circuito (flujo laminar o la cabina de seguridad del área de pre-PCR) no se detectaron amplicones, aunque sí se hallaron en las mesadas y heladeras. En el resto del circuito, la distribución de los amplicones fue variable. De esta forma se demuestra la importancia de la realización de un control de contaminación ambiental periódico en laboratorios que realizan la técnica de PCR, pudiendo detectar contaminación de forma precoz y tomar acciones correctivas a tiempo.


Polymerase chain reaction (PCR) is the most important technique in Molecular Biology. Its main deficiency is susceptibility to contamination of the sample, either with amplification products generated in previous reactions or with genetic material from other samples. Contamination is understood as the undesired presence of DNA or RNA molecules, which may act as annealing in amplification reactions. The objective of this work is to demonstrate the importance of implementing a periodic environmental pollution control by monitoring the integrity of the PCR. To do this, different areas of work were swabbed and the presence of amplicons were investigated by Real Time PCR in Lighcycler 2.0, Roche® and Ampliprep Cobas s201, Roche®. At critical points in the circuit (laminar flow or pre-PCR area booth) no amplicons were detected, although they were found in countertops and refrigerators. In the rest of the circuit, the distribution of the amplicons was variable. Thus, the importance of conducting a periodic environmental contamination control in laboratories that perform the PCR technique is demonstrated, enabling early detection contamination and corrective actions being taken on time.


A reação em cadeia da polimerase (PCR) é a técnica mais importante em Biologia Molecular. Sua principal deficiência é a suscetibilidade à contaminação da amostra, seja com produtos de amplificação gerados em reações prévias ou com material genético em reações prévias ou com material genético proveniente de outras amostras. Entende-se por contaminação a presença não desejada de moléculas de DNA ou ARN, que podem agir como temperados em reações de amplificação. O objetivo do trabalho foi demonstrar a importância da implementação de um controle de contaminação ambiental periódico monitorando a integridade do circuito de PCR. Para isso, se fez esfregaço de pontos correspondentes a diferentes áreas de trabalho e foi investigada a presença de amplicons mediante PCR Real Time em Lighcycler 2.0, Roche® e Ampliprep Cobas s201, Roche®. Em pontos críticos do circuito (fluxo laminar ou a cabine de segurança da área de pré-PCR), não foram detectados amplicons, apesar de serem encontrados nas bancadas e refrigeradores. No resto do circuito, a distribuição dos amplicons foi variável. Assim mostra a importância da realização de um controle de contaminação ambiental periódico em laboratórios que realizam a técnica de PCR, podendo detectar a contaminação de forma precoce e tomar ações corretivas a tempo.


Subject(s)
Quality Management , DNA-Directed DNA Polymerase , Environmental Pollution , Molecular Biology , Laminar Flow , Equipment and Supplies , Products Distribution , Control , Laboratories
4.
Protein & Cell ; (12): 365-379, 2018.
Article in English | WPRIM | ID: wpr-756937

ABSTRACT

NEDDylation has been shown to participate in the DNA damage pathway, but the substrates of neural precursor cell expressed developmentally downregulated 8 (NEDD8) and the roles of NEDDylation involved in the DNA damage response (DDR) are largely unknown. Translesion synthesis (TLS) is a damage-tolerance mechanism, in which RAD18/RAD6-mediated monoubiquitinated proliferating cell nuclear antigen (PCNA) promotes recruitment of polymerase η (polη) to bypass lesions. Here we identify PCNA as a substrate of NEDD8, and show that E3 ligase RAD18-catalyzed PCNA NEDDylation antagonizes its ubiquitination. In addition, NEDP1 acts as the deNEDDylase of PCNA, and NEDP1 deletion enhances PCNA NEDDylation but reduces its ubiquitination. In response to HO stimulation, NEDP1 disassociates from PCNA and RAD18-dependent PCNA NEDDylation increases markedly after its ubiquitination. Impairment of NEDDylation by Ubc12 knockout enhances PCNA ubiquitination and promotes PCNA-polη interaction, while up-regulation of NEDDylation by NEDD8 overexpression or NEDP1 deletion reduces the excessive accumulation of ubiquitinated PCNA, thus inhibits PCNA-polη interaction and blocks polη foci formation. Moreover, Ubc12 knockout decreases cell sensitivity to HO-induced oxidative stress, but NEDP1 deletion aggravates this sensitivity. Collectively, our study elucidates the important role of NEDDylation in the DDR as a modulator of PCNA monoubiquitination and polη recruitment.


Subject(s)
DNA Damage , DNA Repair , Genetics , DNA Replication , Genetics , DNA-Binding Proteins , Genetics , DNA-Directed DNA Polymerase , Genetics , Endopeptidases , Genetics , Gene Knockout Techniques , Humans , Hydrogen Peroxide , Toxicity , NEDD8 Protein , Genetics , Oxidative Stress , Genetics , Proliferating Cell Nuclear Antigen , Genetics , Ubiquitin-Conjugating Enzymes , Genetics , Ubiquitin-Protein Ligases , Genetics , Ubiquitination , Genetics , Ultraviolet Rays
5.
Braz. j. infect. dis ; 20(2): 173-178, Mar.-Apr. 2016. tab
Article in English | LILACS | ID: lil-780801

ABSTRACT

Abstract Objective There are a lot of disagreements in the studies on hepatitis B virus (HBV) DNA polymerase mutation rate associated with nucleos(t)ide analogues (NAs) in treatment-naive chronic hepatitis B (CHB) patients. This is the first study aimed to investigate the prevalence of spontaneous HBV resistance mutations in Central China. Methods This study included treatment-naive patients with CHB from June 2012 to May 2015 receiving care at the Institute of Liver Disease in Central China. All patients completed a questionnaire covering different aspects, such as family medical history, course of liver disease, medication history, alcohol use, among others. Mutations in HBV DNA polymerase associated with NAs resistance were detected using INNO-LiPA assay. Results 269 patients were infected with HBV genotype B (81.4%), C (17.9%), and both B and C (0.7%). Mutations in HBV DNA polymerase were detected in 24 patients (8.9%) including rtM204I/V (n = 6), rtN236T (n = 5), rtM250V (n = 2), rtL180M (n = 2), rtT184G (n = 1), rtM207I (n = 1), rtS202I (n = 1), rtM204V/I & rtL180M (n = 5), and rtM204I & rtM250V (n = 1). Conclusion Spontaneous HBV resistance mutations in HBV DNA polymerase were found in treatment-naive patients with CHB in Central China. These findings suggest that we should analyze HBV DNA polymerase resistance mutation associated with NAs before giving antiviral therapy such as lamivudine (LAM), adefovir (ADV), and telbivudine (LdT).


Subject(s)
Humans , Male , Female , Pregnancy , Adult , Middle Aged , Aged , Young Adult , DNA, Viral/genetics , Hepatitis B virus/genetics , Hepatitis B, Chronic/virology , Drug Resistance, Viral/genetics , DNA-Directed DNA Polymerase/genetics , Mutation/genetics , Antiviral Agents/therapeutic use , China , Hepatitis B virus/drug effects , Prevalence , Prospective Studies , Hepatitis B, Chronic/drug therapy , Genotype
6.
Indian J Biochem Biophys ; 2015 Feb; 52 (1): 86-94
Article in English | IMSEAR | ID: sea-157959

ABSTRACT

DNA polymerase λ (DNA pol λ) is the only reported X-family DNA polymerases in plants and has been shown to play a significant role in dry quiescent seeds, growth, development and nuclear DNA repair. cDNA for DNA pol λ has been reported in Arabidopsis and japonica rice cultivar and has been characterized from E. coli expressed protein, but very little is known about its activity at protein level in plants. The enzymatic activity of DNA pol λ was studied in dry, imbibed and during different germination stages of indica rice IR-8 (salt sensitive) by in-gel activity assay to determine its physiological role in important stages of growth and development. The upstream sequence was also analyzed using plantCARE database and was found to contain several cis-acting elements, including light responsive elements, dehydration responsive elements, Myb binding sites, etc. Hence, 4-day-old germinating seedlings of IR29, a salt-sensitive, but high yielding indica rice cultivar and Nonabokra, a salt-tolerant, but low yielding cultivar were treated with water (control) or 250 mM NaCl or 20% polyethyleneglycol-6000 for 4 and 8 h. The protein was analyzed by in vitro DNA pol λ activity assay, in-gel activity assay and Western blot analysis. DNA pol λ was not detected in dry seeds, but enhanced after imbibition and detectable from low level to high level during subsequent germination steps. Both salinity and dehydration stress led to the enhancement of the activity and protein level of DNA pol λ, as compared to control tissues. This is the first evidence of the salinity or dehydration stress induced enhancement of DNA pol λ activity in the plumules of rice (Oryza sativa L.) cultivars.


Subject(s)
DNA-Directed DNA Polymerase/analysis , DNA-Directed DNA Polymerase/physiology , Germination/genetics , Oryza/genetics , Oryza/growth & development , Salinity , Seeds/growth & development , Sodium Chloride , Stress, Physiological
7.
Protein & Cell ; (12): 194-210, 2015.
Article in English | WPRIM | ID: wpr-757602

ABSTRACT

DNA primase catalyzes de novo synthesis of a short RNA primer that is further extended by replicative DNA polymerases during initiation of DNA replication. The eukaryotic primase is a heterodimeric enzyme comprising a catalytic subunit Pri1 and a regulatory subunit Pri2. Pri2 is responsible for facilitating optimal RNA primer synthesis by Pri1 and mediating interaction between Pri1 and DNA polymerase α for transition from RNA synthesis to DNA elongation. All eukaryotic Pri2 proteins contain a conserved C-terminal iron-sulfur (Fe-S) cluster-binding domain that is critical for primase catalytic activity in vitro. Here we show that mutations at conserved cysteine ligands for the Pri2 Fe-S cluster markedly decrease the protein stability, thereby causing S phase arrest at the restrictive temperature. Furthermore, Pri2 cysteine mutants are defective in loading of the entire DNA pol α-primase complex onto early replication origins resulting in defective initiation. Importantly, assembly of the Fe-S cluster in Pri2 is impaired not only by mutations at the conserved cysteine ligands but also by increased oxidative stress in the sod1Δ mutant lacking the Cu/Zn superoxide dismutase. Together these findings highlight the critical role of Pri2's Fe-S cluster domain in replication initiation in vivo and suggest a molecular basis for how DNA replication can be influenced by changes in cellular redox state.


Subject(s)
Amino Acid Sequence , Cell Cycle , Cell Proliferation , Chromatin Immunoprecipitation , Cysteine , Genetics , Metabolism , DNA Primase , Genetics , Metabolism , DNA Replication , DNA, Fungal , Genetics , DNA-Directed DNA Polymerase , Metabolism , Immunoblotting , Immunoprecipitation , Iron , Metabolism , Iron-Sulfur Proteins , Metabolism , Molecular Sequence Data , Mutation , Genetics , Oxidative Stress , Protein Binding , Saccharomyces cerevisiae , Genetics , Metabolism , Sequence Homology, Amino Acid , Sulfur , Metabolism
8.
Gut and Liver ; : 103-108, 2015.
Article in English | WPRIM | ID: wpr-61568

ABSTRACT

BACKGROUND/AIMS: To investigate the association between the baseline profiles and dynamics of hepatitis B virus (HBV) DNA polymerase gene mutations and the long-term virological response of lamivudine (LAM)-adefovir (ADV) combination therapy in patients with LAM-resistant chronic hepatitis B. METHODS: Seventy-five patients who received LAM-ADV combination therapy for more than 12 months were analyzed. Restriction fragment mass polymorphism assays were used to detect and monitor the dynamics of LAM- and ADV-resistant mutations. RESULTS: The median duration of LAM-ADV combination therapy was 26 months (range, 12 to 58 months). The baseline mutation profiles, rtM204I (p=0.992), rtM204I/V (p=0.177), and rtL180M (p=0.051), were not correlated with the cumulative virological response, and the baseline HBV DNA level (p=0.032) was the only independent predictive factor for cumulative virological response. Tests for LAM- and ADV-resistant mutations were performed in 12 suboptimal responders in weeks 48 and 96. The population of rtM204 mutants persisted or increased in 8 of 12 patients, and rtA181T mutants newly emerged as a minor population in four patients until 96 weeks. Nevertheless, the viral loads progressively decreased during rescue therapy, and these dynamics did not correlate with virological response. CONCLUSIONS: The baseline profile and dynamics of LAM-resistant mutations during LAM-ADV combination therapy are not associated with a virological response.


Subject(s)
Adenine/administration & dosage , Adult , Aged , Aged, 80 and over , Antiviral Agents/administration & dosage , DNA-Directed DNA Polymerase/genetics , Drug Resistance, Viral/genetics , Drug Therapy, Combination , Female , Hepatitis B virus/genetics , Hepatitis B, Chronic/drug therapy , Humans , Lamivudine/administration & dosage , Male , Middle Aged , Organophosphonates/administration & dosage , Treatment Outcome , Viral Load/drug effects , Young Adult
9.
Indian J Hum Genet ; 2013 July-Sept ;19 (3): 320-324
Article in English | IMSEAR | ID: sea-156577

ABSTRACT

Mitochondria contains a single deoxyribonucleic acid (DNA) polymerase, polymerase gamma (POLG) mapped to long arm of chromosome 15 (15q25), responsible for replication and repair of mitochondrial DNA. Exon 1 of the human POLG contains CAG trinucleotide repeat, which codes for polyglutamate. Ten copies of CAG repeat were found to be uniformly high (0.88) in different ethnic groups and considered as the common allele, whereas the mutant alleles (not -10/not -10 CAG repeats) were found to be associated with oligospermia/oligoasthenospermia in male infertility. Recent data suggested the implication of POLG CAG repeat expansion in infertility, but are debated. The aim of our study was to explore whether the not -10/not -10 variant is associated with spermatogenic failure. As few study on Indian population have been conducted so far to support this view, we investigated the distribution of the POLG CAG repeats in 61 infertile men and 60 normozoospermic control Indian men of Tamil Nadu, from the same ethnic background. This analysis interestingly revealed that the homozygous wild type genotype (10/- 10) was common in infertile men (77% - 47/61) and in normozoospermic control men (71.7% - 43/60). Our study failed to confirm any influence of the POLG gene polymorphism on the efficiency of the spermatogenesis.


Subject(s)
Azoospermia/genetics , Humans , India , DNA-Directed DNA Polymerase/genetics , Infertility, Male/epidemiology , Infertility, Male/etiology , Infertility, Male/genetics , /genetics
10.
Article in Chinese | WPRIM | ID: wpr-312596

ABSTRACT

<p><b>OBJECTIVE</b>To investigate single-molecule detection and characterization of DNA replication.</p><p><b>METHODS</b>Single-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication.</p><p><b>RESULTS</b>The designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis.</p><p><b>CONCLUSIONS</b>The combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.</p>


Subject(s)
Biotinylation , DNA , Chemistry , DNA Replication , DNA, Single-Stranded , Chemistry , DNA-Directed DNA Polymerase , Electrophoresis, Agar Gel , Microscopy, Atomic Force , Nucleic Acid Hybridization , Streptavidin
11.
Rev. Inst. Med. Trop. Säo Paulo ; 55(6): 401-406, Nov-Dec/2013. tab, graf
Article in English | LILACS | ID: lil-690351

ABSTRACT

SUMMARY The use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens. .


RESUMO O propósito deste estudo foi avaliar 6 polimerases de DNA disponíveis comercialmente que são resistentes aos inibidores do PCR para uma amplificação potencial de DNA de amostras de sangue total. O DNA genômico do parasita humano da malária, Plasmodium falciparum, foi analisado sob condições que incluíram os componentes inibidores do sangue extraído de sangue ressacado em papel de filtro. Nossos resultados sugerem que a polimerase KOD FX DNA é superior a outras polimerases. .


Subject(s)
Humans , DNA, Protozoan/genetics , DNA-Directed DNA Polymerase/genetics , Malaria, Falciparum/diagnosis , Plasmodium falciparum/genetics , Polymerase Chain Reaction/methods , DNA, Protozoan/blood , DNA-Directed DNA Polymerase/blood , Reagent Kits, Diagnostic , Reproducibility of Results , Sensitivity and Specificity
12.
Indian J Hum Genet ; 2013 July-Sept ;19 (3): 320-324
Article in English | IMSEAR | ID: sea-156584

ABSTRACT

Mitochondria contains a single deoxyribonucleic acid (DNA) polymerase, polymerase gamma (POLG) mapped to long arm of chromosome 15 (15q25), responsible for replication and repair of mitochondrial DNA. Exon 1 of the human POLG contains CAG trinucleotide repeat, which codes for polyglutamate. Ten copies of CAG repeat were found to be uniformly high (0.88) in different ethnic groups and considered as the common allele, whereas the mutant alleles (not -10/not -10 CAG repeats) were found to be associated with oligospermia/oligoasthenospermia in male infertility. Recent data suggested the implication of POLG CAG repeat expansion in infertility, but are debated. The aim of our study was to explore whether the not -10/not -10 variant is associated with spermatogenic failure. As few study on Indian population have been conducted so far to support this view, we investigated the distribution of the POLG CAG repeats in 61 infertile men and 60 normozoospermic control Indian men of Tamil Nadu, from the same ethnic background. This analysis interestingly revealed that the homozygous wild type genotype (10/- 10) was common in infertile men (77% - 47/61) and in normozoospermic control men (71.7% - 43/60). Our study failed to confirm any influence of the POLG gene polymorphism on the efficiency of the spermatogenesis.


Subject(s)
DNA-Directed DNA Polymerase/genetics , Humans , India/epidemiology , Infertility, Male/genetics , Male , Polymorphism, Genetic/genetics , Spermatozoa/abnormalities , Trinucleotide Repeat Expansion/genetics
13.
Chinese Journal of Virology ; (6): 432-436, 2013.
Article in Chinese | WPRIM | ID: wpr-339932

ABSTRACT

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.


Subject(s)
Bunyaviridae Infections , Diagnosis , Virology , Cell Line , DNA Ligases , Metabolism , DNA, Complementary , Genetics , DNA-Directed DNA Polymerase , Metabolism , Dengue , Diagnosis , Virology , Dengue Virus , Genetics , Genome, Viral , Genetics , Humans , Phlebovirus , Genetics , RNA, Viral , Genetics , Reference Standards , Reverse Transcriptase Polymerase Chain Reaction , Methods , Viral Load
14.
Article in English | WPRIM | ID: wpr-147324

ABSTRACT

Obesity gives vent to many diseases such as type 2 diabetes, hypertension, and hyperlipidemia, being considered as the main causes of mortality and morbidity worldwide. The pathogenesis and pathophysiology of metabolic syndrome can well be understood by studying the molecular mechanisms that control the development and function of adipose tissue. In human body, exist two types of adipose tissue, the white and the brown one, which are reported to play various roles in energy homeostasis. The major and most efficient storage of energy occurs in the form of triglycerides in white adipose tissue while brown adipose tissue actively participates in both basal and inducible energy consumption in the form of thermogenesis. Recent years have observed a rapid and greater interest towards developmental plasticity and therapeutic potential of stromal cells those isolated from adipose tissue. The adipocyte differentiation involves a couple of regulators in the white or brown adipogenesis. Peroxisome proliferators-activated receptor-gamma actively participates in regulating carbohydrate and lipid metabolism, and also acts as main regulator of both white and brown adipogenesis. This review based on our recent research, seeks to highlight the adipocyte differentiation.


Subject(s)
Adipocytes , Adipocytes, Brown , Adipogenesis , Adipose Tissue , Adipose Tissue, Brown , Adipose Tissue, White , DNA-Directed DNA Polymerase , Genes, Homeobox , Homeostasis , Human Body , Humans , Hyperlipidemias , Hypertension , Lipid Metabolism , Obesity , Peroxisomes , Stromal Cells , Thermogenesis , Triglycerides
15.
Article in Chinese | WPRIM | ID: wpr-234676

ABSTRACT

A common DNA polymerase can replicate DNA which functions normally. However, if DNA suffers damage, the genome can not be replicated by a common DNA polymerase because DNA lesions will block the replication apparatus. Another kind of DNA polymerases in organism, Y-family DNA polymerases which is also called translesion synthesis (TLS) polymerases, can deal with this problem. Their main functions are bypassing the lesions in DNA, replicating the genome and saving the dying cells. This thesis presents a historical review of the literature pertinent to the structure, functions and roles of Y-family DNA polymerases.


Subject(s)
DNA Damage , DNA Repair , DNA Replication , DNA-Directed DNA Polymerase , Classification , Metabolism , Physiology , Humans , Mutagenesis , Mutagens , Proliferating Cell Nuclear Antigen , Genetics
16.
Article in Chinese | WPRIM | ID: wpr-285979

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the drug-resistant genes at hepatitis B virus (HBV) polymerase region during entecavir (ETV) treatment.</p><p><b>METHODS</b>Serum samples from chronic hepatitis B patients with virologic breakthrough during enticavir therapy were studied. The resistant mutation patterns in the polymerase gene of hepatitis B virus were analyzed using the polymerase chain reaction (PCR)-sequencing method.</p><p><b>RESULTS</b>ETV resistance was detected from 19 out of 29 ETV-refractory patients, among whom 16 (84.2%) had a history of lamivudine-refractory. The mutation patterns were diverse, while rtL180 + rtM204 + rtT184 (58.6%, 17/29) was most common in patients with ETV genotype resistance. Four of 7 patients (7/29, 24.1%) with genotype B were detected to have ETV genotype resistance, while 15 of 22 patients (22/29, 75.9%) with genotype C were detected to have ETV genotype resistance. The rate of ETV genotype resistance was 57.1% (4/7) and 68.2% (15/22) in patients with genotype B and genotype C,while no statistical difference was found(P = 0.665).</p><p><b>CONCLUSIONS</b>ETV genotype resistance is more common in patients who have been refractory to ETV and lamivudine sequential treatment. rtM204+rtL180+rtT184 mutation is common in genotype B and C ETV resistance patients.</p>


Subject(s)
Adult , Antiviral Agents , Therapeutic Uses , DNA, Viral , Blood , Genetics , DNA-Directed DNA Polymerase , Genetics , Drug Resistance, Viral , Genetics , Female , Guanine , Therapeutic Uses , Hepatitis B virus , Genetics , Hepatitis B, Chronic , Drug Therapy , Virology , Humans , Male , Middle Aged , Mutation , Viral Proteins , Genetics , Young Adult
17.
Protein & Cell ; (12): 864-874, 2012.
Article in English | WPRIM | ID: wpr-757842

ABSTRACT

In addition to DNA repair pathways, cells utilize translesion DNA synthesis (TLS) to bypass DNA lesions during replication. During TLS, Y-family DNA polymerase (Polη, Polκ, Polı and Rev1) inserts specific nucleotide opposite preferred DNA lesions, and then Polζ consisting of two subunits, Rev3 and Rev7, carries out primer extension. Here, we report the complex structures of Rev3-Rev7-Rev1(CTD) and Rev3-Rev7-Rev1(CTD)-Polκ(RIR). These two structures demonstrate that Rev1(CTD) contains separate binding sites for Polκ and Rev7. Our BIAcore experiments provide additional support for the notion that the interaction between Rev3 and Rev7 increases the affinity of Rev7 and Rev1. We also verified through FRET experiment that Rev1, Rev3, Rev7 and Polκ form a stable quaternary complex in vivo, thereby suggesting an efficient switching mechanism where the "inserter" polymerase can be immediately replaced by an "extender" polymerase within the same quaternary complex.


Subject(s)
Binding Sites , Crystallography, X-Ray , DNA Repair , DNA-Binding Proteins , Chemistry , Genetics , Metabolism , DNA-Directed DNA Polymerase , Chemistry , Genetics , Metabolism , Fluorescence Resonance Energy Transfer , Humans , Mad2 Proteins , Nuclear Proteins , Chemistry , Genetics , Metabolism , Nucleotidyltransferases , Chemistry , Genetics , Metabolism , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Proteins , Chemistry , Genetics , Metabolism , Recombinant Proteins , Chemistry , Genetics
18.
Article in Chinese | WPRIM | ID: wpr-305068

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the distribution of hepatitis B virus (HBV) genotypes in Qingdao, and the relationship of HBV genotypes with the serum HBV-DNA levels and HBV YMDD spontaneous mutation of patients, then to discuss the clinical significance.</p><p><b>METHODS</b>Hepatitis B virus genotypes and YMDD spontaneous mutation of 144 patients were detected by real time PCR (Taqman probe), then the results were analyzed by statistical method.</p><p><b>RESULTS</b>Of the 144 patients, 130 (90.3%) were genotype C, 12 (8.3%) were genotype B, and 2 (1.4%) were neither genotype B nor genotype C; 33 (22.9%) were detected to have YMDD mutation, and 25 (75.5%) were YVDD positive, 3 (9.1%) were YIDD positive, 5 (15.2%) were YVDD and YIDD positive. There were no significant differences between clinical diagnosis, serum HBV-DNA levels, YMDD spontaneous mutation and HBV genotypes (P > 0.05).</p><p><b>CONCLUSION</b>Genotype C is the dominant position for HBV genotype in Qingdao. Untreated patients with chronic hepatitis B have YMDD spontaneous mutation. HBV genotypes have no association with YMDD spontaneous mutation and the development of diseases.</p>


Subject(s)
Adult , Aged , Aged, 80 and over , China , DNA-Directed DNA Polymerase , Chemistry , Genetics , Metabolism , Female , Genotype , Hepatitis B virus , Classification , Genetics , Humans , Male , Middle Aged , Mutation , Viral Proteins , Chemistry , Genetics , Metabolism , Young Adult
19.
Article in Chinese | WPRIM | ID: wpr-231134

ABSTRACT

<p><b>OBJECTIVE</b>In an attempt to study the moleculr characterization and epidemiology of simian adenoviruses in nonhuman primate (NHP) populations.</p><p><b>METHODS</b>We examined a colony of captively bred rhesus macaques (Macaca mulatta) in China for the presence of adenoviral DNA in stool samples. This was done by using the PCR method that targeted the adenovirus polymerase gene, and the PCR positive fragments were cloned for sequencing and phylogenetic analyses.</p><p><b>RESULTS</b>Among the 57 animals analyzed, fecal samples from 12 animals were positive for the presence of adenoviral DNA. The results suggested that the viral DNA clones were primarily segregated into two large groups: SAdV-6 (2 non-redundant sequences) and SAdV-7 (9 non-redundant sequences). In addition, there were three clones with more similarity to SAdV-1, SAdV-3 and HAdV-52 respectively.</p><p><b>CONCLUSION</b>Our data confirmed the prevalence of adenoviral DNA in the feces of NHPs and revealed the adenoviruses in the gastrointestinal tract of the study animals. heterogeneity and phylogenetics of the adenoviruses in the gastrointestinal tract of the study animals.</p>


Subject(s)
Adenoviridae , Classification , Genetics , Animals , DNA-Directed DNA Polymerase , Genetics , Feces , Virology , Female , Macaca mulatta , Virology , Male , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA , Viral Proteins , Genetics
20.
Chinese Journal of Hepatology ; (12): 423-426, 2011.
Article in Chinese | WPRIM | ID: wpr-330735

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the relationship between the mutation patterns of rtM204V/I (methionine to valine or isoleucine at position rt204 of reverse transcriptase domain) in hepatitis B virus (HBV) polymerase gene and HBV genotypes.</p><p><b>METHODS</b>A total of 2849 HBV complete genome sequences were retrieved from the GenBank/EMBL/DDBJ. HBV genotypes were determined by using MEGA4 software. The amino acid sequences of the reverse transcriptase (RT) domain were aligned. Data were analyzed using SPSS 13.0. RESULTS Among the 2849 HBV complete genome sequences, 217 strains with Y (I/V) DD were identified. Of them, 120 had YIDD mutation and the genotype/subgenotype distribution was as follows: A (2), B(B2 19), C(C1 1, C2 78, C5 1), D(17), E(1), G(1); 97 had YVDD mutation and the genotype/subgenotype distribution was as follows: A(17), B(B2 22), C(C1 3, C2 48), D(3), G(3), H(1). There is a significant difference in the mutation patterns of Y (I/V) DD among genotypes of A-D, A-C, and between genotype A and B, P < 0.01.There is a difference in the mutation pattern of Y (I/V) DD among genotypes of B-D, between genotype C and D, P < 0.05. Genotype A has a higher tendency to develop YVDD mutation, whereas genotype D has a higher frequency to develop YIDD mutation. The rtM204V-rtL180M mutations were more frequently found in subgenotype B2 than in subgenotype C2 while the rtM204V-rtL180M-rtV173L mutations were more associated with subgenotype C2 (P < 0.01).</p><p><b>CONCLUSION</b>Different HBV genotype/subgenotype may select different mutation pattern in the YMDD domain. Subgenotype C2 is more diversity and complexity than other HBV genotypes/subgenotypes.</p>


Subject(s)
Antigenic Variation , DNA Mutational Analysis , DNA, Viral , Genetics , DNA-Directed DNA Polymerase , Genetics , Genotype , Hepatitis B virus , Genetics , Viral Proteins , Genetics
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