RESUMO
L-asparaginase (L-ASN) is widely applied in the treatment of malignant tumor and low-acrylamide food production, however, the low expression level hampers its application. Heterologous expression is an effective strategy to increase the expression level of target enzymes, and Bacillus is generally used as the host for efficient production of enzymes. In this study, the expression level of L-asparaginase in Bacillus was enhanced through optimization of expression element and host. Firstly, five signal peptides (SPSacC, SPAmyL, SPAprE, SPYwbN and SPWapA) were screened, among which SPSacC showed the best performance, reaching an activity of 157.61 U/mL. Subsequently, four strong promoters (P43, PykzA-P43, PUbay and PbacA) from Bacillus were screened, and tandem promoter PykzA-P43 showed the highest yield of L-asparaginase, which was 52.94% higher than that of control strain. Finally, three Bacillus expression hosts (B. licheniformis Δ0F3 and BL10, B. subtilis WB800) were investigated, and the maximum L-asparaginase activity, 438.3 U/mL, was reached by B. licheniformis BL10, which was an 81.83% increase compared with that of the control. This is also the highest level of L-asparaginase in shake flask reported to date. Taken together, this study constructed a B. licheniformis strain BL10/PykzA-P43-SPSacC-ansZ capable of efficiently producing L-asparaginase, which laid the foundation for industrial production of L-asparaginase.
Assuntos
Bacillus licheniformis/metabolismo , Asparaginase/genética , Bacillus/genética , Sinais Direcionadores de Proteínas , Regiões Promotoras Genéticas/genética , Bacillus subtilis/genética , Proteínas de BactériasRESUMO
Bacillus subtilis is recognized as a generally-regarded-as-safe strain, and has been widely used in the biosynthesis of high value-added products, including N-acetylneuraminic acid (NeuAc) which is widely used as a nutraceutical and a pharmaceutical intermediate. Biosensors responding to target products are widely used in dynamic regulation and high-throughput screening in metabolic engineering to improve the efficiency of biosynthesis. However, B. subtilis lacks biosensors that can efficiently respond to NeuAc. This study first tested and optimized the transport capacity of NeuAc transporters, and obtained a series of strains with different transport capacities for testing NeuAc-responsive biosensors. Subsequently, the binding site sequence of Bbr_NanR responding to NeuAc was inserted into different sites of the constitutive promoter of B. subtilis, and active hybrid promoters were obtained. Next, by introducing and optimizing the expression of Bbr_NanR in B. subtilis with NeuAc transport capacity, we obtained an NeuAc-responsive biosensor with wide dynamic range and higher activation fold. Among them, P535-N2 can sensitively respond to changes in intracellular NeuAc concentration, with the largest dynamic range (180-20 245) AU/OD. P566-N2 shows a 122-fold of activation, which is 2 times of the reported NeuAc-responsive biosensor in B. subtilis. The NeuAc-responsive biosensor developed in this study can be used to screen enzyme mutants and B. subtilis strains with high NeuAc production efficiency, providing an efficient and sensitive analysis and regulation tool for biosynthesis of NeuAc in B. subtilis.
Assuntos
Ácido N-Acetilneuramínico/metabolismo , Bacillus subtilis/metabolismo , Regiões Promotoras Genéticas/genética , Sítios de Ligação , Técnicas BiossensoriaisRESUMO
Promoter is an important genetic tool for fine-tuning of gene expression and has been widely used for metabolic engineering. Corynebacterium glutamicum is an important chassis for industrial biotechnology. However, promoter libraries that are applicable to C. glutamicum have been rarely reported, except for a few developed based on synthetic sequences containing random mutations. In this study, we constructed a promoter library based on the native promoter of odhA gene by mutating the -10 region and the bystanders. Using a red fluorescent protein (RFP) as the reporter, 57 promoter mutants were screened by fluorescence imaging technology in a high-throughput manner. These mutants spanned a strength range between 2.4-fold and 19.6-fold improvements of the wild-type promoter. The strongest mutant exhibited a 2.3-fold higher strength than the widely used strong inducible promoter Ptrc. Sequencing of all 57 mutants revealed that 55 mutants share a 1-4 bases shift (4 bases shift for 68% mutants) of the conserved -10 motif "TANNNT" to the 3' end of the promoter, compared to the wild-type promoter. Conserved T or G bases at different positions were observed for strong, moderate, and weak promoter mutants. Finally, five promoter mutants with different strength were employed to fine-tune the expression of γ-glutamyl kinase (ProB) for L-proline biosynthesis. Increased promoter strength led to enhanced L-proline production and the highest L-proline titer of 6.4 g/L was obtained when a promoter mutant with a 9.8-fold higher strength compared to the wild-type promoter was used for ProB expression. The use of stronger promoter variants did not further improve L-proline production. In conclusion, a promoter library was constructed based on a native C. glutamicum promoter PodhA. The new promoter library should be useful for systems metabolic engineering of C. glutamicum. The strategy of mutating native promoter may also guide the construction of promoter libraries for other microorganisms.
Assuntos
Corynebacterium glutamicum/metabolismo , Biblioteca Gênica , Engenharia Metabólica , Regiões Promotoras Genéticas/genéticaRESUMO
The effect of altering the promoter region of ubiquitous chromatin-opening element (UCOE) and matrix attachment region (MAR) on stable and efficient expression of genes was investigated. Four different promoters were tested, namely, oct4 containing an enhancer region, sox2 having a CpG island, nanog having no regulatory elements, and CMV containing a CpG island and an enhancer region. Eight reporter plasmids were constructed: pOCT4-UCOE, pOCT4-MAR, pSOX2-UCOE, pSOX2-MAR, pNANOG-UCOE, pNANOG-MAR, pCMV-UCOE, and pCMV-MAR. Stable and efficient expression was observed when UCOE combined with the oct4 promoter, whereas the sox2 was the best promoter suited for MAR. Comparison of the stable clones of oct4-UCOE and sox2-MAR showed that UCOE-regulated expression is more stable and efficient than MAR-regulated expression. When CpG island-containing promoter is linked with UCOE, stable and efficient expression could be observed. These data suggest that an enhancer region in the promoter leads to high, yet unstable expression when combined with UCOE, whereas CpG islands stabilize expression. In conclusion, UCOE and MAR interact with regulatory elements on the promoter by altering the chromatin open state and chromatin loop to regulate gene expression.
Assuntos
Cromatina/genética , Ilhas de CpG/genética , Expressão Gênica , Regulação da Expressão Gênica , Regiões Promotoras Genéticas/genéticaRESUMO
Background: Matrix metalloproteinase 12 (MMP12), a member of MMPs, can take lots of roles including extracellular matrix component degradation, viral infection, inflammation, tissue remodeling and tumorigenesis. To explore the transcriptional regulation of MMP12 gene, a sensitive luciferase reporter HEK293 cell line for endogenous MMP12 promoter was generated by CRISPR/Cas9 technology. Results: The HEK293-MMP12-T2A-luciferase-KI cell line was successfully established by CRISPR/Cas9 technology. The sequencing results indicated that one allele of the genome was proven to have a site-directed insertion of luciferase gene and another allele of the genome was confirmed to have additional 48 bp insertion in this cell line. The cell line was further demonstrated to be a sensitive reporter of the endogenous MMP12 promoter by applying transcription factors STAT3, AP-1 and SP-1 to the cell line. The reporter cell line was then screened with bioactive small molecule library, and a small molecule Tanshinone I was found to significantly inhibit the transcriptional activity of MMP12 gene in HEK293-MMP12-T2A-luciferase-KI cell line by luciferase activity assay, which was further confirmed to inhibit the expression of MMP12 mRNA in wild-type HEK293 cells. Conclusions: This novel luciferase knock-in reporter system will be helpful for investigating the transcriptional regulation of MMP12 gene and screening the drugs targeting MMP12 gene.
Assuntos
Humanos , Metaloproteinase 12 da Matriz/genética , Sistemas CRISPR-Cas , Luciferases/genética , Transcrição Gênica , Comunicação Celular , Linhagem Celular , Regiões Promotoras Genéticas/genética , Técnicas de Cultura de Células , Matriz Extracelular , Técnicas de Introdução de Genes , Repetições Palindrômicas Curtas Agrupadas e Regularmente EspaçadasRESUMO
ABSTRACT Objectives: This observational study analyzed telomerase reverse transcriptase (pTERT) mutations in 45 fine-needle aspiration (FNA) specimens obtained from thyroid nodules followed by postoperatively confirmation of papillary thyroid cancer (PTC) diagnosis, examining their relationship with clinicopathologic aspects and the BRAFV600E mutation. Subjects and methods: Clinical information was collected from patients who presented to Ribeirao Preto University Hospital for surgical consultation regarding a thyroid nodule and who underwent molecular testing between January 2010 to October 2012. Tests included a DNA-based somatic detection of BRAFV600E and pTERT mutations. Results: We found coexistence of pTERTC228T and BRAFV600E mutations in 8.9% (4/45) of thyroid nodules. All nodules positive for pTERT mutations were BRAFV600E positives. There was a significant association between pTERTC228T/BRAFV600E with older age and advanced stage compared with the group negative for either mutation. Conclusions: This series provides evidence that FNA is a reliable method for preoperative diagnosis of high-risk thyroid nodules. pTERTC228T/BRAFV600E mutations could be a marker of poor prognosis. Its use as a personalized molecular medicine tool to individualize treatment decisions and follow-up design needs to be further studied.
Assuntos
Humanos , Masculino , Feminino , Adolescente , Adulto , Pessoa de Meia-Idade , Idoso , Adulto Jovem , Neoplasias da Glândula Tireoide/genética , Nódulo da Glândula Tireoide/genética , Telomerase/genética , Proteínas Proto-Oncogênicas B-raf/genética , Câncer Papilífero da Tireoide/genética , Prognóstico , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/patologia , Análise Mutacional de DNA , Valor Preditivo dos Testes , Fatores Etários , Regiões Promotoras Genéticas/genética , Nódulo da Glândula Tireoide/diagnóstico , Nódulo da Glândula Tireoide/patologia , Biópsia por Agulha Fina , Período Pré-Operatório , Câncer Papilífero da Tireoide/diagnóstico , Câncer Papilífero da Tireoide/patologia , Metástase Linfática/diagnóstico , Mutação/genética , Estadiamento de NeoplasiasRESUMO
OBJECTIVE@#To explore the polymorphisms of T149C and T950C gene in osteoprotectin (OPG) promoter sites and the levels of serum OPG and soluble nuclear factor-ΚB receptor activator ligand (sRANKL) and the incidence of coronary heart disease (CHD).@*METHODS@#528 patients in Tianjin suspected of CHD and underwent coronary angiography (CAG) who admitted to the department of cardiology of Tianjin Chest Hospital from April 2017 to December 2018 were enrolled. According to the CAG results, they were divided into two groups: CHD group (n = 302) and non-CHD group (n = 226). The gender, age, history of hypertension, family history of CHD, diabetes, levels of blood lipid parameters in serum and other clinical data of patients were recorded. The levels of serum OPG and sRANKL were measured by enzyme-linked immunosorbent assay (ELISA). T149C and T950C gene polymorphisms were analyzed by polymerase chain reaction-restriction endonuclease fragment length polymorphism (PCR-RFLP) methods. Hardy-Weinberg genetic balance test was performed for alleles. Binomial classification multivariate non-conditional Logistic regression method was used to analyze the relationship between T149C and T950C gene polymorphisms, serum levels of OPG and sRANKL and CHD.@*RESULTS@#All patients were enrolled in the final analysis. The serum level of OPG in CHD group was significantly higher than that in non-CHD group (μg/L: 1.76±0.49 vs. 1.47±0.29, P < 0.01), the serum level of sRANKL was significantly lower than that in non-CHD group (ng/L: 342.14±121.38 vs. 376.63±108.66, P < 0.05). Logistic regression analysis showed that after adjusting for age, gender, blood lipid parameters, diabetes and other factors, the increase in serum OPG level was an independent risk factor for CHD [odds ratio (OR) = 1.995, 95% confidence interval (95%CI) = 1.935-2.066, P = 0.012]. PCR-RFLP results showed that TT, TC and CC genotypes were found in T149C and T950C of OPG promoter. According to Hardy-Weinberg equilibrium test, the polymorphisms of OPG T149C and T950C accorded with Hardy-Weinberg law, achieving genetic balance with representative of the population. The frequencies of TT, TC, CC and alleles T and C in T149C genotypes of non-CHD group were 53.5%, 42.9%, 3.6%, 75.0% and 25.0%, respectively, and they were 43.1%, 50.3%, 6.6%, 68.2% and 31.8%, respectively in CHD group. There were statistically significant differences in genotype and allele frequencies between the two groups (all P < 0.05). It was shown by Logistic regression analysis that the risk of CHD in TC+CC genotype of T149C was 1.86 of TT genotype (OR = 1.86, 95%CI = 1.24-2.78, P = 0.003). It was suggested that C allele might be a susceptible gene for CHD. In non-CHD group, the frequencies of TT, TC, CC, and alleles T and C in T950C genotypes were 39.8%, 46.5%, 13.7%, 63.1% and 36.9%, respectively. They were 39.4%, 43.4%, 17.2%, 61.1% and 38.9%, respectively in CHD group. There were no significant differences in genotype and allele frequencies between the two groups (all P > 0.05). Logistic regression analysis showed that TC+CC genotype of T950C was not related with CHD.@*CONCLUSIONS@#The increased level of serum OPG was closely related with CHD and could be used as a risk factor for CHD. The cases carried OPG T149C TC+CC genotype might have the risk suffering CHD. C allele is might be a susceptible gene.
Assuntos
Feminino , Humanos , Masculino , China/epidemiologia , Doença das Coronárias/epidemiologia , Osteoprotegerina/genética , Polimorfismo Genético , Regiões Promotoras Genéticas/genética , Fatores de RiscoRESUMO
Human papillomavirus infection is associated with the development of malignant and benign neoplasms. Approximately 40 viral types can infect the anogenital mucosa and are categorized into high- and low-risk oncogenic human papillomavirus, depending on their association with the development of cervical carcinoma. High-risk human papillomavirus 16 and 18 are detected in 55% and 15% of all invasive cervical squamous cell carcinomas worldwide, respectively. Low-risk human papillomavirus 6 and 11 are responsible for 90% of genital warts and are also associated with the development of recurrent respiratory papillomatosis. Human papillomavirus preferentially infects mitotic active cells of the basal layer from both mucosal and cutaneous epithelium through microabrasions. The viral life cycle synchronizes with the epithelial differentiation program, which may be due, in part, to the binding of differentially expressed cellular transcription factors to the long control region throughout the various epithelial layers. This review aimed to summarize the current knowledge regarding the mechanisms by which viral gene expression is regulated and the influence of human papillomavirus heterogeneity upon this phenomenon. A better understanding of the regulatory mechanisms may elucidate the particularities of human papillomavirus-associated pathogenesis and may provide new tools for antiviral therapy.
Assuntos
Humanos , Papillomaviridae/genética , Fatores de Transcrição/genética , Regulação Viral da Expressão Gênica , Infecções por Papillomavirus/virologia , Papillomaviridae/fisiologia , Proteínas Oncogênicas Virais/genética , Regiões Promotoras Genéticas/genéticaRESUMO
Introducción. Una parte de los aislamientos de Mycobacterium tuberculosis multirresistente también presenta resistencia a la etionamida. Es importante determinar si la resistencia a la isoniacida es independiente o se cruza con la resistencia a la etionamida, ya que si sucede lo segundo habría que reevaluar el tratamiento antituberculoso de segunda línea. La prueba molecular GenoType MTBDR plus ® detecta las mutaciones asociadas con la resistencia a isoniacida y podría detectar la resistencia cruzada a la etionamida. Objetivo. Evaluar la prueba GenoType MTBDR plus ® y comparar su desempeño con el de la secuenciación, en la detección de mutaciones en el gen katG y en el promotor inhA en aislamientos clínicos de M. tuberculosis multirresistente. Materiales y métodos. Se utilizaron el estuche comercial GenoType MTBDR plus 1.0 ® y la secuenciación para evaluar mutaciones en el gen katG y en el promotor inhA en 30 aislamientos de M. tuberculosis multirresistente con resistencia a la etionamida. La cepa de laboratorio H37Rv y tres aislamientos sensibles a los medicamentos de primera línea, sirvieron de control. Resultados. Al comparar los resultados de la secuenciación y de la prueba GenoType MTBDR plus ® , el índice kappa fue de 1. Todos los aislamientos resistentes a la isoniacida y la etionamida tenían las mutaciones detectadas con la prueba GenoTypeMTBDR plus ® en el gen katG, y 40 % de ellos, las detectadas en el promotor inhA. Mediante secuenciación se encontraron, además, mutaciones en katG en posiciones diferentes a las detectadas por la prueba GenoType MTBDR plus ® . Conclusión. La prueba GenoTypeMTBDR plus ® tiene la capacidad de detectar rápidamente la resistencia a isoniacida. Además, los resultados del estudio sugieren que también podría utilizarse como prueba de tamización para detectar la resistencia cruzada a etionamida.
Introduction: A variable proportion of isolates of multidrug-resistant Mycobacterium tuberculosis also presents resistance to ethionamide. It is important to determine whether resistance to isoniazid is independent or crossed with resistance to ethionamide, given that this could lead to the re-evaluation of second-line anti-tuberculosis treatment. The GenoType MTBDR plus ® molecular test is used for the detection of MDR-MTB, as it identifies mutations associated with resistance to isoniazide and could detect cross-resistance with ethionamide. Objective: To evaluate the performance of GenoType MTBDR plus ® in comparison with sequencing in the detection of mutations in gene katG and promotor inhA in clinical isolates of multidrug-resistant M. tuberculosis . Materials and methods: The GenoType MTBDR plus 1.0 ® commercial kit and sequencing were used to evaluate mutations in gene katG and promotor inhA in 30 multidrug-resistant M. tuberculosis isolates that were resistant to ethionamide. The laboratory strain H37Rv and three pan-sensitive isolates acted as controls. Results: The kappa index for the comparison between the results of sequencing and GenoType MTBDR plus ® was 1. All the isolates resistant to isoniazid and ethionamide had the mutations detected by GenoTypeMTBDR plus ® in the katG gene and 40% of them in promotor inhA. Sequencing also revealed katG mutations in positions different to those detected by GenoType MTBDR plus ® . Conclusion: GenoType MTBDR plus ® is able to detect resistance to isoniazid rapidly. Our results suggest that it could also be used to screen for cross-resistance with ethionamide.
Assuntos
Humanos , Oxirredutases/genética , Proteínas de Bactérias/genética , Catalase/genética , Técnicas de Tipagem Bacteriana/métodos , Análise de Sequência de DNA/métodos , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Etionamida/farmacologia , Técnicas de Genotipagem , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Antituberculosos/farmacologia , DNA Bacteriano/genética , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas/genética , Etionamida/metabolismo , Genótipo , Isoniazida/metabolismo , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Antituberculosos/metabolismoRESUMO
Assuntos
Arabidopsis/microbiologia , Proteínas da Membrana Bacteriana Externa/genética , Brassica/microbiologia , Doenças das Plantas/microbiologia , Pseudomonas syringae/genética , Pseudomonas syringae/patogenicidade , Sequência de Bases , Meios de Cultura , Elementos de DNA Transponíveis/genética , Genes Bacterianos , Mutação/genética , Folhas de Planta/microbiologia , Regiões Promotoras Genéticas/genéticaRESUMO
The experiment aimed to specifically monitor the passage of lactobacilli
Assuntos
Animais , Duodeno/microbiologia , Cabras/microbiologia , Lactobacillus plantarum/fisiologia , Rúmen/microbiologia , Administração Oral , Eletroporação , Fluorescência , Proteínas de Fluorescência Verde/genética , Lactobacillus plantarum/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genéticaRESUMO
BackgroundThe hepatitis C virus has been recognized as the leading cause of chronic liver disease in the world. Host genetic factors have been implicated in the persistence of hepatitis C virus infection. Single nucleotide polymorphisms at positions -607 C/A (rs1946518) and -137 G/C (rs187238) in the IL-18 gene promoter have been suggested to be associated with delayed hepatitis C virus clearance and persistence of the disease.ObjectiveIdentify these polymorphisms in a population infected with hepatitis C virus from the Brazilian Amazon region.MethodsIn a cross-sectional analytical study conducted in Belém, Pará, Brazil, 304 patients infected with hepatitis C virus were divided into two groups: group A, patients with persistent infection; group B, patients with spontaneous clearance. The control group consisted of 376 volunteers not infected with hepatitis C virus. Samples were analyzed by RT-PCR for the detection of viral RNA and by RFLP-PCR to evaluate the presence of the -137 G/C and -607 C/A IL-18 gene promoter polymorphisms.ResultsComparison of polymorphism allele frequencies between the patient and control groups showed a higher frequency of allele C at position -607 among patients (P=0.02). When the association between the polymorphisms and viral infection was analyzed, patients carrying genotype C/A at position -607 were found to be at higher risk of persistent hepatitis C virus infection (P=0.03).ConclusionThe present results suggest a possible role of the -607 IL-18 gene promoter polymorphism in the pathogenesis of hepatitis C virus infection.
ContextoO vírus da hepatite C é reconhecido como a maior causa de doença hepática crônica no mundo. Fatores genéticos do hospedeiro têm sido implicados na persistência da infecção pelo vírus da hepatite C. Polimorfismos de nucleotídeos únicos na posição -607 C/A (rs1946518) e -137 G/C (rs187238) na região promotora do gene da IL-18 têm sido evidenciados em alguns estudos que sugerem sua associação ao atraso na depuração do vírus da hepatite C e na persistência da doença.ObjetivoO presente estudo pretende identificar esses polimorfismos em uma população da região da Amazônia Brasileira infectada pelo vírus da hepatite C.MétodosEstudo do tipo transversal analítico no município de Belém-PA foi realizado em 304 pacientes infectados pelo vírus da hepatite C, divididos em: grupo A, pacientes com infecção persistente; grupo B, pacientes que apresentaram clareamento viral. Como grupo controle participaram 376 voluntários não infectados pelo vírus da hepatite C. As amostras foram submetidas à RT-PCR, para detecção do RNA viral e, posteriormente, à RFLP-PCR para avaliação do polimorfismo na região promotora do gene da IL-18, nas posições -137 G/C e -607 C/A.ResultadosA comparação das frequências dos polimorfismos no grupo de pacientes com o grupo controle demonstrou uma maior frequência do alelo C na posição -607 entre os pacientes (P=0,02) que no grupo controle. Na análise da associação dos polimorfismos com a infecção viral foi obtido que portadores do genótipo C/A na posição -607 possuíam maior risco de infecção persistente pelo vírus da hepatite C (P=0,03).ConclusãoNossos resultados sugerem uma possível implicação do polimorfismo da região promotora -607 do gene IL-18 na patogenia da infecção pelo vírus da hepatite C.
Assuntos
Feminino , Humanos , Masculino , Hepatite C/genética , /genética , Polimorfismo de Nucleotídeo Único , Brasil , Estudos Transversais , DNA Viral/análise , Predisposição Genética para Doença , Genótipo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Regiões Promotoras Genéticas/genética , RNA ViralRESUMO
Paracoccidioidomycosis (PCM) is caused by dimorphic fungi from theParacoccidioides brasiliensis complex. Previous studies have demonstrated that the severity of disease is associated with a T-helper 2 immune response characterised by high interleukin (IL)-4 production. In the present study we analysed two polymorphisms in the IL-4gene (-590 C/T and intron-3 microsatellite) in 76 patients with PCM and 73 control subjects from an endemic area. The production of IL-4 by peripheral blood mononuclear cells after antigen or phytohaemagglutinin stimulation was determined by ELISA. A significant correlation was observed between the RP2/RP2 intron-3 genotype and infection with Paracoccidioides sp.(p = 0.011), whereas the RP1/RP1 genotype was correlated with resistance. No significant correlation was observed for the IL-4promoter polymorphism. Furthermore, the low IL-4 expression observed in the control group compared with patients was associated with the RP1/RP1 genotype. These results suggest that IL-4polymorphisms might be associated with the ability of the host to control Paracoccidioides sp.infection. The relevance of this polymorphism is supported by the observation that patients with disease produce high levels of IL-4 following mitogen or antigen stimulation. The IL-4gene is located in the cytokine cluster region of chromosome 5 where other polymorphisms have also been described.
Assuntos
Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Doenças Endêmicas , Predisposição Genética para Doença , /genética , /metabolismo , Paracoccidioidomicose/imunologia , Polimorfismo Genético/imunologia , Ensaio de Imunoadsorção Enzimática , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Repetições de Microssatélites , Paracoccidioidomicose/epidemiologia , Regiões Promotoras Genéticas/genética , Estatísticas não ParamétricasRESUMO
Pseudomonas syringae pv. maculicola is a natural pathogen of members of the Brassicaceae plant family. Using a transposon-based mutagenesis strategy in Pseudomonas syringaepv. maculicola M2 (PsmM2), we conducted a genetic screen to identify mutants that were capable of growing in M9 medium supplemented with a crude extract from the leaves of Arabidopsis thaliana. A mutant containing a transposon insertion in the hrpZ gene (PsmMut8) was unable to infect adult plants from Arabidopsis thaliana or Brassica oleracea, suggesting a loss of pathogenicity. The promotorless cat reporter present in the gene trap was expressed if PsmMut8 was grown in minimal medium (M9) supplemented with the leaf extract but not if grown in normal rich medium (KB). We conducted phylogenetic analysis using hrpAZB genes, showing the classical 5-clade distribution, and nucleotide diversity analysis, showing the putative position for selective pressure in this operon. Our results indicate that the hrpAZB operon from Pseudomonas syringaepv. maculicola M2 is necessary for its pathogenicity and that its diversity would be under host-mediated diversifying selection.
Assuntos
Arabidopsis/microbiologia , Brassica/microbiologia , Doenças das Plantas/microbiologia , Proteínas da Membrana Bacteriana Externa/genética , Pseudomonas syringae/genética , Pseudomonas syringae/patogenicidade , Elementos de DNA Transponíveis/genética , Folhas de Planta/microbiologia , Genes Bacterianos , Meios de Cultura , Mutação/genética , Regiões Promotoras Genéticas/genética , Sequência de BasesAssuntos
Animais , Ratos , Fator Natriurético Atrial/metabolismo , Cálcio/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Poli(ADP-Ribose) Polimerases/metabolismo , Angiotensina II/farmacologia , Animais Recém-Nascidos , Fator Natriurético Atrial/genética , Cálcio/metabolismo , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , /metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Contração Miocárdica/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos Sprague-Dawley , Ribose/metabolismoRESUMO
Gastric cancer is one of the most common cancers in the world. The aims of this study were to evaluate the association between polymorphisms in TFF gene family, TFF1, TFF2, and TFF3 and the risk of gastric cancer (GC) and GC subgroups in a Korean population via a case-control study. The eight polymorphisms in TFF gene family were identified by sequencing and genotyped with 377 GC patients and 396 controls by using TaqMan genotyping assay. The rs184432 TT genotype of TFF1 was significantly associated with a reduced risk of GC (odds ratio, [OR) = 0.45; 95% confidence interval, [CI] = 0.25-0.82; P = 0.009), more protective against diffuse-type GC (OR = 0.20; 95% CI = 0.05-0.89; P = 0.035) than GC (OR = 0.34; 95% CI = 0.14-0.82; P = 0.017) in subjects aged < 60 yr, and correlated with lymph node metastasis negative GC and diffuse-type GC (OR = 0.44; 95% CI = 0.23-0.86; P = 0.016 and OR = 0.20; 95% CI = 0.05-0.87; P = 0.031, respectively). In addition, a decreased risk of lymph node metastasis negative GC and diffuse-type GC was observed for rs225359 TT genotype of TFF1 (OR = 0.46, 95% CI = 0.24-0.88; P = 0.020 and OR = 0.21, 95% CI = 0.05-0.88; P = 0.033, respectively). These findings suggest that the rs184432 and rs225359 polymorphisms in TFF1 have protective effects for GC and contribute to the development of GC in Korean individuals.
Assuntos
Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Biomarcadores Tumorais/genética , Marcadores Genéticos/genética , Predisposição Genética para Doença/epidemiologia , Incidência , Peptídeos/genética , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes , República da Coreia/epidemiologia , Medição de Risco/métodos , Sensibilidade e Especificidade , Neoplasias Gástricas/epidemiologia , Proteínas Supressoras de Tumor/genéticaRESUMO
Short telomeres are known as one of the risk factors for human cancers. The present study was conducted to evaluate the association between 6 polymorphisms, which were related with short telomere length in the Korean population, and lung cancer risk using 1,100 cases and 1,096 controls. Among the 6 polymorphisms, TERT rs2853669 was significantly associated with increased lung cancer risk under a recessive model (odds ratio [OR]=1.38, 95% confidence interval [CI]=1.05-1.81, P=0.02). The effect of rs2853669 on lung cancer risk was significant in younger individuals (OR=1.73, 95% CI=1.18-2.54, P=0.005) and adenocarcinoma (OR=1.50, 95% CI=1.07-2.07, P=0.02). Our results suggest that a common functional promoter polymorphism, TERT rs2853669, may influence both telomere length and lung cancer risk in the Korean population.
Assuntos
Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adenocarcinoma/epidemiologia , Estudos de Casos e Controles , Frequência do Gene/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Neoplasias Pulmonares/epidemiologia , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , República da Coreia/epidemiologia , Telomerase/genética , Telômero/fisiologia , Homeostase do Telômero/genéticaRESUMO
PURPOSE: Although the polymorphisms of erythrocyte complement receptor type 1 (CR1) in patients with malaria have been extensively studied, a question of whether the polymorphisms of CR1 are associated with severe malaria remains controversial. Furthermore, no study has examined the association of CR1 polymorphisms with malaria in Chinese population. Therefore, we investigated the relationship of CR1 gene polymorphism and malaria in Chinese population. MATERIALS AND METHODS: We analyzed polymorphisms of CR1 gene rs2274567 G/A, rs4844600 G/A, and rs2296160 C/T in 509 patients with malaria and 503 controls, using the Taqman genotyping assay and PCR-direct sequencing. RESULTS: There were no significant differences in the genotype, allele and haplotype frequencies of CR1 gene rs2274567 G/A, rs4844600 G/A, and rs2296160 C/T polymorphisms between patients with malaria and controls. Furthermore, there was no association of polymorphisms in the CR1 gene with the severity of malaria in Chinese population. CONCLUSION: These findings suggest that CR1 gene rs2274567 G/A, rs4844600 G/A, and rs2296160 C/T polymorphisms may not be involved in susceptibility to malaria in Chinese population.
Assuntos
Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Alelos , Povo Asiático , Estudos de Casos e Controles , China , Eritrócitos/parasitologia , Predisposição Genética para Doença , Genótipo , Haplótipos , Malária/etnologia , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Receptores de Complemento/sangue , Taq PolimeraseRESUMO
The Wnt signaling pathway has regulatory roles in cell proliferation, differentiation, and polarity. Aberrant Wnt pathway regulation can lead to abnormal cell proliferation and cancer, and loss of Wnt7a expression has been demonstrated in lung cancer cell lines. E-cadherin keeps intercellular integrity and prevents metastasis. Therefore, E-cadherin has been known as a prognostic factor in cancer. In the present study, we investigated the E-cadherin expression status by immunohistochemical stain and the Wnt7a promoter methylation status in human non-small cell lung carcinoma (NSCLC) by methylation-specific PCR. We also analyzed their correlations with clinicopathological factors. Methylation of the Wnt7a gene promoter was detected in the lung tissues of 32 of 121 (26.4%) patients with NSCLC. Wnt7a promoter methylation was correlated with advanced tumor stage (P = 0.036) and distant metastasis (P = 0.037). In addition, Wnt7a promoter methylation showed correlation with loss of E-cadherin expression (P < 0.001). However, Wnt7a promoter methylation was not closely related with gender, age, histological type, or smoking habit. Even though Wnt7a methylation could not show significant correlation with the long term survival of the patients with limited follow up data, these findings suggest that loss of the Wnt7a gene induced by promoter methylation might be another prognostic factor for NSCLC and that restoration of Wnt7a may be a promising treatment for NSCLC.
Assuntos
Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Caderinas/biossíntese , Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA/genética , Neoplasias Pulmonares/genética , Metástase Neoplásica/genética , Estadiamento de Neoplasias , Regiões Promotoras Genéticas/genética , República da Coreia , Biomarcadores Tumorais/genética , Proteínas Wnt/genéticaRESUMO
The Wnt signaling pathway has regulatory roles in cell proliferation, differentiation, and polarity. Aberrant Wnt pathway regulation can lead to abnormal cell proliferation and cancer, and loss of Wnt7a expression has been demonstrated in lung cancer cell lines. E-cadherin keeps intercellular integrity and prevents metastasis. Therefore, E-cadherin has been known as a prognostic factor in cancer. In the present study, we investigated the E-cadherin expression status by immunohistochemical stain and the Wnt7a promoter methylation status in human non-small cell lung carcinoma (NSCLC) by methylation-specific PCR. We also analyzed their correlations with clinicopathological factors. Methylation of the Wnt7a gene promoter was detected in the lung tissues of 32 of 121 (26.4%) patients with NSCLC. Wnt7a promoter methylation was correlated with advanced tumor stage (P = 0.036) and distant metastasis (P = 0.037). In addition, Wnt7a promoter methylation showed correlation with loss of E-cadherin expression (P < 0.001). However, Wnt7a promoter methylation was not closely related with gender, age, histological type, or smoking habit. Even though Wnt7a methylation could not show significant correlation with the long term survival of the patients with limited follow up data, these findings suggest that loss of the Wnt7a gene induced by promoter methylation might be another prognostic factor for NSCLC and that restoration of Wnt7a may be a promising treatment for NSCLC.