Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 25
Filtrar
1.
Front Public Health ; 9: 710209, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34805062

RESUMO

Most cervical cancers were closely associated with human papillomavirus (HPV) infections. Therefore, understanding the ecological diversity of HPV prevalence and genotype distribution among various populations in different geographical regions was essential for optimizing HPV vaccination and maximizing the vaccination effects. A total of 12,053 patient data from the three-level hospitals in Hengyang city were retrospectively analyzed. In this study, the HPV prevalence was 10.16% overall, and the multiple-type infection rate was 1.83%. The HR-HPV infection rate was 8.52%. The top six HPV genotypes were as follows in descending order: HPV16, HPV58, HPV52, HPV39, HPV51, and HPV53. The HPV prevalence in the group above 60 years old was the most, and their HR-HPV infection rate corresponded to the most too. The infection rates of HPV and HR-HPV among outpatients were both lower than those among the hospitalized-patients, respectively. Among the hospitalized-patients, the infection rates of HPV and HR-HPV among the 50-60 years group were the most in both. The HR-HPV ratio-in-positive among HPV-positive patients with the histopathologic examination was higher than that among those patients without. Among 52 HPV-positive patients with cervical squamous carcinoma, the ratio-in-positive of HPV16 was 61.54%. This study demonstrated that the HPV prevalence varied with age among women from Hengyang district of Hunan province in China and showed that HPV16, HPV58, HPV52, HPV39, HPV51, and HPV53 genotypes were more popularly distributed in this region, which could provide the experimental basis for Chinese public health measures on cervical cancer prevention.


Assuntos
Infecções por Papillomavirus , China/epidemiologia , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/prevenção & controle , Prevalência , Estudos Retrospectivos
2.
Int J Med Microbiol ; 308(7): 776-783, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29891193

RESUMO

Mycoplasma pneumoniae (M. pneumoniae), as an obligate parasite, has evolved a protective strategy for coping with oxidative challenges caused by M. pneumoniae itself as well as the host immune system. However, to date, few antioxidant enzymes have been identified in mycoplasmas. In this report, we identified a protein encoded by the mpn668 gene from M. pneumoniae with a putative function as an organic hydroperoxide reductase (Ohr). The results indicated that the recombinant 140 amino acid protein, designated rMPN668, displayed hydroperoxidase activity towards both organic (tert-butyl hydroperoxide) and inorganic (hydrogen peroxide) hydroperoxides in the presence of a reducing agent such as dithiothreitol. Moreover, the expression of mpn668 in M. pneumoniae is upregulated in response to oxidative stress. Additionally, homology modeling of MPN668 and a molecular dynamics simulation suggest that both Cys55 and Cys119 form part of the active site of the protein. Mutants in which Cys55 or Cys119 were replaced with a serine lack antioxidant activity, indicating that MPN668 is a Cys-based peroxidase, consistent with it representing a new member of the Ohr family.


Assuntos
Farmacorresistência Bacteriana/genética , Peróxido de Hidrogênio/farmacologia , Mycoplasma pneumoniae/genética , Peroxirredoxinas/genética , terc-Butil Hidroperóxido/farmacologia , Sequência de Aminoácidos , Regulação Bacteriana da Expressão Gênica , Simulação de Dinâmica Molecular , Mycoplasma pneumoniae/efeitos dos fármacos , Mycoplasma pneumoniae/enzimologia , Estresse Oxidativo/fisiologia , Homologia de Sequência de Aminoácidos
3.
BMC Microbiol ; 17(1): 153, 2017 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-28693414

RESUMO

BACKGROUND: Chlamydia pneumoniae (C. pneumoniae) is pathogenic to humans, by causing pulmonary inflammation or bronchitis in both adolescents and young adults. However, the molecular signals linking C. pneumoniae components to inflammation remain elusive. This study was to investigate the effect of Chlamydia-specific Cpn0423 of C. pneumoniae on C. pneumoniae-mediated inflammation. RESULTS: Cpn0423 was detected outside of C. pneumoniae inclusions, which induced production of several cytokines including macrophage inflammatory protein-2 (MIP-2) and interleukins (ILs). Production of the Cpn0423-induced cytokines was markedly reduced in cells pretreated with NOD2-siRNA, but not with negative control oligonucleotides. Mice treated with Cpn0423 through intranasal administration exhibited pulmonary inflammation as evidenced by infiltration of inflammatory cells, increased inflammatory scores in the lung histology, recruitment of neutrophils and increased cytokines levels in the BALF. CONCLUSION: Cpn0423 could be sensed by NOD2, which was identified as an essential element in a pathway contributing to the development of C. pneumoniae -mediated inflammation.


Assuntos
Proteínas de Bactérias/imunologia , Infecções por Chlamydophila/imunologia , Chlamydophila pneumoniae/imunologia , Mediadores da Inflamação/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Pneumonia Bacteriana/microbiologia , Animais , Proteínas de Bactérias/genética , Quimiocina CXCL2/genética , Quimiocina CXCL2/imunologia , Infecções por Chlamydophila/genética , Infecções por Chlamydophila/microbiologia , Chlamydophila pneumoniae/genética , Humanos , Interleucinas/genética , Interleucinas/imunologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Adaptadora de Sinalização NOD2/genética , Pneumonia Bacteriana/genética , Pneumonia Bacteriana/imunologia
4.
Cell Mol Biol Lett ; 20(5): 788-97, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26540225

RESUMO

Death domain associated protein (Daxx), a multi-functional protein, plays an important role in transcriptional regulation, cell apoptosis, carcinogenesis, anti-virus infection and so on. However, its regulatory mechanisms for both cell survival and apoptosis remain largely obscure. Our review of recent studies shows that Daxx has many interesting functional dualities and can provide a reference for further research on Daxx.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Cromatina/metabolismo , Proteínas Correpressoras , DNA Helicases/química , DNA Helicases/metabolismo , Humanos , Chaperonas Moleculares , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Telomerase/metabolismo , Proteína Nuclear Ligada ao X
5.
Biomed Res Int ; 2015: 652416, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25685800

RESUMO

Chlamydophila psittaci (C. psittaci) is a human zoonotic pathogen, which could result in severe respiratory disease. In the present study, we investigated the role and mechanism of the type III secretion system (T3SS) of C. psittaci in regulating the inflammatory response in host cells. C. psittaci-infected THP-1 cells were incubated with the specific T3SS inhibitor INP0007, inhibitors of ERK, p38, or JNK, and the levels of inflammatory cytokines were analyzed using Q-PCR and ELISA. The levels of ERK, p38, and JNK phosphorylation were analyzed by Western blot. Our results verified that INP0007 inhibited chlamydial growth in vitro, but the coaddition of exogenous iron completely reversed the growth deficit. INP0007 inhibited the growth of C. psittaci and decreased the levels of IL-8, IL-6, TNF-α, and IL-1ß. Exogenous iron restored the chlamydial growth but not the production of inflammatory cytokines. These results demonstrated that the expression of inflammatory cytokines during infection was associated with the T3SS which reduced by incubation with ERK and JNK inhibitors, but not with p38 inhibitor. We concluded that the T3SS elicited inflammatory responses by activating the JNK or ERK signaling pathways in the infection of C. psittaci.


Assuntos
Sistemas de Secreção Bacterianos , Chlamydophila psittaci/metabolismo , MAP Quinase Quinase 4/metabolismo , Sistema de Sinalização das MAP Quinases , Psitacose/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linhagem Celular Tumoral , Chlamydophila psittaci/genética , Citocinas/genética , Citocinas/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/genética , Psitacose/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética
6.
Biomed Environ Sci ; 28(1): 25-35, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25566860

RESUMO

OBJECTIVE: A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis). METHODS: 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay. RESULTS: The sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively. CONCLUSION: Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.


Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Immunoblotting/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/farmacologia , Genótipo , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Fatores de Tempo
7.
Environ Toxicol ; 30(9): 1082-90, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24616003

RESUMO

Perfluorooctanyl sulfonate (PFOS), a cardiac toxicity compound, has been widely detected in the environment and in organisms. However, the toxic mechanism is not clear. Our previous study indicated that prenatal PFOS exposure led to swollen mitochondrial with vacuolar structure and loss of cristae in offsping's heart. The purpose of this study was to investigate the effect of PFOS on the apoptosis in developing heart and mitochondria-mediated apoptosis pathway. Pregnant Sprague-Dawley (SD) rats were exposed to PFOS at doses of 0.1, 0.6, and 2.0 mg/kg-d and 0.05% Tween 80 as control by gavage from gestation day 2 (GD 2) to GD 21. Apoptosis, as well as expression of apoptosis related genes associated with mitochondrial-mediated apoptosis pathway, including p53, bcl-2, bax, cytochrome c, caspase-9, and caspase-3 were analyzed in heart tissues from weaned (postnatal day 21, PND 21) offspring. The results showed that prenatal PFOS exposure resulted in apoptosis in the offspring's heart. The mRNA and protein expression levels of p53, bax, cytochrome c, caspase-9, and caspase-3 in the offspring's heart were enhanced in various PFOS-treated groups, meanwhile, the bcl-2 expression levels were decreased. Our results indicated that prenatal PFOS exposure induced the apoptosis of weaned offspring rat heart tissue via mitochondria-mediated apoptotic pathway.


Assuntos
Ácidos Alcanossulfônicos/toxicidade , Apoptose/efeitos dos fármacos , Fluorocarbonos/toxicidade , Coração/efeitos dos fármacos , Mitocôndrias/metabolismo , Animais , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Feminino , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Desmame , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
8.
Biomed Environ Sci ; 26(3): 169-75, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23425799

RESUMO

OBJECTIVE: To prepare antibodies against pORF5 plasmid protein of Chlamydia trachomatis and develop double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) for the detection of genital C. trachomatis infections. METHODS: The pORF5 protein was expressed in Escherichia coli and used to immunize BALB/c mice and New Zealand rabbits to produce monoclonal antibodies (mAbs) and polyclonal antibody (pAb) for DAS-ELISAs. Clinical samples from 186 urogenital infection patients (groups I) and 62 healthy donors (groups II) were detected in parallel by the DAS-ELISAs developed in this study and by IDEIA PCE commercial ELISA. RESULTS: Two hybridoma cell lines, named 2H4 and 4E6, stably secreting specific mAbs against pORF5 were obtained. The mAb 2H4 was recognized by 32 (17.20%, positive recognition rate) and 25 (13.44%), mAb 2H4 by 0 (0%) and 2 (3.22%) samples from groups I and II, respectively. The sensitivities of mAbs 2H4 and 4E6 were 92.11% and 77.78% and the specificities were 100% and 96.88%, respectively in relation to the IDEIA PCE commercial ELISA. The sensitivities of detection for the DAS-ELISAs were 10 ng/mL (based on 2H4) and 18 ng/mL (based on 4E6). CONCLUSION: Two DAS-ELISAs were developed in this study that provided a feasible and effective assay that could be considered alternative tools for the serodiagnosis of C. trachomatis infection.


Assuntos
Infecções por Chlamydia/diagnóstico , Chlamydia trachomatis/patogenicidade , Ensaio de Imunoadsorção Enzimática/métodos , Sistema Urogenital/microbiologia , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
9.
Biomed Environ Sci ; 26(3): 190-200, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23425802

RESUMO

OBJECTIVE: To study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure. METHODS: FP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and immunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples (159 patients with Lyme disease and 292 controls), all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index. RESULTS: Criteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively. CONCLUSION: Establishment of WB criteria for B. afzelii is important in validating the diagnostic assays for Lyme disease in China.


Assuntos
Western Blotting/métodos , Grupo Borrelia Burgdorferi/patogenicidade , Doença de Lyme/diagnóstico , Doença de Lyme/microbiologia , China , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos
10.
Biomed Environ Sci ; 25(6): 690-6, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23228839

RESUMO

OBJECTIVE: This paper aims to develop a monoclonal antibodies (MAbs)- based ELISA for detecting Chlamydophila pneumoniae (C. pneumoniae) antigens in humans with the variable domains (VD) 2 and 3 of the major outer membrane protein (MOMPVD2-VD3) and to assess its sensitivity and specificity by comparing with a widely used MAb that is able to recognize the elementary bodies of C. pneumoniae. METHODS: MOMPVD2-VD3 were overexpressed in Escherichia coli and purified by affinity chromatography. Mice were immunized with the recombinant antigen, and hybridomas secreting MAbs were screened. Three stable hybridomas clones were selected and named 5D6, 7G3, and 8C9. The MAbs-based ELISA was scrutinized for species-specific recognition with a number of human throat swab samples from Group I (156 patients with typical respiratory illness clinically confirmed before) and Group II (57 healthy donors). RESULTS: In Group I, 55 positive cases were detected by anti-EB MAb-based ELISA, 51 cases were positive by MAbs 5D6-based ELISA, and 33 and 38 cases were positive by MAb 8C9 and 7G3-based ELISA respectively. Of the 57 samples from Group II "healthy donors", 5 were positive and 52 were negative with both anti-EB and 5D6-based tests, while 2 and 3 positive cases were identified by the other two MAb-based ELISAs respectively. CONCLUSION: The novel MOMPVD2-VD3 MAb-based assay may have higher specificity than the anti-EB MAb, which may possibly be used as an alternative tool for the diagnosis of C. pneumoniae infection.


Assuntos
Anticorpos Monoclonais , Proteínas da Membrana Bacteriana Externa/imunologia , Infecções por Chlamydophila/microbiologia , Chlamydophila pneumoniae/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Animais , Infecções por Chlamydophila/diagnóstico , Humanos , Camundongos , Estrutura Terciária de Proteína
11.
J Mol Model ; 18(3): 1009-13, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21656240

RESUMO

The biological toxicity of uranyl ion (UO (2) (2+) ) lies in interacting with proteins and disrupting their native functions. The structural and functional consequences of UO (2) (2+) interacting with cytochrome b (5) (cyt b (5)), a small membrane heme protein, and its heme axial ligand His39Ser variant, cyt b (5) H39S, were investigated both experimentally and theoretically. In experiments, although cyt b (5) was only slightly affected, UO (2) (2+) binding to cyt b (5) H39S with a K (D) of 2.5 µM resulted in obvious alteration of the heme active site, and led to a decrease in peroxidase activity. Theoretically, molecular simulation proposed a uranyl ion binding site for cyt b (5) at surface residues of Glu37 and Glu43, revealing both coordination and hydrogen bonding interactions. The information gained in this study provides insights into the mechanism of uranyl toxicity toward membrane protein at an atomic level.


Assuntos
Citocromos b5/química , Citocromos b5/genética , Urânio/química , Sítios de Ligação , Domínio Catalítico , Variação Genética , Heme , Ligação de Hidrogênio , Íons , Cinética , Modelos Moleculares , Mutação de Sentido Incorreto , Ligação Proteica , Urânio/toxicidade
12.
J Mol Model ; 18(4): 1591-6, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21805122

RESUMO

Sperm whale myoglobin (swMb) is a well-studied heme protein, both experimentally and theoretically. Comparatively, little attention has been paid to another member of Mb family, Aplysia limacina myoglobin (apMb). swMb and apMb have the same overall structure and perform the same biological function, i.e., O(2) carrier, while using a distinct heme active site. To provide insights into the structure-function relationship for these two Mbs, we herein made a comparison in terms of their dynamics properties using molecular dynamics simulation. We analyzed the overall structure and protein motions, as well as the intramolecular contacts, namely salt-bridges and hydrogen bonds, especially the interactions between the heme propionate groups and the polypeptide chain. The internal cavities in apMb were also compared to the well-known xenon and other cavities in swMb. Based on current simulations, we propose a unique "arginine gate" for apMb, which has a similar function to the histidine gate observed for swMb in previous studies. This study provides valuable information for understanding the homology of heme proteins, and also aids in rational design of structural and functional heme proteins by alternating the heme active site.


Assuntos
Heme/química , Hemeproteínas/química , Mioglobina/química , Animais , Aplysia/química , Domínio Catalítico , Heme/metabolismo , Hemeproteínas/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Simulação de Dinâmica Molecular , Mioglobina/metabolismo , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Cachalote , Relação Estrutura-Atividade
13.
J Mol Model ; 18(4): 1553-60, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21805125

RESUMO

Bovine liver cytochrome b (5) (cyt b (5)), with heme bound noncovalently, has been converted into a cyt c-like protein (cyt b (5) N57C) by constructing a thioether linkage between the heme and the engineered cysteine residue. With no X-ray or NMR structure available, we herein performed a molecular modeling study of cyt b (5) N57C. On the other hand, using amino acid sequence information for a newly discovered member of the cyt b (5) family, domestic silkworm cyt b (5) (DS cyt b (5)), we predicted the protein structure by homology modeling in combination with MD simulation. The modeling structure shows that both Cys57 in cyt b (5) N57C, and Cys56, a naturally occurring cysteine in DS cyt b (5), have suitable orientations to form a thioether bond with the heme 4-vinyl group, as the heme is in orientation A. In addition to providing structural information that was not previously obtained experimentally, these modeling studies provide insight into the formation of cyt c-like thioether linkages in cytochromes, and suggest that c-type cyt b (5) maturation involves a b-type intermediate.


Assuntos
Citocromos b5/química , Citocromos c/química , Sequência de Aminoácidos , Animais , Bombyx/enzimologia , Domínio Catalítico , Bovinos , Citocromos b5/metabolismo , Citocromos c/metabolismo , Fígado/enzimologia , Modelos Químicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Alinhamento de Sequência , Relação Estrutura-Atividade , Sulfetos
14.
Nan Fang Yi Ke Da Xue Xue Bao ; 31(11): 1830-4, 2011 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-22126759

RESUMO

OBJECTIVE: To clone the plasmid protein pORF8 of Chlamydia trachomatis and localize its expression in Chlamydia-infected cells. METHODS: pORF8 gene was amplified and cloned into pGEX-6p vector, and the pORF8 fusion protein was expressed in E.coli XL1 Blue. After purification with glutathione-conjugated agarose beads, the pORF8 fusion protein was used to immunize BALB/c mice to generate polyclonal antibodies against pORF8 protein. The antibodies obtained were used to localize the plasmid protein pORF8 in Chlamydia-infected cells with immunofluorescence assay (IFA). RESULTS: The pORF8 gene 744 bp in length was successfully cloned and the GST fusion protein with a relative molecular mass of 54 000 was obtained. The cellular distribution pattern of the plasmid protein pORF8 was similar to that of the major outer membrane protein (MOMP), a known C. trachomatis inclusion body protein, but not to that of chlamydial protease-like activity factor (CPAF, a secreted protein). CONCLUSION: The plasmid protein pORF8 is localized on the bacterial organism as an inclusion body protein in C. trachomatis-infected cells. The cellular location of pORF8 protein can potentially provide important insights into the pathogenesis of C. trachomatis.


Assuntos
Proteínas de Bactérias/biossíntese , Chlamydia trachomatis/genética , Plasmídeos/biossíntese , Animais , Anticorpos/imunologia , Proteínas de Bactérias/genética , Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/química , Chlamydia trachomatis/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos/genética , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia
15.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 27(9): 954-8, 2011 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-21906466

RESUMO

AIM: To expresse the Chlamydia pneumoniae Cpn0810 in E.coli BL21, and to study weather could it inducing proinflamatory cytokines including TNF-α and IL-6 in human monocytic (THP-1) and cell apoptosis. METHODS: Polymerase chain reaction(PCR) was used to amplify the Cpn0810 gene, PCR products were purified and cloned into the prokaryotic expression vector pGEX6p-2. The restriction plasmids pGEX6p-2/Cpn0810 confirmed by PCR and sequencing was transformed into E.coli BL21. The recombinant protein was purified with glutathione S-transferase (GST) resin chromatography of Novagen after renaturation. THP-1 cells were stimulated by different concentrations of Cpn0810 and for various durations to test the production and the expression of TNF-α and IL-6 by ELISA. Cell apoptosis was detected in C.pneumoniae Cpn0810 cells by Hoechst33258 fluorescence staining and Cell apoptosis was detected in THP-1 cells by Annexin-V-FITC-propidiu-m iodide (PI) staining. RESULTS: The restriction enzymes cleavage analysis and nucleotide sequencing showed the target gene was successfully inserted into pGEX6p-2 prokaryotic expression vector. Cpn0810 stimulated THP-1 cell to produce proinflamatory cytokines including TNF-α and IL-6 in a dose and time-dependent manner. After THP-1 cells were treated with 10 mg/L Cpn0810 for 24 h, apoptosis with nuclear chromatin fragmentation as well as cell shrinkage was observed by fluorescent staining and microscopy; apoptosis of cell was detected after 24 h in THP-1 cells treated with Cpn0810. CONCLUSION: Cpn0810 recombinant protein could stimulate THP-1 cell to produce and express proinflamatory cytokines including TNF-α and IL-6; After THP-1 cells were treated with 10 mg/L Cpn0810 for 24 h, apoptosis of cell was detected after 24 h in THP-1 cells treated with Cpn0810.


Assuntos
Apoptose/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Chlamydophila pneumoniae/imunologia , Citocinas/biossíntese , Mediadores da Inflamação/metabolismo , Linhagem Celular , Chlamydophila pneumoniae/genética , Humanos , Interleucina-6/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Fator de Necrose Tumoral alfa/metabolismo
16.
Chemotherapy ; 57(2): 128-33, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21430380

RESUMO

PURPOSE: It was the aim of our study to evaluate the in vitro activities of tetracycline (TET), erythromycin (ERY) and levofloxacin (LVX) alone and in dual combinations against ureaplasmas. METHODS: The minimum inhibitory concentrations (MICs) of 51 ureaplasmal strains were determined by microdilution assay. RESULTS: TET was the most active when the antibiotics were used alone. The combinations resulted in significantly decreased MICs for every agent compared with the use of single antibiotics (p < 0.05, respectively), except for ERY in the ERY-LVX pair (p > 0.05), and decreased the MICs more significantly in the strains with an MIC ≥4 mg/l compared with MIC <4 mg/l, except for the TET-ERY pair. The ERY-LVX pair increased ERY MICs significantly in the MIC <4 mg/l group (p < 0.05). The combinations resulted in more beneficial MICs in strains where both agents had an MIC ≥4 mg/l compared with those where either had an MIC ≥4 mg/l, as well as in strains where either agent had an MIC <4 mg/l compared with those where both had an MIC <4 mg/l. CONCLUSIONS: Drugs in dual combinations always give more beneficial MICs against ureaplasmas than one agent alone. Combinational benefits prefer strains with a higher initial MIC.


Assuntos
Antibacterianos/farmacologia , Eritromicina/farmacologia , Levofloxacino , Ofloxacino/farmacologia , Tetraciclina/farmacologia , Ureaplasma/efeitos dos fármacos , Combinação de Medicamentos , Quimioterapia Combinada , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Infecções por Ureaplasma/tratamento farmacológico , Infecções por Ureaplasma/microbiologia
17.
J Zhejiang Univ Sci B ; 10(1): 67-76, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19198025

RESUMO

Mycoplasmas, the smallest free-living, self-replicating bacteria with diameters of 200 to 800 nm, have been reported to be associated with human diseases. It is well known that the mycoplasma lipoprotein/peptide is able to modulate the host immune system, whose N-terminal structure is an important factor in inducing immunity and distinguishing Toll-like receptors (TLRs). However, there is still no clear elucidation about the pathogenic mechanism of mycoplasma lipoprotein/peptide and the signaling pathway. Some researchers have focused on understanding the structures of these proteins and the relationships between their structure and biological function. This review provides an update on the research in this field.


Assuntos
Lipoproteínas/química , Lipoproteínas/metabolismo , Modelos Biológicos , Mycoplasma/química , Mycoplasma/metabolismo , Receptores Toll-Like/química , Receptores Toll-Like/metabolismo
18.
Wei Sheng Wu Xue Bao ; 47(5): 899-904, 2007 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-18062270

RESUMO

Designed to investigate the potential pathogenicity of Mycoplasma genitalium (M. genitalium) and its molecular mechanisms responsible for the induction of proinflammatory cytokines gene expression in human monocytic cells (THP-1) stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. genitalium. THP-1 cells were stimulated with LAMPs to analyze the production of proinflammatory cytokines and the expression of mRNA was detected by RT-PCR. Cell apoptosis was detected in THP-1 cells by Annexin V-propidium iodide staining. The activity of transcriptional factors, NF-kappaB, was examined in THP-1 cells treated with LAMPs by EMSA. The effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, on the production of proinflammatory cytokines, the expression of mRNA and apoptosis were also examined in THP-1 cells treated with LAMPs. M. genitalium LAMPs stimulate THP-1 cells to produce TNF-alpha, IL-1beta and IL-6 in dose- and time-dependent manner. The mRNA levels and cell apoptosis are also downregulated in response to LAMPs stimulation and inhibited by PDTC treatment. M. genitalium LAMPs are found to trigger NF-kappaB activation, a possible mechanism for the induction of mRNA expression and the cell apoptosis. This study demonstrated that M. genitalium may be an important etiological factor of certain disease due to the ability of LAMPs to stimulated the expression of mRNA and apoptosis, which is probably mediated through the activation of NF-kappaB.


Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Bactérias/toxicidade , Citocinas/biossíntese , Proteínas de Membrana/toxicidade , Monócitos/efeitos dos fármacos , Mycoplasma genitalium/patogenicidade , NF-kappa B/metabolismo , Células Cultivadas , Citocinas/genética , Humanos , Lipídeos , Monócitos/citologia , Monócitos/imunologia , RNA Mensageiro/análise
19.
Wei Sheng Wu Xue Bao ; 47(3): 512-6, 2007 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-17672316

RESUMO

To express the recombinant protein MOMP(VD2-VD3) of Chlamydia pneumoniae, and research on the immunocompetence of the MOMP(VD2-VD3) to support serodiagnosis,PCR and gene recombinant technique was used to clone the targeted DNA fragment from a strain AR-39. The recombinant plasmid was induced in E. coli BL21 after having constructed the prokaryotic expression system, then the immunocompetence of the expression product was analyzed by Western blot and indirected ELISA which is based on the animal experimentation. A group of control sera and 126 sera from patients with coronary heart disease were examined by using ELISAs based on the recombinant protein (MOMP(VD2-VD3), and then the results were evaluated comparing with a commercial ELISAs kit. The results of the Western blot and indirected ELISA showed ompA(VD2-VD3) gene inserted in pET30a could express a recombinant protein with the molecular weight of 24kDa in BL21 and specifically reacted with the antibodies against the MOMP. Specific humoral response was elicited after immune the BALB/c mouse with protein and the specific antibody titer was more than 1:20480. Using a panel of control sera, the participation of the recombinant antigen, the sensitivity and the specificity of the indirected ELISAs were 100% respectively. Comparisons between two methods in detecting 126 sero samples, the concordance of two tests was 96.3%. The results reported here show that the recombinant protein with excellent immunocompetence could benefit the research on the serodiagnosis to Chlamydia pneumoniae.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Infecções por Chlamydophila/diagnóstico , Chlamydophila pneumoniae/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Motivos de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Infecções por Chlamydophila/imunologia , Infecções por Chlamydophila/microbiologia , Chlamydophila pneumoniae/química , Chlamydophila pneumoniae/genética , Escherichia coli/metabolismo , Humanos , Imunoglobulina G/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
20.
Wei Sheng Wu Xue Bao ; 46(5): 807-11, 2006 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-17172033

RESUMO

The aim was to investigate the molecular mechanisms responsible for the inducible nitric oxide synthase (iNOS) gene expression stimulated by lipid associated membrane proteins (LAMPs) of Ureaplasma urealyticum (Uu). Mouse macrophages were stimulated by Ureaplasma urealyticum LAMPs to analyze the production of nitric oxide (NO) and the expression of iNOS detected by RT-PCR and Western blot. The activation of NF-kappaB was examined in mouse macrophages treated with LAMPs by indirect immunofluorescence (IFA), immunocytochemistry and Western blot. The effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB and of cycloheximide (CHX), a protein synthase inhibitor, on the expression of iNOS and on the activation of NF-kappaB were also investigated in mouse macrophages treated with LAMPs. Results showed Ureaplasma urealyticum LAMPs stimulated mouse macrophages to express iNOS and thus produce NO in dose- and time-dependent manners by activating nuclear factor kappaB. The activation of NF-kappaB and the expression of iNOS were inhibited by LAMPs combination with PDTC or CHX. In conclusion, these findings suggested Ureaplasma urealyticum may be an important pathogenic factor due to the ability of LAMPs to stimulate the expression of iNOS, which is probably medicated by the activation of NF-kappaB.


Assuntos
Macrófagos/enzimologia , Proteínas de Membrana/fisiologia , NF-kappa B/fisiologia , Óxido Nítrico Sintase Tipo II/genética , Ureaplasma urealyticum/fisiologia , Animais , Cicloeximida/farmacologia , Regulação Enzimológica da Expressão Gênica , Lipídeos , Camundongos , Óxido Nítrico/biossíntese , Prolina/análogos & derivados , Prolina/farmacologia , Tiocarbamatos/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA