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1.
Int J Tuberc Lung Dis ; 20(12): 1681-1688, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28000588

RESUMO

SETTING: Mexico City, Mexico. OBJECTIVE: To identify proteins synthetised by Mycobacterium tuberculosis in hypoxic culture, which resemble more closely a granuloma environment than aerobic culture, and to determine if they are recognised by antibodies from patients with active pulmonary tuberculosis (PTB). DESIGN: Soluble extracts from M. tuberculosis H37Rv cultured under aerobic or hypoxic conditions were analysed using two-dimensional polyacrylamide gel electrophoresis, and proteins over-expressed under hypoxia were identified by mass spectrometry. The presence of immunoglobulin (Ig) G, IgA and IgM antibodies against these proteins was determined in the serum of 42 patients with active PTB and 42 healthy controls. RESULTS: We selected three M. tuberculosis H37Rv proteins (alpha-crystallin protein [Acr, Rv2031c], universal stress protein Rv2623 and isocitrate lyase [ICL, RV0467]) that were over-expressed under hypoxia. Titres of anti-Acr and anti-ICL IgA antibodies were higher in patients than in healthy controls, with an area under the receiver operating characteristic curve of 0.71 for anti-ICL IgA antibodies. CONCLUSION: ICL could be used in combination with other M. tuberculosis antigens to improve the sensitivity and specificity of current serological TB diagnostic methods.


Assuntos
Anticorpos Antibacterianos/sangue , Imunoglobulina A/sangue , Isocitrato Liase/imunologia , Tuberculose Pulmonar/diagnóstico , alfa-Cristalinas/imunologia , Adulto , Idoso , Antígenos de Bactérias/sangue , Estudos de Casos e Controles , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , México , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Sensibilidade e Especificidade , Tuberculose Pulmonar/sangue , Adulto Jovem
2.
Immunol Invest ; 43(1): 28-40, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24063571

RESUMO

Dengue virus (DENV) RNA replication requires 2 viral proteins, non-structural protein 3 (NS3) and NS5. NS5 consists of 2 functional domains: a methyltransferase (MTase) domain involved in RNA cap formation and located in the amino terminal region and a RNA-dependent RNA polymerase domain essential for virus replication and located in the carboxyl terminal region. To gain additional insight into the structural interactions between viral proteins and cellular factors involved in DENV RNA replication, we generated a panel of rat monoclonal antibodies (mAbs) against the NS5 MTase domain. Six rat mAbs were selected from 41 clones, of which clone 13G7 was further characterized. The specificity of this antibody for NS5 was demonstrated by western blot of DENV-infected cells, which revealed that this antibody recognizes all 4 DENV serotypes. Furthermore, Western blotting analysis suggested that this antibody recognizes a sequential epitope of the NS5 protein. Positive and specific staining with 13G7 was detected predominantly in nuclei of DENV-infected cells, similarly a pattern was observed in both in human and monkey cells. Furthermore, the NS5 staining co-localized with a Lamin A protein (Pierson index: 0.7). In summary, this monoclonal antibody could be used to identify and evaluate different cellular factors that may interact with NS5 during DENV replication.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , RNA Polimerases Dirigidas por DNA/imunologia , Vírus da Dengue/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Especificidade de Anticorpos/imunologia , Linhagem Celular , Chlorocebus aethiops , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/metabolismo , Vírus da Dengue/classificação , Vírus da Dengue/genética , Humanos , Lamina Tipo A/metabolismo , Ligação Proteica/imunologia , Transporte Proteico , Ratos , Sorotipagem , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificação , Proteínas não Estruturais Virais/metabolismo
3.
Res Vet Sci ; 94(1): 9-21, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22884173

RESUMO

Spoligotyping is the most frequently used method for genotyping isolates of Mycobacterium bovis worldwide. In the current work, we compared spoligotypes from 1684 M. bovis isolates from Argentina (816), Brazil (412), Chile (66), Mexico (274) and Venezuela (116), obtained from cattle, humans, pigs, wild boars, farmed deer, goats, buffaloes, cats, and wild animals. A total of 269 different spoligotypes were found: 142 (8.4%) isolates presented orphan spoligotypes, whereas 1542 (91.6%) formed 113 different clusters. In cattle, SB0140 was the most representative spoligotype with 355 (24.6%) isolates, followed by SB0121 with 149 (10.3%) isolates. Clustering of spoligotypes ranged from 95.2% in Argentina to 85.3% in Mexico. Orphan spoligotypes were also variable, ranging from 23.7% in Mexico to 4.1% in Brazil. A large proportion of spoligotypes were common to the neighboring countries Argentina, Brazil and Chile. In conclusion, despite the diversity of spoligotypes found in the five countries studied, there are major patterns that predominate in these neighboring countries. These clusters may reflect a long-lasting active transmission of bovine tuberculosis or common historical origins of infection.


Assuntos
Mycobacterium bovis/genética , Tuberculose Bovina/microbiologia , Animais , Animais Selvagens/microbiologia , Argentina , Brasil , Búfalos/microbiologia , Gatos/microbiologia , Bovinos/microbiologia , Humanos , México , Tipagem Molecular/veterinária , Sus scrofa/microbiologia , Suínos/microbiologia , Tuberculose/veterinária , Venezuela
4.
J Hosp Infect ; 80(1): 74-6, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22055394

RESUMO

This study examined the frequency of occurrence of non-tuberculous mycobacteria (NTM) in potable water samples from a main trauma hospital in Mexico City. Sixty-nine potable water samples were collected, 23 from each source: cistern, kitchen tap and bathroom showers. Of the 69 samples, 36 harboured NTM species. Twenty-nine of the 36 isolates were Mycobacterium mucogenicum, two Mycobacterium rhodesiae, one Mycobacterium peregrinum, one Mycobacterium fortuitum and three were Mycobacterium spp. Hospital potable water harbouring NTM represents a potential source for nosocomial infections, therefore we suggest that hospital potable water microbiological guidelines should include testing for NTM species.


Assuntos
Água Potável/microbiologia , Mycobacterium/classificação , Mycobacterium/isolamento & purificação , Hospitais , Humanos , México , Infecções por Mycobacterium não Tuberculosas/prevenção & controle
5.
Eur J Clin Microbiol Infect Dis ; 27(6): 451-9, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18246375

RESUMO

Recently, the incidence of human mycobacterial infections due to species other than M. tuberculosis has increased worldwide. Since disease control depends on appropriate antimicrobial therapy, the precise identification of these species of clinical importance has become a major public health concern. Identification of mycobacteria has been hampered because of the lack of specific, rapid, and inexpensive methods. Therefore, we aimed at designing and validating a bacterial lysate-based polymerase chain reaction identification scheme. This scheme can classify clinical isolates into: (1) the genus Mycobacterium, (2) the M. tuberculosis complex, (3) the nontuberculous mycobacteria, and (4) the species M. avium, M. intracellulare, M. abscessus, M. chelonae, M. fortuitum and M. bovis of clinical importance, and M. gordonae, the most commonly encountered nonpathogenic species in clinical laboratories. By using M. fortuitum and M. avium lysates as models, the method sensitivity was determined to be 372 pg of DNA. In a blind parallel comparison between our approach and conventional biochemical tests, both assays correctly categorized 75 patient's mycobacterial isolates. However, our approach only required 4-9 h for categorization compared with at least 15 days by conventional tests. Furthermore, our methodology could also detect M. fortuitum and M. avium from liquid cultures, after only 2 and 6 days, respectively, of incubation. Our new identification scheme is therefore sensitive, specific, rapid, and economic. Additionally, it can help to provide proper treatment to patients, to control these diseases, and to improve our knowledge of the epidemiology of mycobacteriosis, all urgently needed, particularly in developing countries.


Assuntos
Bactérias/genética , Extratos Celulares/genética , Mycobacterium/patogenicidade , Sensibilidade e Especificidade , Técnicas de Tipagem Bacteriana , Humanos , Mycobacterium/classificação , Mycobacterium/genética , Mycobacterium/isolamento & purificação , Infecções por Mycobacterium/microbiologia , Infecções por Mycobacterium/fisiopatologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Especificidade da Espécie
6.
Epidemiol Infect ; 130(3): 485-90, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12825733

RESUMO

Bovine tuberculosis is a zoonotic disease that not only causes huge economic losses but also poses an important risk for human infection. The definitive identification of a clinical isolate relies on time-consuming, highly specialized and laborious biochemical tests. We have developed a method for the rapid and reliable identification of Mycobacterium bovis and for its simultaneous differentiation from other members of the M. tuberculosis complex. Furthermore, the technique also allowed us to distinguish M. tuberculosis complex members from other Mycobacterial species. The method comprises both a single PCR and a multiplex-PCR and can be confidently applied to samples of both veterinary and human origin.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Mycobacterium bovis/classificação , Reação em Cadeia da Polimerase/métodos , Tuberculose Bovina/diagnóstico , Tuberculose/diagnóstico , Animais , Bovinos , Primers do DNA , DNA Bacteriano/análise , Humanos
7.
Scand J Immunol ; 57(5): 439-45, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12753500

RESUMO

Assessment of cytokine expression has become crucial to understand host responses to infections as well as autoimmunity. Several approaches including Northern blot, RNase protection assay and enzyme-linked immunosorbent assay have been used for this purpose, but they are time consuming, labour intense, and relatively large quantity of the samples is usually required. Recently, a technique termed real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to determine genetic expression with great sensitivity and specificity; however, specialized instrumentation and costly reagents are usually needed. We aimed at using low-cost reagents for real-time PCR. This was achieved by adapting a conventional RT-PCR protocol to the quantitative real-time format, by the addition of the SYBR Green I reagent. We validated the approach by assessing the cytokine gene expression of murine splenocytes upon stimulation with phorbol 12-myristate 12-acetate (PMA)-ionomycin. The results using this technique were compared with those obtained with the well-established gene array method. We conclude that the use of the SYBR Green I reagent during real-time RT-PCR provides a highly specific and sensitive method to quantify cytokine expression with accuracy and no post-PCR manipulation.


Assuntos
Citocinas/biossíntese , Corantes Fluorescentes/análise , Perfilação da Expressão Gênica/métodos , Compostos Orgânicos , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/biossíntese , Actinas/biossíntese , Actinas/genética , Animais , Benzotiazóis , Sistemas Computacionais/economia , Análise Custo-Benefício , Custos e Análise de Custo , Citocinas/genética , Diaminas , Feminino , Perfilação da Expressão Gênica/economia , Indicadores e Reagentes/economia , Interferon gama/biossíntese , Interferon gama/genética , Interleucina-12/biossíntese , Interleucina-12/genética , Subunidade p40 da Interleucina-12 , Interleucina-2/biossíntese , Interleucina-2/genética , Ionomicina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase/economia , Subunidades Proteicas/biossíntese , Subunidades Proteicas/genética , Quinolinas , RNA Mensageiro/análise , Sensibilidade e Especificidade , Baço/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética
8.
J Bacteriol ; 184(4): 1078-88, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11807068

RESUMO

Mycobacteria are thought to have either one or two rRNA operons per genome. All mycobacteria investigated to date have an operon, designated rrnA, located downstream from the murA gene. We report that Mycobacteriun fortuitum has a second rrn operon, designated rrnB, which is located downstream from the tyrS gene; tyrS is very close to the 3' end of a gene (3-mag) coding for 3-methylpurine-DNA-glycosylase. The second rrn operon of Mycobacterium smegmatis was shown to have a similar organization, namely, 5' 3-mag-tyrS-rrnB 3'. The rrnB operon of M. fortuitum was found to have a single dedicated promoter. During exponential growth in a rich medium, the rrnB and rrnA operons were the major and minor contributors, respectively, to pre-rRNA synthesis. Genomic DNA was isolated from eight other fast-growing mycobacterial species. Samples were investigated by Southern blot analysis using probes for murA, tyrS, and 16S rRNA sequences. The results revealed that both rrnA and rrnB operons were present in each species. The results form the basis for a proposed new scheme for the classification of mycobacteria. The approach, which is phylogenetic in concept, is based on particular properties of the rrn operons of a cell, namely, the number per genome and a feature of 16S rRNA gene sequences.


Assuntos
Genes de RNAr , Mycobacterium fortuitum/genética , RNA Bacteriano , Óperon de RNAr , Sequência de Bases , DNA Bacteriano , DNA Intergênico , Dados de Sequência Molecular , Mycobacterium/genética , Mycobacterium fortuitum/classificação , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Ribossômico 16S/química , Homologia de Sequência do Ácido Nucleico , Sítio de Iniciação de Transcrição
9.
Methods Mol Med ; 54: 105-24, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-21341072

RESUMO

he synthesis of proteins is an essential process in cell growth and cell proliferation. The DNA sequence of a gene is first copied (transcribed) into an RNA sequence that is translated into a particular amino acid sequence. One strand of the gene, the RNA-like strand (or sense strand) has a nucleotide sequence equivalent to that of the RNA transcript (mRNA) with thymine replacing uracil. The complementary strand is often termed the template strand (or antisense strand), reflecting its role in the transcription process. Transcription takes place starting from the 5'-end and proceeding toward the 3'-end of the RNA product. The transcription process is usually described in three stages, namely, initiation, chain elongation, and termination. This chapter focuses on the initiation of transcription.

10.
J Bacteriol ; 181(15): 4617-27, 1999 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10419962

RESUMO

All mycobacteria studied to date have an rRNA operon, designated rrnA, located downstream from a single copy of the murA gene, which encodes an enzyme (EC 2.5.1.7) important for peptidoglycan synthesis. The rrnA operon has a promoter, P1(A), located within the coding region of murA, near the 3' end. Samples of RNA were isolated from Mycobacterium tuberculosis at different stages of the growth cycle and from Mycobacterium smegmatis grown under different conditions. RNase protection assays were used to investigate transcripts of both murA and rrnA. Transcription of murA was found to continue into the 16S rRNA gene, as if murA and rrnA form a hybrid (protein coding-rRNA coding) operon. During the growth of M. tuberculosis, the hybrid operon contributed approximately 2% to total pre-rRNA. Analysis of M. smegmatis RNA revealed that the level of murA RNA depended on the growth rate and that the patterns of expression during the growth cycle were different for murA and rrnA. M. smegmatis has a second rRNA operon, rrnB, located downstream from a single copy of the tyrS gene, encoding tyrosyl-tRNA synthetase. Transcription of tyrS was found to continue into the 16S rRNA gene rrnB. The hybrid tyrS-rrnB operon contributed 0.2 to 0.6% to rrnB transcripts. The pattern of tyrS expression during the growth cycle matched the pattern of rrnB expression, reflecting the essential role of TyrS and rRNA in protein biosynthesis.


Assuntos
Alquil e Aril Transferases/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Precursores de RNA/biossíntese , RNA Ribossômico 16S/genética , Transcrição Gênica , Tirosina-tRNA Ligase/genética , Sequência de Bases , Meios de Cultura , Primers do DNA , Cinética , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Óperon , Regiões Promotoras Genéticas , Precursores de RNA/genética
11.
J Bacteriol ; 180(21): 5756-61, 1998 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9791129

RESUMO

The roles of multiple promoters in the synthesis of rRNA under different conditions of growth were investigated, using two mycobacterial species as model organisms. When Mycobacterium smegmatis was grown under optimal conditions, its two rRNA operons contributed equally, with two promoters, one from each operon, being responsible for most transcripts. In stationary-phase growth or balanced growth under carbon starvation conditions, one operon (rrnAf) dominated and its three promoters contributed more equally to the generation of transcripts. Mycobacterium tuberculosis has a single operon with two promoters, one of which generated 80% of transcripts, at all stages of growth. We infer that each promoter functions independently according to its intrinsic strength when cells are growing slowly so that one operon with three promoters is roughly equivalent to three operons with one promoter; at high growth rates, occlusion effects reduce the efficiency of multiple promoters to that of a single promoter.


Assuntos
DNA Ribossômico , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Regiões Promotoras Genéticas , Precursores de RNA/biossíntese , RNA Bacteriano/biossíntese , Transcrição Gênica , Meios de Cultura , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium tuberculosis/crescimento & desenvolvimento , RNA Ribossômico/biossíntese , Óperon de RNAr
12.
J Bacteriol ; 179(22): 6949-58, 1997 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9371439

RESUMO

One rRNA operon of all mycobacteria studied so far is located downstream from a gene thought to code for the enzyme UDP-N-acetylglucosamine carboxyvinyl transferase (UNAcGCT), which is important to cell wall synthesis. This operon has been designated rrnAf for fast-growing mycobacteria and rrnAs for slow growers. We have investigated the upstream sequences and promoter activities of rrnA operons of typical fast growers which also possess a second rrn (rrnBf) operon and of the rrnA operons of the fast growers Mycobacterium abscessus and Mycobacterium chelonae, which each have a single rrn operon per genome. These fast growers have a common strategy for increasing the efficiency of transcription of their rrnA operons, thereby increasing the cells' potential for ribosome synthesis. This strategy involves the use of multiple (three to five) promoters which may have arisen through successive duplication events. Thus we have identified a hypervariable multiple promoter region (HMPR) located between the UNAcGCT gene and the 16S rRNA coding region. Two promoters, P1 and PCL1, appear to play pivotal roles in mycobacterial rRNA synthesis; they are present in all of the species examined and are the only promoters used for rRNA synthesis by the pathogenic slow growers. P1 is located within the coding region of the UNAcGCT gene, and PCL1 has a characteristic sequence that is related to but distinct from that of the additional promoters. In fast-growing species, P1 and PCL1 produce less than 10% of rRNA transcripts, so the additional promoters found in the HMPR are important in increasing the potential for rRNA synthesis during rapid growth. In contrast, rrnB operons appear to be regulated by a single promoter; because less divergence has taken place, rrnB appears to be younger than rrnA.


Assuntos
Mycobacterium/genética , Mycobacterium/metabolismo , Regiões Promotoras Genéticas , Ribossomos/metabolismo , Óperon de RNAr , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Genoma Bacteriano , Dados de Sequência Molecular , Estrutura Molecular , Mycobacterium/patogenicidade , Mycobacterium chelonae/genética , Mycobacterium chelonae/metabolismo , Mycobacterium chelonae/patogenicidade , Mycobacterium fortuitum/genética , Mycobacterium fortuitum/metabolismo , Mycobacterium fortuitum/patogenicidade , Mycobacterium phlei/genética , Mycobacterium phlei/metabolismo , Mycobacterium phlei/patogenicidade , Plasmídeos , Reação em Cadeia da Polimerase , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Ribossômico/química , RNA Ribossômico/metabolismo , RNA Ribossômico 16S/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Transcrição Gênica , Virulência/genética
13.
FEMS Microbiol Lett ; 135(1): 71-7, 1996 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-8598279

RESUMO

DNA was isolated from mycobacteria by a simplified procedure. Cells were suspended in 6 M guanidinium chloride, the suspension was cooled to -70 degrees C, then incubated at 65 degrees C for 10 min, cooled in ice, deproteinized by chloroform and DNA was recovered from the supernatant. The procedure was used to obtain DNA from several mycobacteria (1 x 10(9) or more cells) including Mycobacterium neoaurum, M. fortuitum, M. phlei and M. smegmatis. Each of the species was shown to have two ribosomal RNA operons per genome, and preliminary evidence was obtained which suggests that one of these operons is homologous with one of the operons of M. smegmatis.


Assuntos
DNA Bacteriano/isolamento & purificação , Mycobacterium/genética , Sequência de Bases , Enzimas de Restrição do DNA/análise , Guanidina , Guanidinas , Dados de Sequência Molecular , Reação em Cadeia da Polimerase
14.
J Mol Biol ; 202(1): 161-8, 1988 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-3172209

RESUMO

Physarum polycephalum (strain M3CVIII) contains four unlinked actin gene loci, each with two alleles (ardA1, ardA2, ardB1, ardB2, ardC1, ardC2, ardD1 and ardD2). The 4800 base HindIII fragment of the ardC2 allele was previously isolated as a recombinant phage lambda. We now report the structure of the actin gene sequences (C-actin gene). The gene, which contains four intervening sequences, codes for the principal actin isotype of plasmodia and it is expressed in both the haploid myxamoebal and diploid plasmodial phases of the life cycle. The C-actin isotype is closely related to actins of Dictyostelium, Acanthamoebae, Drosophila, sea urchin and mammalian cytoplasmic actin, and more distantly related to actins of yeast, Entamoebae and Tetrahymena. The ardC1 and ardC2 alleles differ by a 700(+/- 100) base-pair insertion/deletion in the vicinity of the 3' end of the transcribed region of the gene.


Assuntos
Actinas/genética , Genes Fúngicos , Physarum/genética , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular
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