Deteção de Mycobacterium tuberculosis em amostras clínicas por reação em cadeia da polimerase utilizando primers baseados na região intergênica plcB-plcC / Using polymerase chain reaction with primers based on the plcB-plcC intergenic region to detect Mycobacterium tuberculosis in clinical samples

OBJECTIVE: To develop a system for the molecular diagnosis of tuberculosis by polymerase chain reaction (PCR), constructing primers based on the difference in gene organization of the intergenic region of phospholipase C (plcB-plcC region), which differentiates Mycobacterium tuberculosis from other mycobacteria. METHODS: A PCR product of the expected size (432 bp) was obtained from M. tuberculosis and M. africanum only. A total of 33 mycobacterial isolates and 273 clinical samples from patients suspected of having tuberculosis were examined. These were used in the comparative study of the PCR technique versus culture. RESULTS: For PCR versus culture, the data showed 93.8 percent accuracy (p < 0.0001), 93.1 percent sensitivity (CI: 88.7-96.0), and 96.4 percent specificity (CI: 96.1-99.4). The Kappa value (0.82) shows that there was a near-perfect concordance between the two tests. CONCLUSION: The use of the plcB-plcC region in PCR amplification was found to be an important and reliable tool for the specific diagnosis of tuberculosis in the samples analyzed.

OBJETIVO: Desenvolver um sistema para o diagnóstico molecular da tuberculose por reação em cadeia da polimerase, do inglês polymerase chain reaction (PCR), pela construção de primers baseados na diferença da organização de uma região intergênica da fosfolipase (phospholipase) C (região plcB-plcC), que diferencia Mycobacterium tuberculosis das outras micobactérias. MÉTODOS: Um produto de PCR com o tamanho esperado (432 pb) foi obtido somente de M. tuberculosis e M. africanum. Um total de 33 isolados micobacterianos e 273 amostras clínicas de pacientes com suspeita de tuberculose foram examinados. Estes foram submetidos ao estudo comparativo da técnica de PCR contra o cultivo. RESULTADOS: Os dados mostraram 93,8 por cento de exatidão para PCR contra o cultivo (p < 0,0001), 93,1 por cento de sensibilidade (IC: 88,7-96,0) e especificidade de 96,4 por cento (IC: 96,1-99,4). O valor de Kappa foi de 0,82, demonstrando um alinhamento perfeito para a verificação do grau de concordância entre os testes. CONCLUSÃO: O uso da região plcB-plcC para a amplificação por PCR é mostrado como uma ferramenta importante e de confiança para o diagnóstico específico de tuberculose nas amostras clínicas analisadas.

Phosphorylation of glycogen synthase kinase-3beta at serine-9 by phospholipase Cgamma1 through protein kinase C in rat 3Y1 fibroblasts

Phospholipase Cgamma1 (PLCgamma1) plays an important role in controlling cellular proliferation and differentiation. PLCgamma1 is overexpressed in some tumors, and its overexpression induces solid tumors in nude mice. However, the regulatory mechanisms underlying PLCgamma1-induced cell proliferation are not fully understood. Here we show that overexpression of PLCgamma1 highly phosphorylated glycogen synthase kinase-3beta (GSK-3beta) at serine-9 in 3Y1 fibroblasts. Inhibition of protein kinase C (PKC)s with GF109203X abrogated GSK-3beta phosphorylation by PLCgamma1. We also found that steady-state level of cyclin D1 protein, but not cyclin D1 mRNA, was highly elevated in response to serum stimulation in PLCgamma1-transfected cells as compared with vector-transfected cells. Since GSK-3beta is involved in cyclin D1 proteolysis in response to mitogenic stimulation, PLCgamma1-mediated GSK-3beta phosphorylation may function as a regulation of cyclin D1 accumulation in PLCgamma1-overexpressing cells.

Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of ß-galactosidase activity. ß-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis

Molecular cloning of a membrane bound tyrosine-specific protein kinase from rat spleen.

Tyrosine-specific protein phosphorylation is believed to play an important (though poorly understood) role in various cellular functions in many normal and malignant cells. In order to understand the function of tyrosine-specific protein kinases in normal cells, it is necessary, as an initial step, to identify genes (and proteins) for these enzymes. For this purpose cDNA libraries were constructed in plasmid vector pGEM-3Z and lambda gt11 using mRNA from rat spleen. From these cDNA libraries, cDNA clones coding for a src-related tyrosine-specific protein kinase were isolated. The largest clone (L115) was 1.94 kb in size. Various restriction fragments of this clone were subcloned in plasmid vector for sequencing. The complete nucleotide sequence of the largest clone showed an open reading frame coding for a protein of 503 amino acids. The presence of a glycine at position 2 and an arginine at position 7 indicated that this protein is likely to be acylated at glycine 2 and therefore associated with plasma membrane. This gene showed high homology to human and mouse hck and hence it is perhaps the rat homologue of hck. Moderate level of expression of this gene was observed only in the adult rat spleen and not in other tissues. These results suggest that this kinase gene is expressed in a tissue specific manner.