Isoniazid resistance in Mycobacterium tuberculosis (MBT) is associated with point mutations in codon 315 of the katG gene. Two PCR technique were developed for detection of point mutations in codon 315. Most frequent point mutations (AGC → ACC and AGC → AGA) were identified in codon 315 by using two sets of primers, either of which included an additional competitive blocking primer with a 3′-terminal phosphate group in order to prevent nonspecific amplification. PCR with a set of two primers, one of which contained five locked nucleic acid monomers (LNA), permits one to detect any of six known mutations in codon 315 of katG and, thereby, discriminate between isoniazid-sensitive and resistant MBT isolates. The structure and purity of the 17-nt long LNA-containing oligonucleotides were characterized by MALDI-TOF mass spectrometry; and the 17 bp duplex formed by two LNA-containing complementary oligonucleotides was analyzed by thermal denaturation.

This study was aimed to investigate the effect of sodium valproate(VPA) on human myelodysplastic syndrome cell line SKM-1 and its mechanism. The cell proliferation was determined by MTT assay, cell apoptosis was analyzed by flow cytometry. The expressions of c-flipl, c-flips and dlk1 mRNA were detected by RT-PCR. The results showed that VPA could inhibited the growth of SKM-1 cells in dose- and time-dependent manners. The flow cytometric analysis indicated that VPA could induce cell apoptosis, apoptosis rate increased in dose-dependent manner. The expressions of c-flipl, c-flips and dlk1 mRNA in SKM-1 cell treated with VPA decreased using of VPA. It is concluded that VPA can induce apoptosis and inhibited proliferation of SKM-1 cells. In this process, the decreasing of c-flipl, c-flips and dlk1 mRNA expression may play important roles.

Objective To evaluate the protective efficacy of MPT64 and ESAT6 DNA vaccines from M tuberculosis Methods BALB/c mice were randomly divided into five groups and subjected to the following treatments respectively, i.e immunized with saline (A), plasmid vector (B), M bovis BCG (C), MPT64 DNA vaccine (D) or ESAT6 DNA vaccine (E); and then infected by intraperitoneal injection with M tuberculosis H 37 Rv The lung histopathological changes were observed 5 or 10 weeks after infection by microscopy Results At 5 weeks after infection, the lung lesions in the mice of group A and B had inflammatory infiltration with epithelial cell granulomas In the mice of group C, main pathological changes were epithelial cell granulomas with moderate granulate hyperplasia in alveolar walls The lung lesions of 3 mice in group D and 1 mice in group E were similar to those seen in the mice in group A and B The lung lesions of 2 mice in group D and 4 mice in group E were similar to those seen in the mice in group C At 10 weeks after infection, their tuberculous pneumonia tended to recovery For the mice in groups A, B and D, their lung pathology exhibited tuberculous granulomas consisted of numerous macrophages, lymphocytes and a few epithelial cells with moderate granulate hyperplasia in alveolar walls For the mice in groups of C and E, their lung developed epithelial cell and lymphocytic granulomas with moderate to severe granulate hyperplasia in alveolar walls Lung tissue necrosis was not observed in any mouse Conclusions MPT64 and ESAT6 DNA vaccines from M tuberculosis could enhance immunity against M tuberculosis The protective efficacy of ESAT6 DNA vaccine is stronger than that of MPT64 DNA vaccine, but not stronger than that of Mycobacterium bovis BCG.

OBJECTIVE:To explore the protein expression level of the lymphocyte function-related antigen CD11a, CD18, CD54 of breast cancer patients' blood, cancer tissue and drainage lymph node, and the effect of chemotherapy as well as Yiqi Yangxue Granule on its expression level.METHODS:The immunohistochemical assay and flow cytometry were used to determine the change of the protein expression level of CD11a, CD18, CD54 on the lymphocyte of blood, cancer tissue and lymph node between before and after chemotherapy and chemotherapy plus Yiqi Yangxue Granule in 72 patients suffering from breast cancer.RESULTS:In breast cancer patients, the level of CD54 was increased more obviously than that of control group, the CD11a, CD18, CD54 of lymphocytes in cancer tissue increased remarkably, while the CD54 expression positive rate reached 100%, the expression level of CD11a, CD18, CD54 which increased significantly in negative lymph node obviously higher than that of positive lymph node.CONCLUSION:The level of adherence cell was markedly abnormal in breast cancer patients, whose immune adherence function disturbed. Although chemotherapy kills the tumor cells, it damages the immune adherence function, Yiqi Yangxue Granule could improve the immune function and enhance the anti-cancer effect of patients.

OBJECTIVE:To understand the mutations of pncA gene in M. tuberculosis isolates, and to evaluate their clinical value.METHODS:Analyzing the pncA genes in 74 M. tuberculosis clinical isolates with PCR-SSCP and PCR-AS. M. tuberculosis strain H37Rv was used as control.RESULTS:32 drug-sensitive isolates all displayed normal pncA SSCP profile. Of 20 non-pyrazinamide-resistant isolates, 4 had abnormal pncA SSCP profile, in which 2 isolates were sequenced, one was TTC-->TTA mutation at codon 58, another was TGG-->CGG mutation at codon 68. Of 22 pyrazinamide-resistant isolates, 10 displayed abnormal pncA SSCP profile, in which 2 isolates sequenced had TTC-->TTA mutation at codon 58.CONCLUSIONS:The mutation of pncA gene is a pyrazinamide-resistant molecular mechanism in M. tuberculosis. Detecting the mutations of pncA genes might diagnose pyrazinamide resistance rapidly in some M. tuberculosis isolates.

Objective To study the molecular mechanisms of drug resistance in Mycobacterium (M). tuberculosis, to evaluate the value of the beta subunit of RNA polymerase (rpoB), the ribosomal sit protein (rpsL), 16Sr RNA (rrs), catalase-peroxidase gene (katG) genes, and inhA regulatory sequence as genetic markers for rifampin (RFP), streptomycin (SM), isoniazid (INH) resistance, and to develop new methods for detecting the drug resistance.Method The rpoB, rpsL, rrs, katG genes, and inhA regulatory sequence in 85 M. tuberculosis isolates were analyzed with polymerase chain reaction (PGR), PCR-single-stranded conformation polymorphism analyses (SSCP), PCR-nucleotide sequence analyses ( NS) and PCR-restriction fragment length polymorphism (RFLP).Results The sensitivity of amplifying the drug-resistant genes with PCR was 1-10 pg DNA. Twenty-eight drug-sensitive strains had no alterations in the rpoB, rpsL, rrs, katG genes, and inhA regulatory sequences. 93.3% of 45 M. tuberculosis RFP-resistant (RFPr) isolates had rpoB mutations. Codon 531 and 526 of the rpoB are the most common sites of nucleotide substitutions. 72.5% of 40 SM-resistant (SMr) isolates had an identical mutation at codon 43 of the rpsL gene. No isolates had a mutation at codon 88 of the rpsL. Only 7.5% of these SMr isolates had A-to-C transversions at position 513 of the rrs gene. Of 34 INH-resistant (INHr) isolates, 11.8% had complete katG deletions, 55.9% had mutations in the selected region of katG. Only 8.8% had alterations in the inhA regulatory sequences. 60.9% of RFPr, INHr, and SMr isolates had mutations in genetic markers for these drug resistance.Conclusions Most drug resistance in M. tuberculosis was due to simple mutations occurring in chromosomally encoded genes. Alterations in rpoB, rpsL and katG gene may be the important mechanism of M, tuberculosis resistance to RFP, SM, and INH. PCR, PCR-SSCP, PCR-NS, and PCR-RFLP are going to become the simple, rapid and reliable diagnostic tests for drug resistance in M. tuberculosis.

OBJECTIVE:To investigate the mutations of ahpC genes in M. tuberculosis isoniazid-resistant isolates, and to study the correlation between ahpC alterations and isoniazid resistance.METHOD:Analyzing the ahpC coding sequences and ahpC promoters in 62 M. tuberculosis clinical isolates with PCR-SSCP method. M. tuberculosis strain H37RV was used as control.RESULT:The ahpC coding sequences showed normal SSCP profiles in 62 clinical isolates. AhpC promoter mutations were identified in 5 of 12 high isoniazid-resistant strains, and were not altered in 18 low isoniazid-resistant and 32 drug-sensitive isolates.CONCLUSION:The mutation of ahpC coding gene was not correlated with isoniazid-resistant M. tuberculosis isolates. AhpC promoter mutation was a compensatory change in katG-defective and catalase-negative isolates, may be used as a indirect marker for high resistance to isoniazid.

OBJECTIVE:To understand mutation of rifampicin-resistant genes in M. tuberculosis clinical isolates, and to develop a new method for detecting drug resistance.METHOD:Analyzing the rpoB genes in 50 M. tuberculosis clinical isolates with polymerase chain reaction-single-stranded conformation polymorphism (PCR-SS-CP) and PCR-direct sequencing(PCR-DS) techniques. M. tuberculosis strain H37Rv was used as control.RESULT:Of 3 drug-sensitive isolates and 2 non-rifampicin-resistant isolates selected, only 1 drug-sensitive isolate with abnormal rpoB SSCP profile had TCG-->TTG mutation at codon 531. Of 45 rifampicin-resistant isolates, 35 isolates with abnormal SSCP profiles had mutations in rpoB sequences, in which 14 isolates displayed TCG-->TTG or TGG or TAC mutations at codon 531, 14 had CAC-->TAC or GAC or CCC or CTC or GTC mutations at codon 526, 2 had GAC-->GTC or TAC mutations at codon 516, 2 had two point mutations, and 3 had different rpoB sequences from that of M. tuberculosis.CONCLUSION:Resistance to rifampicin in most M. tuberculosis isolates is due to the mutations on the genes encoding the RNA polymerase subunit (rpoB). PCR-SSCP and PCR-DS techniques might become simple, rapid and reliable diagnostic tests for rifampicin resistance in clinical isolates.

OBJECTIVE:To observe the mutations of rpsL gene in M. tuberculosis streptomycin-resistant isolates, and to develop a new method for detecting drug resistance.METHOD:Detecting the rpsL genes with PCR-SSCP and analyzing their codons 43 by PCR-RFLP.RESULTS:Strain H37Rv was used as a control. In 22 M. tuberculosis clinical isolates, the rpsL PCR fragments from 5 drug-susceptible isolates had no differences in the SSCP profiles with strain H37Rv, and were restricted by Mbo II. 11 of the 13 streptomycin-resistant isolates showed apparent differences in the SSCP profiles and were not digested with Mbo II. 1 of the 4 other drug-resistant isolates also had apparent SSCP differences and was not digested by Mbo II.CONCLUSIONS:The results suggested that the rpsL gene mutation was frequently observed in M. tuberculosis streptomycin-resistant isolates, and usually situated at codon 43, PCR-SSCP and PCR-RFLP might become a simple, rapid and reliable diagnostic test for drug resistance.