Selection and application of peptide mimotopes of MPT64 protein in Mycobacterium tuberculosis.

Antibody responses can be useful markers of tuberculosis (TB) infection, especially in the screening of extra-pulmonary TB. MPT64 is an important antigen in Mycobacterium tuberculosis (MTB) infection and is used in serological diagnosis. However, large variability in the diagnostic accuracy of MPT64 as a serological tool has limited its application. Phage-displayed random peptide libraries have emerged as a powerful technique to select peptides (epitopes) or mimotopes that may serve as surrogate diagnostic markers in serological tests. In the present study, this method was employed to identify mimotopes of the MPT64 protein of MTB by screening a linear heptapeptide library with rabbit antibodies raised against MPT64 protein. Two antigenic mimotopes (M2 and M6) resembling B-cell epitopes of MPT64 were identified that bound the affinity purified anti-MPT64 polyclonal antibodies and competed with MPT64 for antibody binding. From the results of sequence alignment and a structure modelling figure of MPT64, the sequence of the 2nd to 5th amino acids (DSML) of M2 was totally consistent with the sequence of the 224th to 227th amino acids of MPT64 and the peptide is located on the surface of the space structure of MPT64, suggesting that it might be a linear epitope of MPT64. The recognition of both phage-displayed and synthetic peptides of M2 by the anti-MPT64 polyclonal antibodies also supported this. Although no recurring sequence and no analogue to MPT64 of M6 were found for sequence alignment, the recognition of both phage-displayed and synthetic peptides of M6 by the anti-MPT64 polyclonal antibodies indicated that it might be a mimotope of a conformational epitope of MPT64. According to the results of the reactivity of human sera with synthetic M2 and M6 peptides and MPT64, M2 showed a significantly higher AUC and sensitivity than M6 and MPT64, especially for the sera from sputum-negative TB patients, suggesting that the M2 mimotope may be useful in serological diagnostic testing for TB.

[Identification of the Mycobacterium tuberculosis complex by detecting recombinant secretory protein MPT64].

OBJECTIVE: To identify the Mycobacterium tuberculosis complex by detecting the secretory protein MPT64. METHODS: The gene mpt64 was amplified by polymerase chain reaction (PCR) from the genome of Mycobacterium tuberculosis H(37)Rv strain and cloned into expression vector. Immune sera from rabbits by recombinant proteins MPT64, were used to make enzyme-labeled antibodies and coated antibodies. A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was established to detect the secretory protein MPT64 of the culture supernatants in Mycobacterium strains. Results of ELISA were compared to those of the gene mpt64 amplified by PCR. RESULTS: A recombinant vector was constructed. The minimum detectable concentration of MPT64 was 0.01 mg/L. A total of 27 reference strains and 170 clinical isolate strains were evaluated. PCR for Mycobacterium tuberculosis reference strain, Mycobacterium bovis reference strain and Mycobacterium africanum reference strain was positive, but that for other reference stains was negative, consistent with the results of ELISA. In the 170 clinical isolate stains, the positive result of PCR and ELISA was 98.2% (111/113) and 97.3% (110/113) respectively, while the specificity of PCR and ELISA was both 100%, no positive result in non-tuberculosis mycobacterium strains. CONCLUSION: Identification of the Mycobacterium tuberculosis complex by detecting secretory protein MPT64 is rapid, sensitive, and specific, which can be used routinely in clinical laboratories.

Detection of mutations associated with isoniazid resistance in Mycobacterium tuberculosis isolates from China.

Nine structural genes (furA, katG, inhA, kasA, Rv0340, iniB, iniA, iniC, and efpA) and two regulatory regions (the oxyR-ahpC intergenic region and the promoter of mabA-inhA) in 87 isoniazid (INH)-monoresistant and 50 INH-susceptible Mycobacterium tuberculosis isolates collected from five provinces of China were analyzed by sequencing. Eighty-two (94.3%) INH-resistant isolates had mutations in the katG gene, with the katG Ser315Thr mutation predominant (55.2%). No mutation at codon 463 of katG was detected among the 50 INH-susceptible isolates with different IS6110 fingerprints. In addition, there were 35 (40.2%) INH-resistant isolates that had a mutation at codon 463 of katG. Of the INH-resistant strains, 20 (23.0%) isolates harbored double mutations at two separate loci of katG. Mutations in the inhA promoter region occurred in 13 (14.9%) isolates; 4.6% of the isolates had inhA structural gene mutations, and 11.5% harbored mutations in the oxyR-ahpC intergenic region. Drug resistance-associated mutations were detected in the iniBAC region and efpA.

Clinical research of heart rate turbulence on predictive value in patients with acute myocardial infarction / 中华心血管病杂志

<p><b>OBJECTIVE</b>To assess the predictive value of heart rate turbulence (HRT) in patients with acute myocardial infarction.</p><p><b>METHODS</b>One hundred and twenty-five patients with acute myocardial infarction were enrolled in this study. During the period from 6 to 21 days after onset of acute myocardial infarction, they were undergone 24-hour Holter recordings to collect the mean RR interval and heart rate variability (HRV) SDNN. The Holter files were processed with software of "HRT! View V0.60-1" to obtain the value of Turbulence Onset (TO) and Turbulence Slope (TS) and the value of "heart rate variability (HRV) SDNN". LVEF and EDD were measured by Ultrasonic Cardiography. Endpoint of follow-up was cardiac death. According to the results, patients were divided into two groups (the "survivors" and the "nonsurvivors"). The predictive value for high-risk patients with acute myocardial infarction was assessed by variables between the two groups.</p><p><b>RESULTS</b>In the period of follow-up (mean 225.4 +/- 99.8 days), 14 patients died and 111 patients survived. In the univariate Cox regression analysis, "TS" was a strong univariate predictor of mortality (hazard ratio 11.46, P < 0.01); "TO" was a relatively weak predictor and the hazard ratio was 2.76 (P > 0.05). Combination of abnormal TO and abnormal TS was the strongest mortality predictor (hazard ratio 26.70, P < 0.01); in the multivariate Cox regression analysis, TS < or = 2.5 ms/RR and EDD > or = 5.6 cm were the independent predictors of mortality with hazard ratios 9.49 (P < 0.01) and 3.64 (P < 0.05), respectively.</p><p><b>CONCLUSIONS</b>The absence of the heart rate turbulence after ventricular premature beats is a very potent post-infarction risk predictor which is independent of and stronger than other known risk predictors.</p>

Regulation of aroP expression by tyrR gene in Escherichia coli.

tyrR gene encodes a global regulatory protein (TyrR), which plays an important role in the transcriptional regulation of eight transcription units (including tyrR gene itself) whose protein products catalyze key steps in aromatic amino acid biosynthesis and/or transport. The aroP gene encodes an integral membrane protein (AroP) that transports aromatic amino acids through the cell membrane. The transcription of aroP was reported to be repressed by TyrR. In this work, aroP(p) (aroP gene carrying its own promoter), aroP (aroP gene without promoter) and tyrR genes were amplified by PCR from genomic DNA of E. coli K12 and introduced into E. coli WT5. The expression of aroP and tyrR were detected and the activities of AroP and TyrR were determined. The introduction of either aroP(p) or aroP elevated the strain's transport activity by 1.40 or 1.46-fold respectively. Transformant carrying tyrR gene showed an ATPase activity 1.69-fold compared with the control. When the genes were linked in tandem and co-expressed in a plasmid, the relative AroP transport activity of the strain harboring aroP(p) -tyrR (0.95) was significantly lower than that of aroP-tyrR (1.31). The results indicated that TyrR might be able to reduce the expression of aroP gene by binding with the aroP promoter region in E.coli.

[Knockout of tyrR gene in Escherichia coli and its effects on the phenylalanine biosynthesis].

TyrR is a global regulation protein encoded by tyrR gene which controls several transcriptional units involving biosynthesis and transportation of aromatic amino acids in Escherichia coli. In this work, the tyrR gene was knocked out by a double-cross homologous recombination. The tyrR- mutant was verified by structural identification by PCR and sequencing, and functional demonstration using lacZ reporter gene. In tyrR- mutant, the activities of two key enzymes in the phenylalanine biosynthesis pathway, whose expression was controlled by TyrR, DAHPS and AT, had been shown to elevate by a 1.08-fold and 2.70-fold compared with the parent strain, respectively. The yield of phenylalanine biosynthesis in the mutant was 1.59 times higher than that of wild type strain. The repression on the transcription of aroP, encoding an aromatic amino acid permease, was eliminated, resulting in a 70.2% increase of the aromatic amino acid transportation in tyrR- mutant strain.