Activity of aminosidine (paromomycin) for Mycobacterium tuberculosis and Mycobacterium avium.
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Drug Resistance, Microbial↗
Biomedical subjects
M V Reddy
Publications and source records attributed to M V Reddy.
In vivo activity of paromomycin against susceptible and multidrug-resistant Mycobacterium tuberculosis and M. avium complex strains.
Encouraged by in vitro results, we have assessed the in vivo activity of paromomycin (PRM) against Mycobacterium tuberculosis, multidrug-resistant (MDR) M. tuberculosis (resistant to isoniazid, rifampin, and streptomycin), and Mycobacterium avium complex in C57BL/6 mice and their beige counterparts. In all these experiments, PRM was effective in preventing mortality from a mycobacterial infection and was significantly more active than the drug-free control (P < 0.0005) in reducing the CFU relative to the mean log CFU in the lungs, livers, and spleens of infected animals. In the drug-susceptible M. tuberculosis experiment, PRM given at 100 and 200 mg/kg of body weight was significantly less active than isoniazid at 25 mg/kg (P < 0.0005) in reducing the mean log CFU in the lungs, livers, and spleens of infected mice. In the MDR M. tuberculosis experiment, PRM given at 200 mg/kg was effective, relative to the drug-free control, in reducing the mean log CFU of an isolate of M. tuberculosis resistant to isoniazid, rifampin, and streptomycin. In the M. avium complex experiment, PRM given at 200 mg/kg was as effective as amikacin at 50 mg/kg in reducing the mean log CFU in the lungs, livers, and spleens of infected mice. On the basis of our experiments, we believe that PRM has promising activity in vivo in the treatment of infections caused by M. tuberculosis, MDR M. tuberculosis, and M. avium complex.
Animals↗
Comparison of effects of yoga & physical exercise in athletes.
The effect of pranayama a controlled breathing practice, on exercise tests was studied in athletes in two phases; sub-maximal and maximal exercise tests. At the end of phase I (one year) both the groups (control and experimental) achieved significantly higher work rate and reduction in oxygen consumption per unit work. There was a significant reduction in blood lactate and an increase in P/L ratio in the experimental group, at rest. At the end of phase II (two years), the oxygen consumption per unit work was found to be significantly reduced and the work rate significantly increased in the experimental group. Blood lactate decreased significantly at rest in the experimental group only. Pyruvate and pyruvate-lactate ratio increased significantly in both the groups after exercise and at rest in the experimental group. The results in both phases showed that the subjects who practised pranayama could achieve higher work rates with reduced oxygen consumption per unit work and without increase in blood lactate levels. The blood lactate levels were significantly low at rest.
Adult↗
Detection of tuberculous IgG antibodies using Mycobacterium tuberculosis H37Ra, excretory secretory antigen and tuberculin-purified protein derivative.
Different antigen preparations, viz. excretory-secretory antigen (ES Ag), phosphate buffer saline soluble antigen (PBS-S Ag) and sodium dodecyl sulphate soluble antigen (SDS-S Ag) of M. tuberculosis (M.tb) H37Ra strain along with tuberculin purified protein derivative (PPD) were employed in stick indirect ELISA to detect IgG antibodies in sera of sputum positive pulmonary tuberculosis cases. Sera from healthy individuals and individuals with diseases other than tuberculosis (cross-reacting diseases) were used as controls. ES antigen and PPD showed higher antibody titres in tuberculosis cases (GMT-1378 each) compared to PBS-S Ag (GMT-454) and SDS-S Ag (GMT-974). Thereafter, an extensive study was done analysing higher number of sera in each group for the detection of tuberculous IgG antibodies using ES Ag and PPD. The ES Ag showed better sensitivity (87%) and specificity (85%) compared to the sensitivity (73%) and specificity (78%) achieved with PPD. The ES Ag also showed higher IgG antibody titre (GMT-1068) than PPD (GMT-721). From the present study it can be envisaged that ES Ag has high diagnostic potential in tuberculosis.
Antibodies, Bacterial↗
C18 thin-layer chromatographic enhancement of the 32P-postlabeling assay for aromatic or bulky carcinogen-DNA adducts: evaluation of adduct recoveries in comparison with nuclease P1 and butanol methods.
The suitability of C18 reversed-phase thin-layer chromatography (TLC) for enrichment of adducts in the 32P-postlabeling assay was investigated for structurally diverse classes of DNA adducts derived from benzo[a]pyrene, 2-acetylaminofluorene, benzoquinone, safrole, and mitomycin C. The TLC enrichment involved retention of adducts to the C18 phase followed by elution with organic solvent-water. Adduct patterns obtained by the C18 purification were qualitatively similar to those obtained by the nuclease P1 and butanol procedures, the two commonly used enrichment methods. Adduct recoveries by the C18 method varied for different adducts and were significantly lower than those obtained by the other two techniques.
2-Acetylaminofluorene↗
32P-postlabelling analysis of small aromatic and of bulky non-aromatic DNA adducts.
The 32P-postlabelling methodology for analysis of DNA adducts derived from carcinogens containing one aromatic ring (e.g., safrole, styrene oxide, benzene metabolites, 1-nitrosoindole-3-acetonitrile) or a bulky non-aromatic moiety (e.g., mitomycin C, diaziquone) is reviewed. Six steps are involved: digestion of DNA to 3'-nucleotides, enrichment of adducts, 32P-labelling of adducts, separation of labelled adducts by TLC, detection, and quantitation. The first step, DNA digestion with micrococcal nuclease and spleen phosphodiesterase, is applicable to DNA modified with most carcinogens independent of their size and structure. Of the two commonly used procedures for enrichment of aromatic adducts in DNA digests, the nuclease P1 treatment is substantially more effective than butanol extraction for small aromatic and bulky non-aromatic adducts. For initial purification of these adducts from unadducted material after 32P-labelling, multi-directional polyethyleneimine (PEI)-cellulose TLC using 1 M sodium phosphate, pH 6.0, as the D1 solvent is not suitable, because they are not retained on PEI-cellulose under these conditions. A higher concentration of sodium phosphate (e.g., 2.3 M) or development with D1 and D3 solvents in the same direction helps to retain adducts of safrole and of benzene metabolites. Also, transfer of adducts from multiple cut-outs above the origin after D1 chromatography, as adopted for analysis of I-compounds, is potentially applicable. However, initial purification by reverse-phase TLC, followed by in situ transfer and resolution by PEI-cellulose TLC has been found to be most effective for these adducts. Reverse-phase TLC at 4 degrees C or in a stronger salt solution further improves retention of some adducts (e.g., mitomycin C and diaziquone adducts). For adduct separation by PEI-cellulose TLC, salt solutions with or without urea are used.
Carcinogens↗
Diagnostic use of filarial antibody and antigen isolated from hydrocele fluid for human filariasis.
Paired samples of serum and hydrocele fluid of filarial patients associated with hydrocele were analysed for filarial antibody and antigen. Sera samples showed higher titers of filarial antibody and antigen compared to their corresponding hydrocele fluid samples. HFIgG isolated from hydrocele fluid was equally useful as FSIgG isolated from serum and detected filarial antigen in 23 out of 26 microfilaraemic sera, 7 out of 10 chronic sera and 3 out of 18 endemic normal sera by inhibition ELISA. Filarial antigen was isolated from hydrocele fluid. In inhibition ELISA antigen fraction, HFA-9 (20-25 kDa) isolated by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis showed high reciprocal antigen titer of 2048. This active antigen fraction was evaluated for its diagnostic utility in comparison with Wuchereria bancrofti microfilariae excretory-secretory antigen (Wb mf ES Ag) in inhibition ELISA. Both antigen preparations detected filarial antigen in about 80% of microfilaraemic sera, 60% chronic sera and 20-30% of endemic normal sera. This study showed that antibody and antigen isolated from hydrocele fluid were equally sensitive as FSIgG and Wb mf ES Ag in the detection of filarial antigen by inhibition ELISA. Thus hydrocele fluid may be used as an alternative source for the isolation of antibody and antigen of immunodiagnostic importance.
Animals↗
Heat shock treatment of macrophages causes increased release of superoxide anion.
Heat shock treatment of murine macrophages and the J774 cell line resulted in an enhanced capacity to release superoxide anion (O2-) upon stimulation. There was no concomitant increase in hydrogen peroxide production, and the macrophage microbicidal activity against Mycobacterium tuberculosis, Mycobacterium avium complex, and Staphylococcus aureus was not altered.
Animals↗
Detection of filarial antigen using antibodies raised against Wuchereria bancrofti microfilarial SDS soluble antigen.
Polyclonal antibodies raised in mouse ascitic fluid against Wuchereria bancrofti microfilarial antigens (Wb Mf SDS S Ag) were studied for their diagnostic use in bancroftian filariasis using a dip stick, enzyme-linked immunosorbent assay. In sandwich ELISA, 100% of microfilaremic sera (30 out of 30) 53% of acute filarial sera (7/13), 40% of subacute filarial sera (6 out of 15), 13% of chronic filarial sera (2/15) and 20% of endemic area normal sera (3/15) showed the presence of filarial antigen. Determination of filarial antigen titer in microfilaremic sera showed an apparent positive correlation between microfilarial density and antigen titer. The antibody raised against Wb Mf SDS S Ag was found to be cross reactive with phosphorylcholine epitopes. The filarial antigen detected by anti Wb Mf SDS S Ag antibodies in sandwich ELISA is possibly associated with the active stage (microfilaremia) of infection.
Animals↗
Functional analysis of the N-terminal domain of Tat protein of the human immunodeficiency virus type 1.
The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent trans-activator of the viral long terminal repeat (LTR). The N-terminal region of Tat is rich in proline and acidic residues analogous to the activation domains of other transcription factors such as GAL-4 and CTF/NF-1. Several basic residues are also present in this region. To investigate the role of these structural features in the Tat-mediated trans-activation, we have chemically synthesized and evaluated Tat analogs with alanine or glutamine replacing one or more of these amino acid residues. Our data show that substitution of Glu-2, His-13, or all the proline in the Pro-Xaa3-Pro triad drastically reduced activity. In contrast, changes at Arg-7, Lys-12 and any one proline residue in the triad moderately reduced, and substitution of Lys-19 showed little effect on, activity. These results show that the native structure of the N-terminal 19 amino acid sequence is essential for Tat function, and that the overall topology of this domain and not the acidic residues alone appears necessary for trans-activation.
Amino Acid Sequence↗
Mechanisms associated with the generation of biologically active human immunodeficiency virus type 1 particles from defective proviruses.
The human immunodeficiency virus (HIV) is the etiological agent of acquired immunodeficiency syndrome (AIDS). HIV exhibits extensive genetic diversity and it is apparent that an infected individual contains different populations of distinct viral strains, a large proportion of which has been found surprisingly to be defective for replication. A similar phenomenon has also been observed with some cell lines that are known to produce infectious viral particles but harbor defective proviral genomes. Here, we investigated the molecular basis of this phenomenon by cloning proviral genomes of HIV from a cell line that was capable of producing high titers of biologically active HIV particles that readily induced syncytia with CD4+ cell lines and peripheral blood lymphocytes. This cell line was found to contain five proviral genomes, all of which, when tested individually, failed to produce replication-competent viruses upon transfection into human cells. However, when a specific combination of two proviral genomes was used in such transfection studies, it was possible to obtain biologically active, replication-competent viral particles that infected and replicated in CD4+ cell lines and induced syncytia characteristic of HIV. Such a result may be due to homologous recombination between proviral DNAs occurring in cells after transfection and/or complementation of replication-defective proviral DNAs. The diploid nature of the viral RNA genome present in the viral particle may enable the persistence of defective HIV genomes.
Cell Line↗
Postlabeling analysis of polycyclic aromatic hydrocarbon-DNA adducts in white blood cells of foundry workers.
Blood samples were obtained from 61 volunteers working in a Finnish iron foundry who were exposed to carcinogenic polycyclic aromatic hydrocarbons and from 19 control subjects not known to be exposed to these chemicals. Foundry workers were categorized into high, medium, or low exposure groups, based on their exposure levels to airborne benzo[a]pyrene (BaP) (high greater than 0.2, medium 0.05-0.2, low less than 0.05 micrograms BaP/m3 air). Aromatic adducts were determined in white blood cell DNA from exposed and unexposed subjects using 32P-postlabeling. There was a highly significant correlation between the estimated exposure and adduct levels as determined by analysis of variance. The levels of adducts found in the high and medium group samples ranged from 5 to over 20 adducts in 10(8) DNA nucleotides. No effects due to age, sex, or the smoking habits of the subjects were observed. Within each group, the SEM appeared rather small. This study demonstrates the utility of the 32P-postlabeling assay to assess human exposure to known and unknown environmental aromatic genotoxicants.
Adult↗
Nuclease S1-mediated enhancement of the 32P-postlabeling assay for aromatic carcinogen-DNA adducts.
Treatment of DNA digests with nuclease P1 prior to 32P-labeling of adducts has previously been shown to enhance the sensitivity of the 32P-postlabeling assay for the detection of aromatic carcinogen-DNA adducts. The enhancement was based on the ability of nuclease P1 to remove the 3'-phosphate from normal nucleotides but not the corresponding phosphate from most aromatic adducted nucleotides. We investigated the utility of another 3'-dephosphorylating enzyme, nuclease S1, for this purpose, and found it to be as effective as nuclease P1. The recovery of DNA adducts derived from benzo[a]-pyrene (B[a]P), benzoquinone (BQ) and 2-acetylaminofluorene (AAF) was comparable after enhancement with either enzyme. Some differences were, however, observed. Recovery of a minor B[a]P adduct was 1.5 times higher by the S1 procedure. Among minor adducts of BQ, two showed higher values (2.8- and 6.1-fold) by the S1 procedure and one by the P1 procedure (2.4-fold). The major AAF adduct, deoxyguanosine-C8-AF, exhibited poorer recovery (1-11%) by either procedure, while the minor adducts, deoxyguanosine-N2-AAF and deoxyguanosine-C8-AAF, showed better recovery (2-3 times) than by the enhancement procedure involving extraction of adducts into butanol. Our results show that the nuclease S1 assay can complement the nuclease P1 assay, with improved recoveries for some adducts. Considering the complexity of the postlabeling assay, this additional variant may prove useful in unequivocal detection of DNA adducts.
2-Acetylaminofluorene↗
32P-postlabeling detection of thymine glycols: evaluation of adduct recoveries after enhancement with affinity chromatography, nuclease P1, nuclease S1, and polynucleotide kinase.
Thymine glycol (Tg) is a product of DNA damage by oxygen radicals generated by oxidative mutagens and carcinogens and ionizing radiation. The highly sensitive 32P-postlabeling assay was validated and optimized for the measurement of Tg generated in vitro by the reaction of dTp or calf thymus DNA with osmium tetroxide (OsO4). Adduct detection was enhanced by purification of Tg adducts using phenylboronate affinity chromatography or by preferential dephosphorylation of unmodified 3'-nucleotides with nuclease P1, nuclease S1, or polynucleotide kinase; Tg nucleotides were found to be resistant to limited enzymatic 3'-dephosphorylation. Two adducts were seen with OsO4-modified dTp, which may have been cis-Tg adducts, because they were retained on a phenylboronate column, and because OsO4 selectively forms cis-Tg adducts. With OsO4-modified DNA, several adducts were detected, two major derivatives of which coincided chromatographically with those seen in OsO4-modified dTp. The recoveries of major adducts were similar before and after enrichment by different methods, indicating that Tg adducts were resistant to enzymatic dephosphorylation. The efficacy of labeling of the two major Tg adducts by polynucleotide kinase was optimal at 60 microM ATP and higher, whereas it was about 3%, 50%, and 80% of the optimal rate at 2, 10, and 30 microM, respectively. This was in contrast to our previous finding that only 0.25 microM ATP was needed for optimal labeling of benzoquinone-DNA adducts.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Triphosphate↗
Penicillinase ELISA for detection of tubercular antigen in tuberculosis.
Stick sandwich enzyme linked immunosorbent assay (ELISA) using rabbit anti PPD-RT 23 immunoglobulins and enzyme penicillinase has been explored for detection of tubercular antigen in sera and CSF samples of pulmonary tuberculosis and tubercular meningitis (TBM) respectively. The analysis of sera showed 73.3% of pulmonary tuberculosis cases, 16.2% of healthy controls and 44.4% of Hansen's disease positive for tubercular antigen. The accuracies of positive and negative predictive values were 69% and 82% respectively. The analysis of CSF samples showed the presence of tubercular antigen in 76.4% of TBM, 16.6% of pyogenic meningitis cases, 19.4% of neurological diseases other than meningitis and 16.1% non-neurological disease controls. The accuracies of positive and negative predictive values were 48% and 94% respectively. Hence this simple test using economical and indigenous reagents can be applied for the diagnosis of pulmonary and extra-pulmonary tuberculosis.
Antigens, Bacterial↗
32P-assay of DNA adducts in white blood cells and placentas of pregnant women: lack of residential wood combustion-related adducts but presence of tissue-specific endogenous adducts.
Residential wood combustion (RWC), which has been increasingly used as a heating source, is of health concern because emissions from RWC contain carcinogenic polycyclic aromatic hydrocarbons (PAH). To assess health risk, the possible formation of PAH-DNA adducts in white blood cells (WBC) and placentas of nonsmoking women exposed to RWC smoke during pregnancy was measured by a nuclease P1-enhanced 32P-postlabeling assay having a sensitivity limit of one lesion per 10(9-10) DNA nucleotides. DNA samples isolated from 12 exposed specimens (8 WBC, 4 placentas) and 13 unexposed control specimens (8 WBC, 5 placentas) were hydrolyzed to mononucleotides, which were then 32P-labeled and separated by high-resolution thin-layer chromatography. Comparison of autoradiograms of exposed DNA samples with those of controls failed to show exposure-related adducts. All placental DNA maps exhibited one major (47 +/- 10%) and 12 minor extra spots, however, that were not seen on WBC-DNA maps. These derivatives corresponded to an average of 12 (+/- 6) modifications in 10(9) nucleotides. Similarly, WBC DNA showed four spots that were absent in placental DNA and were not quantified because of their low levels. Neither placental nor WBC DNA adducts coincided chromatographically with the product formed by the reaction of benzo(a)pyrene diol epoxide I with N2 of guanine in DNA. Although these results suggest that RWC smoke does not elicit detectable levels of aromatic DNA adducts in humans, lack of evaluation of RWC exposure levels leaves some uncertainty in this conclusion. The results, however, clearly show that placental and WBC DNA contain covalent modifications that are unrelated to RWC exposure. These DNA derivatives may be caused by ingestion and/or inhalation of, or skin contact with, low levels of environmental genotoxicants or may arise from endogenous electrophiles of as yet unknown origin.
Benzo(a)pyrene↗
Mobility, distribution and persistence of metalaxyl residues in pearl millet (Pennisetum americanum (L.) Leeke.).
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Alanine↗
An improved method for in vitro cultivation of murine splenic macrophages.
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Animals↗