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M V Reddy

Publications and source records attributed to M V Reddy.

At least 19 recordsLinked to original sources
PubMed2001

Detection of DNA adducts using a quantitative long PCR technique and the fluorogenic 5' nuclease assay (TaqMan).

The detection of DNA adducts is an important component in assessing the mutagenic potential of exogenous and endogenous compounds. Here, we report an in vitro quantitative long PCR (XL-PCR) assay to measure DNA adducts in human genomic DNA based on their ability to block and inhibit PCR amplification. Human genomic DNA was exposed to test compounds and then a target sequence was amplified by XL-PCR. The amplified sequence was then quantified using fluorogenic 5' nuclease PCR (TaqMan) and normalized to a solvent-treated control. The extent of DNA adduction was determined based on the reduction in amplification of the target sequence in the treated sample. A 17.7kb beta-globin fragment was chosen as the target sequence for these studies, since preliminary experiments revealed a two-fold increased sensitivity of this target compared to a 10.4kb HPRT fragment for detecting hydrogen peroxide-induced DNA damage. Validation of the XL-PCR assay with various compounds demonstrated the versatility of the assay for detecting a wide range of adducts formed by direct acting or S9-activated mutagens. The same DNA samples were also analyzed using 32P-postlabeling techniques (thin-layer chromatography or high-performance liquid chromatography) to confirm the presence of DNA adducts and estimate their levels. Whereas 32P-postlabeling with nuclease P(1) enrichment was more sensitive for detecting bulky adducts induced by the compounds benzo[a]pyrene, dimethylbenzanthracene, 3-methylindole, indole 3-carbinol, or 2-acetylaminofluorene, the XL-PCR procedure was more sensitive for detecting smaller or labile DNA adducts formed by the compounds methyl methanesulfonate, diethyl nitrosamine, ethylnitrosourea, diepoxybutane, ICR-191, styrene oxide, or aflatoxin B(1). Compounds not expected to form adducts in DNA, such as clofibrate, phenobarbital, chloroform or acetone, did not produce a positive response in the XL-PCR assay. Thus, quantitative XL-PCR provides a rapid, high-throughput assay for detecting DNA damage that complements the existing 32P-postlabeling assay with nuclease P(1) enrichment.

Animals↗
PubMed2001

Asymmetric synthesis of umuravumbolide.

[figure: see text] This first asymmetric synthesis of enantiopure desacetylumuravumbolide and umuravumbolide via asymmetric reduction, allylboration, and ring-closing metathesis confirms their revised structures and configurations. A convenient procedure to upgrade the enantiopurity of alpha,beta-acetylenic alcohols is also described.

Catalysis↗
PubMed2001

Sero-reactivity of two different antigenic protein fractions of M.tb H37Ra excretory-secretory antigen in pulmonary and extra-pulmonary tuberculosis.

Excretory-secretory proteins of Mycobacterium tuberculosis H37Ra, have been of diagnostic interest in pulmonary (PTB) and extrapulmonary tuberculosis (EPTB). Two different excretory-secretory antigenic proteins of M.tbH37Ra viz., EST-DE1 (a 6% TCA soluble and DEAE anion exchange purified antigen) and ESAS-7 (50% ammonium sulphate solubilized and SDS-PAGE fractionated antigen) were studied in stick-indirect penicillinase ELISA for detecting tuberculous IgG antibodies in serum samples of pulmonary as well as extrapulmonary tuberculosis (tuberculous lymphadenopathy (TBLN), tuberculous meningitis (TBM), bone & joint tuberculosis (B&J TB), abdominal tuberculosis (Abd. TB) patients. The ESAS-7 antigen has shown comparatively better seroreactivity (90%) than that of EST-DE1 antigen in pulmonary tuberculosis cases. The overall specificity of 93.2% using ESAS-7 antigen was also found better compared to 86.4% obtained using EST-DE1 antigen. Further, in extra pulmonary tuberculosis group, using ESAS-7 antigen 84% (21/25) of histopathologically confirmed TBLN cases and 90% (9/10) clinically diagnosed and ATT responded TBM cases showed positive reaction for tuberculous IgG antibody. The per cent positivity using EST-DE1 antigen was however comparatively low in TBLN and TBM cases, (76% and 80% respectively). In histopathologically proven bone and joint tuberculosis and abdominal tuberculosis cases EST-DE1 antigen showed better sensitivity of 75% and 83.3% respectively in IgG antibody detection compared to that of ESAS-7 antigen (50% and 66% respectively). From the present study, it can be envisaged that ESAS-7 antigenic fraction has a good potential in the diagnosis of pulmonary and certain extra-pulmonary tuberculosis infection (TBLN & TBM) whereas EST-DE1 was found to be better in detecting specific antibodies in bone & joint and abdominal tuberculosis.

Antibody Formation↗
PubMed2000

Methods of DNA adduct determination and their application to testing compounds for genotoxicity.

At the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC (March 25-26, 1999), a working group considered the uses of DNA adduct determination methods for testing compounds for genotoxicity. When a drug or chemical displays an unusual or inconsistent combination of positive and negative results in in vitro and in vivo genotoxicity assays and/or in carcinogenicity experiments, investigations into whether or not DNA adducts are formed may be helpful in assessing whether or not the test compound is a genotoxin. DNA adduct determinations can be carried out using radiolabeled compounds and measuring radioactive decay (scintillation counting) or isotope ratios (accelerator mass spectrometry) in the isolated DNA. With unlabeled compounds adducts may be measured by (32)P-postlabeling analysis of the DNA, or by physicochemical methods including mass spectrometry, fluorescence spectroscopy, or electrochemical detection, or by immunochemical methods. Each of these approaches has different strengths and limitations, influenced by sensitivity, cost, time, and interpretation of results. The design of DNA binding studies needs to be on a case-by-case basis, depending on the compound's profile of activity. DNA purity becomes increasingly important the more sensitive, and less chemically specific, the assay. While there may be adduct levels at which there is no observable biological effect, there are at present insufficient data on which to set a threshold level for biological significance.

DNA Adducts↗
PubMed2000

Methods for testing compounds for DNA adduct formation.

This article, based on a presentation on DNA adduct detection given at a Genetic Toxicology Association workshop, is an overview of methods used for testing compounds for DNA adduct formation. A DNA adduct study may be initiated on a case by case basis when there are conflicting results within the standard battery of genetic toxicology tests or when tumors are detected in the animal bioassay for nongenotoxic compounds. Methods for adduct detection include the 32P-postlabeling assay, the use of radioactive test chemicals, physicochemical methods, and immunoassays. Of these, the 32P-postlabeling assay and the use of radiochemicals are discussed in greater detail, since only these two methods are readily applicable to test a compound for the formation of uncharacterized DNA adducts. The other methods are applicable to those adducts that have been chemically characterized or that contain a fluorophore or electrochemically active groups. Evaluation of mutagenic and carcinogenic risk from DNA adducts would require the understanding of various parameters, including the chemical nature, quantity and stability of adducts, proliferation rates for target cells to fix adducts into mutations, mutagenic and repair efficiencies of adducts, and the extent of modifications in critical genes. Since such data cannot be readily obtainable, the toxicological risk from uncharacterized adducts is difficult to assess.

Animals↗
PubMed2000

A 43-kDa circulating filarial antigen fraction of Wuchereria bancrofti in immunoprophylaxis against Brugia malayi in jirds.

A 43 kiloDaltan (kDa) antigen fraction (CFA2-6) isolated from microfilaraemic plasma of bancroftian filarial patients showed selective reactivity with sera samples collected from endemic normals. Antibodies raised against this antigen showed a strong reactivity with the surface of Brugia malayi infective larvae as well as microfilariae. Similar antigenic determinants were detected in the parasite extracts, but not in the excretory-secretory products. Further analysis was done on the immunoprophylactic potential of CFA2-6 in inducing immunity against Brugia malayi in Meriones unguiculatus (jird) in vivo. A strong protective response of approximately 84% was observed against the development of the filarial parasite in the jirds immunized with CFA2-6. The immunized jirds also showed a significant clearance (87%) of microfilariae inoculated intravenously. Approximately 65% of infective larvae failed to survive in jirds transferred with anti-CFA2-6 serum compared to the jirds transferred with sera from the control jirds. Passive transfer of anti-CFA2-6 antibody to the jirds followed by intravenous inoculation of microfilariae resulted in the reduction of 77% of circulating microfilariae. This study suggests that the 43-kDa CFA2-6 could stimulate a strong protective immune response against infective larvae and microfilariae in experimental animals.

Animals↗
PubMed2000

Electret ion chambers for estimating alpha particle energy.

Electret ion chambers (EICs) are inexpensive, lightweight, robust, commercially available, passive, charge-integrating devices for measuring different radiations. At Florida International University's Hemispheric Center for Environmental Technology (FIU-HCET), EIC performance was evaluated with the goal of providing the U.S. Department of Energy (DOE) with a low-cost, low-risk means of alpha contamination monitoring using an existing commercial technology. During this evaluation, sensitivity of electrets in 960-mL hemispherical ion chamber (radius: 7.6 cm) was observed to increase with alpha particle energy because alpha particles expended their entire energy in the chamber volume. This observation prompted us to explore another EIC application: estimation of alpha particle energy. Correlation between electret mid-point voltage, alpha particle energy, and response was developed for the 960-mL EIC and was used to determine energy of an unknown source. The response (change in voltage per disintegration) is the ratio of the change of voltage per unit time and alpha particle activity. Since an EIC measures only the change in voltage per minute, to determine the alpha particle energy, the source activity should be accurately determined by another method.

Alpha Particles↗
PubMed2000

Seroreactivity of purified Brugia malayi microfilarial soluble and excretory-secretory antigens in different clinical presentations of bancroftian filariasis.

Brugia malayi microfilarial excretory-secretory (mf ES) and phosphate buffer saline soluble (mf S) antigens were fractionated by fast protein liquid chromatography (FPLC) on superdex 200 HR 10/30 gel filtration column. The active antigen fractions were identified and explored in comparison with whole mf ES and mf S antigens to detect filarial IgG antibodies in different groups viz microfilaraemics, acute, chronic and occult filarial cases of Wuchereria bancrofti infection and endemic and non-endemic normals. One of the fractions of mf ES antigen (ESF-6) and two fractions of mf S antigen (SF-2 & 3) were identified to be useful to detect filarial antibodies. A pooled preparation of these antigen fractions gave a sensitivity of 86.6% (for microfilaraemic cases) and a specificity of 95% to detect filarial IgG antibodies by indirect ELISA. The pooled FPLC purified mf antigens also showed 55-88% of cases of different grades of clinical filariasis and 65% of tropical pulmonary eosinophilia cases as positive for filarial antibodies. The pooled FPLC purified B. malayi mf antigens with higher specificity are preferable to whole mf ES and mf S antigens to detect active filarial infection in microfilaraemia and as well in different clinical entities of bancroftian filariasis.

Animals↗
PubMed1999

Lamellarin alpha 20-sulfate, an inhibitor of HIV-1 integrase active against HIV-1 virus in cell culture.

HIV-1 integrase is an attractive target for anti-retroviral chemotherapy, but to date no clinically useful inhibitors have been developed. We have screened diverse marine natural products for compounds active against integrase in vitro and found a series of ascidian alkaloids, the lamellarins, that show selective inhibition. A new member of the family named lamellarin alpha 20-sulfate (1), the structure of which was determined from spectroscopic data, displayed the most favorable therapeutic index. The site of action of lamellarin alpha 20-sulfate on the integrase protein was mapped by testing activity against deletion mutants of integrase. Inhibition of isolated catalytic domain was detectable though weaker than inhibition of full length integrase; possibly lamellarin alpha 20-sulfate binds a site composed of multiple integrase domains. Lamellarin alpha 20-sulfate also inhibited integration in vitro by authentic HIV-1 replication intermediates isolated from infected cells. Lamellarin alpha 20-sulfate was tested against wild type HIV using the MAGI indicator cell assay and found to inhibit early steps of HIV replication. To clarify the inhibitor target, we tested inhibition against an HIV-based retroviral vector bearing a different viral envelope. Inhibition was observed, indicating that the HIV envelope cannot be the sole target of lamellarin alpha 20-sulfate in cell culture. In addition, these single round tests rule out action against viral assembly or budding. These findings provide a new class of compounds for potential development of clinically useful integrase inhibitors.

Animals↗
PubMed1999

Surgical filariasis: immunoscreening for filarial IgG antibodies using Wuchereria bancrofti microfilarial excretory-secretory antigen.

A clinical study and immunoscreening was conducted on 363 suspected filarial patients attending the surgery out patient division of the MGIMS, Sevegram. The disease was significantly higher in males (86%) than in females (14%). Majority (52.9%) of the cases were in the age group of 11-30 years. The distribution of cases into three different grades of infection showed, 52.6%, 33.3% and 14.1% of the cases having acute (grade I), sub-acute (grade II) and chronic (grade III) stages of infection respectively. While 73% of the cases had genital manifestations, 23% were with lymphatic obstruction in limbs and the rest of the 4% suffered from manifestations like cellulitis, abscesses, haematuria and chyluria. Filarial IgG antibodies against microfilarial excretory-secretory (mf ES) antigen were detected in 89% of cases with genital manifestations, 87% of lymphoedema cases, 67% of lymphadenitis cases and 60% of cases with other clinical manifestations and 3% of endemic normals.

Adolescent↗
PubMed1998

Src kinases and not JAKs activate STATs during IL-3 induced myeloid cell proliferation.

Interaction of IL-3 with its receptor is known to activate STAT-3 via phosphorylation of Tyrosine 701, which facilitates its dimerization and translocation to the nucleus, leading to the transcription of its target genes. In this communication, we have investigated the nature of tyrosine kinases that mediate STAT-3 phosphorylation during IL-3-mediated activation of myeloid cell proliferation. Our results show that interaction of IL-3 with its receptor leads to the activation of c-Src kinase activity, which in turn facilitates the binding of c-Src to STAT-3. This association leads to the phosphorylation of STAT-3, allowing this transcription factor to translocate to the nucleus. Expression of a dominant negative mutant of src (AMSrc) in these cells results in a block to IL-3 mediated phosphorylation of STAT-3, and its ability to bind to DNA. On the other hand, expression of a dominant negative mutant of JAK2 (JAK2KE) had no effect on IL-3-mediated activation of STAT-3. Our results also show that AMSrc does not affect the phosphorylation of JAK2, suggesting that JAK and STAT phosphorylation events are mediated by two independent pathways. Inhibition of c-Src activation by AMSrc, which leads to a block to STAT-3 activation, results in a dramatic inhibition of cell proliferation mediated by IL-3. However, expression of AMSrc does not activate apoptotic pathways. In contrast, expression of JAK2KE results in accelerated apoptosis of 32Dcl3 cells grown in the absence of IL-3 with concomitant down-regulation of Erk-2 kinase activity. These results suggest that Src family kinases mediate the phosphorylation of STATs and play a critical role in signal transduction pathways associated with myeloid cell proliferation while JAK kinases mediate the activation of Erk-2 pathway which appears to provide antiapoptotic signals. Thus the activation of JAKs and STATs appear to be two independent but related events, which dictate two separate biological outcomes, the combination of which results in proliferation and survival of myeloid precursor cells.

Animals↗
PubMed1998

Pictorial CME.

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Echocardiography, Doppler↗
PubMed1997

Correlation of mutagenic potencies of various petroleum oils and oil coal tar mixtures with DNA adduct levels in vitro.

An in vitro system was utilized to measure DNA adduct-forming ability of petroleum oils and oil coal tar mixtures to define correlations between DNA adduct levels and their mutagenic potencies. The system consisted of reaction of dimethyl sulfoxide extracts of oils with calf thymus DNA in the presence of Aroclor-induced hamster liver microsomes for 30 min. Following DNA extraction, DNA adducts were measured by the nuclease P1-enhanced postlabeling assay coupled with two-dimensional polyethyleneimine (PEI)-cellulose TLC. Thin layer plates showed putative aromatic DNA adducts, with levels ranging from 60 to 1400 adducts per 10(9) DNA nucleotides. TLC mobilities suggested adducts to be aromatic compounds containing 4 or more rings. A good correlation (coefficient of correlation = 0.91) was observed between DNA adduct levels and Salmonella mutagenicity for 19 oils. All 19 samples tested produced DNA adducts. To expedite the TLC procedure, adducts were resolved by one-dimensional TLC and the radioactivity measured using a mechanical scanner. Results were comparable to those obtained by two-dimensional TLC and quantification after scraping. Our data show that the in vitro incubation system coupled with the postlabeling adduct assay is a useful screening method to identify mutagenic and potentially carcinogenic oils.

Animals↗
PubMed1997

Luffasterols A-C, 9,11-secosterols from the Palauan sponge Luffariella sp.

The Palauan sponge Luffariella sp. contained manoalide (6) and secomanoalide (7) as the major metabolites. The minor metabolites, luffasterols A-C (3-5) are 9,11-secosterols that contain a 3 beta-acetoxy-5 alpha,6 alpha-epoxy-9-oxo-9,11-secocholest-7-en-11-al ring system joined to three different side chains. The structures of the luffasterols were elucidated by interpretation of spectroscopic data.

Animals↗
PubMed1997

Influence of intensive yoga training on physiological changes in 6 adult women: a case report.

The short-term effects of 4 weeks of intensive yoga practice on physiological responses in six healthy adult female volunteers were measured using the maximal exercise treadmill test. Yoga practice involved daily morning and evening sessions of 90 minutes each. Pre- and post-yoga exercise performance was compared. Maximal work output (Wmax) for the group increased by 21%, with a significantly reduced level of oxygen consumption per unit work but without a concomitant significant change in heart rate. After intensive yoga training, at 154 Wmin(-1) (corresponding to Wmax of the pre-yoga maximal exercise test) participants could exercise more comfortably, with a significantly lower heart rate (p < 0.05), reduced minute ventilation (p < 0.05), reduced oxygen consumption per unit work (p < 0.05), and a significantly lower respiratory quotient (p < 0.05). The implications for the effect of intensive yoga on cardiorespiratory efficiency are discussed, with the suggestion that yoga has some transparently different quantifiable physiological effects to other exercises.

Adult↗
PubMed1996

Chemotherapeutic activity of benzoxazinorifamycin, KRM-1648, against Mycobacterium tuberculosis in C57BL/6 mice.

OBJECTIVE: To investigate the chemotherapeutic activity of benzoxazinorifamycin, KRM-1648, in comparison with rifabutin (RFB) and rifampin (RIF) against experimental tuberculosis. DESIGN: C57BL/6 mice were infected with 10(5)-10(6) colony forming units (CFU) of either drug-susceptible virulent Mycobacterium tuberculosis (H37Rv) or multi-drug resistant (MDR) M. tuberculosis strain (2230) and were treated from the next day (early treatment) or after 2 weeks following infection (established infection) with 20 mg/kg dose of each drug or none (untreated control). The efficacy of chemotherapy was assessed based on prevention of mortality and on CFU levels in the lungs and spleens. RESULTS: All three drugs prevented mortality for up to 28 weeks of observation, while all the untreated control mice died by 4 weeks. Analysis of CFUs revealed superior therapeutic activity of both KRM-1648 and RFB as compared to RIF against the drug-susceptible strain of M. tuberculosis under the early treatment protocol. Twelve weeks' treatment with KRM-1648 or RFB caused complete sterilization of the lungs. However, residual organisms started appearing in the spleens 6 weeks after cessation of treatment with RFB and 16 weeks after KRM-1648 treatment. In mice infected with a MDR strain of M. tuberculosis, which was susceptible in vitro to KRM-1648, the drug did not appear to have any activity. Since the MDR organisms did not multiply in vivo, and did not cause any mortality up to 28 weeks in the RIF-treated control mice, a state of semi-dormancy of the organisms which might prevail in vivo could be responsible for refractoriness to treatment with KRM-1648. CONCLUSIONS: KRM-1648 showed an excellent chemotherapeutic activity, as compared to RFB and RIF, against drug-susceptible tuberculosis. However, all three analogues were ineffective against infection with multi-drug resistant strain of M. tuberculosis.

Animals↗