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Biomedical subjects

R C Gupta

Publications and source records attributed to R C Gupta.

At least 253 records · Page 14Linked to original sources
PubMed1985

Specificities and characteristics of mitochondrial protein antigens assessed by enzyme linked immunosorbent assay for anti-mitochondrial antibodies. Relationship to immunofluorescent and precipitating antibodies.

M-A, M-B and M-C are autoantibodies to mitochondrial proteins frequently found in primary biliary cirrhosis. To study the characteristics and specificities and to isolate antigens reacting with the autoantibodies a sensitive assay (ELISA) was established. Using this technique a significantly elevated level of antibodies was detected in patients with primary biliary cirrhosis (mean OD 460.2, s.d. 145.8) compared to normals (133.7 +/- 49.7). The assay correlated well with the indirect immunofluorescence test for detecting anti-mitochondrial antibodies and the mitochondrial fluorescence could be abolished by absorption of autoantibodies with the mitochondrial fraction. All three types of antibodies could be detected by ELISA; however, sera containing the combination of two or more antibodies yielded higher ELISA values. The ELISA confirmed that the M-A antigen is trypsin and acid (pH 3.0) sensitive but DNAase and RNAase resistant while the M-B antigen is DNAase and trypsin sensitive. The antigens were enriched in the supernatant isolated from the mitochondrial fraction centrifuged at 1,800 g for 60 min and 2% polyethylene glycol precipitates of the mitochondrial fraction. The antigens were found phosphate buffer soluble and therefore could also be enriched by phosphate buffer extraction of the mitochondrial proteins. Thus, ELISA described here provided a sensitive method in the assessment of characteristics and purification of autoantigens related to mitochondrial antibodies.

Antigens↗
PubMed1985

Chronic active hepatitis associated with polymyositis. Association with precipitating mitochondrial M-B antibody.

A 20-year-old black female presented with chronic active hepatitis and polymyositis. Interstitial lung disease and transient polyarticular arthritis were also observed, but other features of systemic lupus erythematosus, scleroderma and Sjögren's syndrome were absent. Both myositis and hepatitis responded to prednisone and azathioprine. The patient's serum contained an unusual autoantibody reacting with mitochondrial proteins (M-B) in immunodiffusion.

Adult↗
PubMed1985

Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen:DNA adducts.

We have previously described a 32P assay for the detection and quantitation of aromatic carcinogen:DNA adducts (R. C. Gupta et al., Carcinogenesis (Lond.), 3: 1081-1092, 1982). The method entails enzymatic digestion of DNA to deoxynucleoside 3'-monophosphates which are then converted to deoxynucleoside 3',5'-[5'-32P]diphosphates by T4 polynucleotide kinase-catalyzed 32P transfer from adenosine [gamma-32P]triphosphate. Labeled adducts are purified and resolved by four-directional thin-layer chromatography. This procedure can detect one adduct in 10(7)-10(8) nucleotides but quantitation of adduct concentrations of one adduct in greater than 5 X 10(6) nucleotides becomes exceedingly difficult. I have now found that isolation of DNA adducts by extraction with 1-butanol in the presence of the phase-transfer agent tetrabutylammonium chloride prior to the labeling allows one to use excess carrier-free adenosine [gamma-32P]triphosphate (100-200 microCi), thus enabling quantitative analysis of a single adduct in 10(9)-10(10) nucleotides when 1-10 micrograms of the DNA are used. Further increase in the sensitivity of the assay requires higher amount of DNA. The four-directional thin-layer chromatography system has been modified so as to analyze simultaneously as many as 35-40 DNA samples. The new protocol, as applied to a number of carcinogenic aromatic amines and polycyclic aromatic hydrocarbons of diverse structure, is capable of detecting and quantitating adducts at the level of one adduct per 10(10) nucleotides.

Adenosine Triphosphate↗
PubMed1984

Intrinsic phosphatase activity of bovine brain calcineurin requires a tightly bound trace metal.

The divalent metal requirement of intrinsic phosphatase activity was investigated using native and trypsinized calcineurin. This was assessed by examining (1) the stimulation of the enzyme by various metals, (2) the inhibition of the enzyme activity by metal chelators (EDTA and EGTA), and (3) the restoration by various metals of the activity of the EDTA-inhibited calcineurin phosphatase. The results supported the view that a tightly bound trace metal is necessary for expression of the phosphatase activity of calcineurin and implicate Mn2+ as the tightly bound metal.

Animals↗
PubMed1984

Effect of subchronic administration of methyl parathion on in vivo protein synthesis in pregnant rats and their conceptuses.

Pregnant rats received daily po doses of the organophosphate methyl parathion (MPTH) from Day 6 through Day 15 or 19 of gestation at doses causing no (1.0 mg/kg) or minimal (1.5 mg/kg) signs of maternal toxicity. Following the dose of MPTH on Day 15 or 19, in vivo protein synthesis was measured 0.5, 1.0, and 2.0 hr after sc injection of L-[1-14C]valine at a dose of 5 microCi/mmol/100 g body wt. The specific activity of [14C]valine in the free amino acid pool and protein bound pool was significantly reduced in various regions of maternal brain and in maternal viscera, placenta, and whole embryos (Day 15), and in fetal brain and viscera (Day 19). The inhibitory effect of MPTH on net protein synthesis was dose dependent, greater on Day 19 than 15 of gestation and more pronounced in fetal than in maternal tissues.

Animals↗
PubMed1984

Nonrandom binding of the carcinogen N-hydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in vivo.

We have examined the distribution of individual adducts in repetitive DNA sequences of rat liver in vivo after a single dose of the carcinogen N-hydroxy-2-acetyl-aminofluorene. Repetitive fragments [82, 125, 179, 225, and 370 base pairs (bp)] were isolated by digestion of hepatic DNA with HindIII restriction endonuclease (EC 3.1.23.21) and gel electrophoresis. As assayed by 32P postlabeling, no qualitative differences were observed between the DNA-bound metabolites in repetitive sequences and total DNA, but preferential binding to these sequences occurred. After 1 day of treatment, the amounts of N-hydroxy-2-acetylaminofluorene-induced adducts were found to be 13.8, 2.0, and 3.0 times higher in 179-, 225-, and 370-bp repeats, respectively, than in total DNA, while 82- and 125-bp repeats showed no differences. The relative distribution of individual adducts varied among the various sequences. After 9 days, all five sequences showed 1.3-1.7 times higher binding as compared to total DNA. In contrast, a random binding was observed when DNA reacted in vitro with an active metabolite, N-acetoxy-2-acetylaminofluorene. Taken together, these results suggest that the enrichment and differential excision of adducts in the repetitive DNA sequences may be a function of the nuclear organization of DNA. This application of the 32P assay constitutes a means to study the DNA damage and excision repair in vivo in chromatin structural components, including transcribed and nontranscribed multiple-copy genes, in a much more sensitive and precise way than has been hitherto possible.

2-Acetylaminofluorene↗
PubMed1984

Acute malathion toxicosis and related enzymatic alterations in Bubalus bubalis: antidotal treatment with atropine, 2-PAM, and diazepam.

Oral administration of malathion (MTH) in sublethal (100 mg/kg) or minimal lethal (125 mg/kg) doses in buffalo calves produced toxicity with an onset within 15-20 min and peak effects including severe tremors and convulsions within 40-60 min. Various antidotal drugs were administered alone or in combination at the time of peak malathion toxicity (within 1 h) and were assessed for their ability to alleviate signs of cholinergic toxicity. Blood cholinesterase and aminotransferases activities were monitored at various times. A combination of atropine sulfate (0.5 mg/kg, 1/4 iv and 3/4 im) and pyridine 2-aldoxime methiodide (2-PAM, 20 mg/kg, iv) reversed the clinical evidence of malathion toxicity within 15 min. The combination of atropine sulfate and diazepam (0.75 mg/kg, iv) prevented death and cholinergic signs of toxicity except for weak muscular fasciculations, which persisted for 30-60 min. Atropine sulfate alone was less effective and also did not reverse malathion-induced biochemical changes. In contrast, administration of either 2-PAM (10-30 mg/kg, iv) or diazepam (0.5-1.0 mg/kg, iv) alone accentuated malathion toxicity. Thus, the combination of atropine sulfate and 2-PAM was the most effective antidotal treatment in acute malathion toxicity.

Animals↗
PubMed1984

32P-postlabeling test for covalent DNA binding of chemicals in vivo: application to a variety of aromatic carcinogens and methylating agents.

Carcinogen--DNA adducts were detected and determined by 32P-postlabeling assay after exposure of mouse or rat tissues in vivo to a total of 28 compounds comprising 7 arylamines and derivatives, 3 azo compounds, 2 nitroaromatics, 12 polycyclic aromatic hydrocarbons, and 4 methylating agents. DNA was isolated from mouse skin, mouse liver, and rat liver after treatment with the individual carcinogens, then digested enzymatically to deoxyribonucleoside 3'-monophosphates, which were converted to 5'-32P-labeled deoxyribonucleoside 3',5'-bisphosphates by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. The nucleotides were resolved by anion-exchange t.l.c. on polyethyleneimine-cellulose and detected by autoradiography. The determination of low levels of DNA binding of the aromatic carcinogens entailed the removal of normal nucleotides prior to the resolution of adduct nucleotides. For this purpose, an alternative procedure employing reversed-phase t.l.c. was devised which offered advantages for the detection of quantitatively minor adducts. The procedures described enabled the detection of 1 aromatic DNA adduct in approximately 10(8) normal nucleotides, while the limit of detection of methylated adducts was 1 adduct in approximately 6 X 10(5) nucleotides. The results show that a great number of carcinogen-DNA adducts of diverse structure are substrates for 32P-labeling by polynucleotide kinase-catalyzed phosphorylation. Because covalent DNA adduct formation in vivo appears to be an essential property of the majority of chemical carcinogens, 32P-postlabeling analysis of carcinogen--DNA adducts in mammalian tissues may serve as a test for the screening of chemicals for potential carcinogenicity.

Alkylating Agents↗
PubMed1984

Formation and removal of DNA adducts in rat liver treated with N-hydroxy derivatives of 2-acetylaminofluorene, 4-acetylaminobiphenyl, and 2-acetylaminophenanthrene.

Formation and removal of hepatic DNA adducts was studied in male Sprague-Dawley rats following single injections of two hepatocarcinogens, N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) and a nonhepatocarcinogen, N-hydroxy-2-acetylaminophenanthrene (N-OH-AAP) at 0.5 h, 1.5 h, 4 h, 24 h, 9 d and 29 d. Using a previously described 32P-postlabeling assay, maximal DNA binding of these compounds was observed at approximately 1.5 h, 0.5 h and 24 h, respectively. In addition to the formation of three already known C8- and N2-acetylated and C8-deacetylated guanine derivatives and several minor unknown adducts with N-OH-AAF, a set of four new major adducts was also detected. These comprised approximately 50% of the total adducts during the first 4 h. The three known adducts amounted to 58, 16 and 6% of the 1.5-h value after 24 h, 9 d and 29 d, respectively, while the bulk (greater than 84%) of the new major adducts were removed from the DNA within 24 h and found only in traces after 9 d. N-OH-AABP formed several unknown minor adducts, in addition to the one major C8-deacetylated and two minor C8- and N2-acetylated guanine derivatives; only the C8-deacetylated and N2-acetylated adducts were detected after 29 d. In the case of N-OH-AAP, two major and several minor adducts were detected, most of which were found to be deacetylated, and as much as 60% of the adducts measured at 24 h were still present after 9 d treatment. These data indicate that certain DNA adducts are repaired rapidly, while others persist for long periods.

2-Acetylaminofluorene↗
PubMed1984

Microbial transformation of primaquine by Candida tropicalis.

The microbial metabolism of primaquine, a 6-methoxy-8-aminoquinoline antimalarial agent, was investigated. The yeast Candida tropicalis was found to convert primaquine to the previously reported N-acetylated derivative. On continued incubation of C. tropicalis in the presence of the N-acetylated derivative, a minor dimeric metabolite was formed. The proposed structure of the metabolite was based primarily on the analysis of its spectroscopic properties (1H and 13C nuclear magnetic resonance spectra and field-desorption mass spectrum). The structure of the metabolite was proven by direct comparison with an authentic sample of the minor dimeric metabolite prepared by treatment of the N-acetylated derivative with formaldehyde in the presence of formic acid in methanol.

Acetylation↗
PubMed1984

Identification of HBsAg determinants in immune complexes from hepatitis B virus-associated vasculitis.

Circulating immune complexes were characterized from 25 sera obtained from five patients with polyarteritis nodosa and three with cutaneous venulitis associated with hepatitis B virus infection. Complexes were isolated by polyethylene glycol and conglutinin-anticonglutinin precipitation methods and were analyzed for HBsAg and anti-HBsAg. Low pH was used to dissociate the complexes, and components were separated into antigen and antibody fractions by using immobilized protein A. In this study, three observations were significant: 1) complexes were frequent and quantitatively more in cutaneous venulitis than in polyarteritis; 2) the levels of HBsAg in the antigen fractions of polyarteritis were greater and correlated with the clinical improvement of the disease; the serum levels of HbsAg remained the same throughout the course of the disease; and 3) complexes from polyarteritis were not completely dissociable at pH 2.6 compared with those from patients with cutaneous venulitis and chronic active hepatitis. The antigen fractions electrophoresed in polyacrylamide gel with SDS demonstrated 6 to 10 protein bands with m.w. ranging between 17,000 and 120,000 daltons. To precisely define the polypeptide antigen moiety involved in the immune complex formation, a transfer blotting technique was used employing human anti-HBsAg globulin as probe. Polypeptides with m.w. 97,000, 49,000, and 23,000 were found to form complexes in both groups of patients.

Adolescent↗
PubMed1984

Precipitating autoantibodies to mitochondrial proteins in progressive systemic sclerosis.

Sera from 88 patients with progressive systemic sclerosis were examined for precipitating mitochondrial antibodies using sonicated rat liver mitochondrial fraction as an antigen source in immunodiffusion. Precipitin lines indicating the presence of anti-mitochondrial antibodies (AMA) in 22 patients were detected. Only six of 22 sera had, additionally, precipitating antibodies to nuclear antigens. Standardized reference sera containing antibodies to mitochondrial antigens (M-A, M-B and M-C systems) were used to further characterize the type of mitochondrial antibodies. M-B antibody was most commonly detected (72.7%) either alone (eight patients) or in combination (eight patients) with M-A and M-C antibodies. M-A antibody was found in 12 patients (54.5%) and M-C antibody was present in three. The antigen related to M-B is DNAase and trypsin sensitive, in contrast to the resistant M-A antigen. AMA were detected in 21 of 22 patients by indirect immunofluorescence. When solid phase ELISA was used to detect AMA using mitochondrial fraction as antigen, a significant difference (P less than 0.005) was noted between sera with and without precipitating mitochondrial antibody. The antibody was frequently present in patients with progressive systemic sclerosis detected 2 or more years earlier (P less than 0.01). Three patients were found to have primary biliary cirrhosis and others had pruritus, hepatomegaly or abnormal liver function tests. The implication of the findings is discussed.

Animals↗
PubMed1984

Incidence of isoniazid acetylation phenotypes in North Indians.

Isoniazid acetylation phenotypes were determined in 110 cases (55 of pulmonary tuberculosis and 55 of nontubercular chest diseases) by sulphadimidine acetylation test (Bratton and Marshall method). Cases with more than 65% of acetylated sulphadimidine in urine at 6th h were classified as fast acetylators, those with less than 50% as slow acetylators and others with values between 50-65% as intermediate acetylators. There were 39.09% fast, 46.36% intermediate and 14.55% slow acetylators. Possible factors for difference in results from other Indian studies have been discussed. Sulphadimidine acetylation showed no variation due to presence of disease, age, sex, religion, place of origin, height, weight and smoking habits. However, 79% of those who took alcohol regularly turned out to be fast acetylators.

Acetylation↗