Towards a system for health care delivery in general practice.
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Australia↗
Biomedical subjects
P Joseph
Publications and source records attributed to P Joseph.
2-Aminofluorene bioactivation by human term placental peroxidase.
Earlier investigations from our laboratory demonstrated that human term placental peroxidase (HTPP) is capable of metabolism of xenobiotics and endogenous compounds. In this study, purified HTPP was found to bioactivate 2-aminofluorene (2-AF) in the presence of H2O2. 2-AF oxidation was studied spectrophotometrically while radiometry was employed to assess the bioactivation. The rate of oxidation and covalent binding to protein and DNA was dependent upon the pH of the reaction medium and the concentration of 2-AF, the enzyme, and H2O2. To observe maximal enzyme velocity of oxidation, the presence of 16.5 microM H2O2, 100 microM 2-AF, 37 micrograms of the enzyme protein/ml, and pH 7.2 was required. Under optimal assay conditions, the range of specific activity between 130 and 165 nmol of 2-AF oxidized/min/mg HTPP was observed. Using similar assay conditions, the magnitude of covalent binding of [3H]-2-AF to protein (BSA) and calf thymus DNA was found to be about 508 pmol bound/min/mg HTPP/mg BSA and 84 pmol bound/min/mg HTPP/mg DNA, respectively. Potassium cyanide and sodium azide, the known inhibitors of different peroxidases, significantly blocked both the oxidation and covalent binding of 2-AF in a dose dependent manner. These results strongly suggest that peroxidase may be one of the important pathways responsible for the bioactivation of arylamines in human term placenta.
Azides↗
Peroxidative xenobiotic oxidation by partially purified peroxidase and lipoxygenase from human fetal tissues at 10 weeks of gestation.
1. Present study reports the ability of partially purified peroxidase and lipoxygenase from human fetal tissues at 10 weeks of gestation to oxidize selected xenobiotics in vitro. 2. Peroxidase was found to oxidize four different chemicals in the presence of H2O2. Sodium azide and potassium cyanide inhibited peroxidase activity towards guaiacol in a concentration-dependent manner. 3. The dioxygenase and co-oxidase activities of lipoxygenase towards linoleic acid and four model xenobiotics, respectively, were observed. Both the catalytic activities of lipoxygenase were significantly inhibited by < 1.0 microM nordihydroguaiaretic acid. 4. These findings suggest that peroxidase and lipoxygenase may be important pathways for peroxidative xenobiotic oxidation in human fetal tissues.
Female↗
Biologically active principles isolated from Salacia oblonga wall.
From the petroleum ether extract of the root bark of Salacia Oblonga wall, two biologically active fractions have been isolated by column and thin layer chromatography. The methanol eluted fraction of the extract absorbed on a column of silica gel at a concentration of 50 micrograms/ml showed 100 percent cytotoxicity on Ehrlich ascites tumour cells. The chloroform eluted fraction of the pet. ether extract and a fluorescent compound separated from it by TLC demonstrated about 60% and 76% hypoglycemic potency of an equal dose of tolbutamide (250 mg/kg) in albino rats. The results indicate the therapeutic importance of S. Oblonga wall.
Animals↗
NAD(P)H:quinone oxidoreductase1 (DT diaphorase) specifically prevents the formation of benzo[a]pyrene quinone-DNA adducts generated by cytochrome P4501A1 and P450 reductase.
Monkey kidney COS1 cells transiently transfected with plasmids pMT2-cytochrome P450 1A1 (CYP1A1), pMT2-cytochrome P450 reductase (P450 reductase), and pMT2-NAD(P)H:quinone oxidoreductase1 (NQO1 or DT diaphorase), individually or in combination, expressed significantly elevated levels of the respective enzyme(s). The transfected cells were homogenized to break cell membranes without affecting the nuclei and incubated with benzo[a]pyrene (BP) to determine the role of cDNA-encoded enzymes in metabolic activation and/or detoxification of BP. These studies were performed by measuring the capacity of the transfected cells to form DNA adducts as determined by 32P postlabeling and protein adduct detection. Cotransfection of the COS1 cells with cDNAs encoding CYP1A1 and P450 reductase resulted in eight distinct BP-DNA adducts. Inclusion of cDNA encoding NQO1 along with CYP1A1 and P450 reductase in transfection reduced the number of DNA adducts to six. The two lost DNA adducts were specifically eliminated due to the presence of cDNA-derived NQO1 activity. Subsequent experiments with BP-1,6-quinone, BP-3,6-quinone, and BP-6,12-quinone identified these two adducts as those of BP quinones. In an in vitro system, BP-3,6-quinone produced two adducts with deoxyguanosine (dG) but not with dA, dC, and dT. Furthermore, the positions of BP-3,6-quinone-dG adducts on TLC plate correspond to those that are prevented by cDNA-derived NQO1, thus identifying these adducts as BP quinones of dG. In addition, NQO1 reduced the amount of protein-BP adducts generated by CYP1A1 and P450 reductase into transfected COS1 cells. These results show that semiquinones can directly bind to DNA and demonstrate that NQO1 activity can specifically reduce the binding of quinone metabolites of BP generated by CYP1A1 and P450 reductase to DNA and protein.
Animals↗
Inhibition of NAD(P)H:quinone oxidoreductase1 in ethacrynic acid-resistant human colon carcinoma cells.
Human colon carcinoma HT29 cells sensitive (WT) and resistant (HT/M and HT/S) to ethacrynic acid (EA) were used to investigate the role of NAD(P)H:quinone oxidoreductase1 (NQO1) in drug resistance. Significant decreases in the levels of NQO1 activity were observed in resistant cells as compared with the sensitive cells. However, the decreased activities of NQO1 in resistant cells were found to be due to inhibition of the enzyme by EA. Human NQO1 cDNA-derived protein in monkey kidney COS1 cell extract was used to demonstrate that in vitro inhibition of NQO1 activity by EA was rapid, reversible and concentration dependent, with an IC50 value of 250 microM. These results suggest that NQO1 may not have a role in EA resistance of human colon carcinoma HT29 cells and that EA is an inhibitor of NQO1 activity.
Animals↗
The role of specific reductases in the intracellular activation and binding of 2-nitroimidazoles.
PURPOSE: To determine the relative effectiveness of specific cellular reductases for the activation and binding of 2-nitroimidazoles in vivo. METHODS AND MATERIALS: Monkey kidney cells were transfected with recombinant plasmids to effect intracellular overexpression of P450 reductase and DT-diaphorase. The covalent binding of 2-nitroimidazoles to cellular macromolecules was measured as a function of time of cell incubation at various oxygen concentrations. The effect of allopurinol on cellular binding of radiolabeled 2-nitroimidazoles was also measured. RESULTS: A 1,000-fold overexpression of DT-diaphorase resulted in a small but significant increase in 2-nitroimidazole binding rate. An 80-fold overexpression of cytochrome P450 reductase resulted in a 5-7-fold increase in the binding rate of 2-nitroimidazole. The inhibition of xanthine oxidase by allopurinol had no effect on 2-nitroimidazole binding rates. The amplification of P450 reductase activity within cells was always much larger than the resultant increase in 2-nitroimidazole binding rate, suggesting an enzyme kinetic process less than first order and possibly of 1/2-order. CONCLUSION: These data suggest that cytochrome P450 reductase is the most important enzyme in these cells for reducing 2-nitroimidazoles to intermediates which can covalently bind to cellular macromolecules. Furthermore, since this cellular process demonstrates approximately 1/2-order kinetics, a tissue's capacity for binding 2-nitroimidazole drug in hypoxia should be proportional to the square root of its intracellular P450 reductase level.
Animals↗
Xenobiotic oxidation during early pregnancy in man: peroxidase catalysed chemical oxidation in conceptual tissues.
1. A membrane-bound peroxidase activity was isolated and purified from pooled samples of human intrauterine tissue (embryo, placenta, amnion, chorion and uterine decidua) of 4-6 weeks of gestation. 2. The H2O2-dependent guaiacol oxidation catalysed by the purified peroxidase exhibited a specific activity of 10.82 +/- 1.81 mumol/min/mg protein. 3. The optimum conditions required for guaiacol oxidation catalysed by the peroxidase included 18 mM guaiacol, 400 microM H2O2, pH 9.0, and 2 micrograms/ml enzyme protein. The guaiacol oxidation was inhibited by KCN and NaN3 with IC50s of 17.6 and 22 microM, respectively. 4. Several other endogenous and exogenous chemicals also underwent H2O2-dependent oxidation catalysed by the purified peroxidase. These results suggest that peroxidase may be one of the major pathways of xenobiotic oxidation present in organogenesis-stage human conceptual tissues influencing the toxicity of chemicals to which the developing embryo is exposed.
Amnion↗
NAD(P)H:quinone oxidoreductase1 (DT-diaphorase): expression, regulation, and role in cancer.
NAD(P)H:quinone oxidoreductase1 (DT-diaphorase or NQO1) is a flavoprotein that promotes obligatory two-electron reduction of quinones, preventing their participation in redox cycling, oxidative stress, and neoplasia. NQO1 is ubiquitously expressed. However, a large amount of variation in NQO1 gene expression was noticed among various human tissues. NQO1 gene is upregulated in livers of hepatocarcinoma patients, and its expression is induced in response to a variety of compounds, including planar aromatic hydrocarbons, phenolic antioxidants/chemoprotectors, tumor promoters, and hydrogen peroxide. Deletion mutagenesis in the NQO1 gene promoter identified several cis-elements including antioxidant response element (ARE), xenobiotic response element, and AP2 element, which regulate the expression and induction of the NQO1 gene. Among these DNA elements, ARE is the most important cis-element required for high basal expression of the NQO1 gene in tumor tissues, as compared to the normal tissues of the same origin, and for its induction in response to xenobiotics and antioxidants. Nucleotide sequence analysis of the ARE indicated presence of three AP1/AP1-like elements and a GCA box. Mutational analysis indicated a requirement of two AP1/AP1-like elements arranged as inverse repeats at the interval of three base pairs for the ARE activity. The GCA box in the ARE was required for optimum basal and induced expression. ARE is a novel cis-element because a single AP1/AP1-like element did not stimulate gene expression in response to xenobiotics and antioxidants. Band shift and supershift assays identified Jun, Fos, and novel proteins in the hARE-nuclear protein complexes that mediate regulation of the NQO1 gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Purification and partial characterization of lipoxygenase with dual catalytic activities from human term placenta.
Lipoxygenase possessing dual catalytic activities, i.e. dioxygenase and hydroperoxidase, was purified from the cytosols of term placentas from non-smoking women. Concanavalin A affinity chromatography followed by phenyl-Sepharose CL-4B chromatography resulted in the separation of one hydrophobic and one non-hydrophobic isoenzyme. The concanavalin A-purified enzyme was used in all subsequent experiments. The dioxygenase activity of the enzyme exhibited a Vmax. of 204.37 +/- 17.66 nmol/min per mg of protein and a Km of 0.79 mM for linoleic acid. The involvement of dioxygen in enzymic linoleic acid oxidation was confirmed by O2 uptake studies. Arachidonic acid and linolenic acid also served as substrates for the dioxygenase activity. The placental lipoxygenase co-oxidized benzidine in the presence of linoleic acid (hydroperoxidase activity). Both the dioxygenase and hydroperoxidase activities were significantly stimulated by Ca2+ (1-100 microM), ATP (10-400 nM) and H2O2 (1-10 nM). Similarly, these two activities were inhibited by nordihydroguaiaretic acid, 5,8,11-eicosatriynoic acid, gossypol, esculetin, butylated hydroxyanisole and butylated hydroxytoluene. Boiled enzyme was without significant dioxygenase and hydroperoxidase activities. Pyrogallol, 3,3'-dimethoxybenzidine, 3,3',5,5'-tetramethylbenzidine, tetramethylphenylenediamine and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) were also co-oxidized by the placental lipoxygenase. These results suggest that: (i) lipoxygenase from human term placenta exhibits both dioxygenase and hydroperoxidase activities, and (ii) this enzyme represents an important pathway for chemical oxidation in the placentas of non-smoking women.
Catalysis↗
Effect of vitamin A on hexachlorocyclohexane (HCH) toxicity in the rat.
1. Technical hexachlorocyclohexane (HCH) depleted hepatic stores of vitamin A in male albino rats to cause secondary vitamin A deficiency. 2. Toxicity of HCH in rats is augmented by dietary vitamin A-deficiency as evidenced by growth retardation, organ hypertrophies and alterations in the serum and liver levels of the marker enzymes of toxicity. 3. Supplementation of dietary vitamin A to the rats either in adequate (2000 IU/kg diet) or in an excess but not hypervitaminotic level (10(5) IU/kg diet) resulted in significant protection against the toxicity of HCH. 4. The activities of the hepatic xenobiotic metabolizing enzymes were generally low (with the exception of glutathione S-transferase) in the vitamin A-deficient rats compared to those of the vitamin A supplemented diet groups. 5. The results indicated that dietary vitamin A influences the response of male albino rats to HCH toxicity possibly by modulating the activities of hepatic xenobiotic metabolizing enzymes.
Animals↗
Inhibition of human term placental and fetal liver glutathione-S-transferases by fatty acids and fatty acid esters.
Glutathione-S-transferase (GST) activity from human term placenta and human fetal liver towards 1-chloro-2,4-dinitrobenzene as the second substrate was significantly inhibited by the saturated fatty acids, stearic (SA) and palmitic (PA) acids and fatty acid esters, ascorbyl stearate (Asc-S) and ascorbyl palmitate (Asc-P). The nature of inhibition of human placental GST was competitive towards CDNB with Ki values of 3.1, 10.0, 13.5 and 18.5 microM for Asc-S, Asc-P, PA and SA, respectively. The inhibitory effect of Asc-S on human term placental GST was reversible. I50 values for Asc-S, Asc-P, SA and PA were 15, 45, 83 and 78 microM, respectively, for partially purified human fetal liver GSTs and 21, 6, 88 and 117 microM, respectively, for partially pure rat liver GSTs. The evidence suggests that Asc-S, Asc-P, SA and PA are potent inhibitors especially of the pi-class of GST.
Animals↗
Role of vitamin A in the haematotoxicity of hexachlorocyclohexane (HCH) in the rat.
The haematotoxicity of technical hexachlorocyclohexane (HCH) (1000 ppm) was investigated in male albino rats fed with diet free of vitamin A or containing vitamin A at 2000 or 10(5) I.U./kg. Assessment of HCH-induced haematotoxicity at the end of the 7 weeks feeding period was done on the basis of haemoglobin content, total count of red blood cells and white blood cells and the differential counts of the white blood cells as well as by parameters such as packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin content, prothrombin time and clotting time. In the rats fed with vitamin A-free diet containing HCH, significant reductions were noticed in the total white blood cells count, clotting time and prothrombin time indicating severe haematotoxicity. Differential count of the white blood cells of these rats revealed a non-significant reduction in the lymphocyte count. The only indication of haematotoxicity caused by hexachlorocyclohexane in the vitamin A supplemented rats was a slight but statistically significant reduction of the total count of white blood cells. These results demonstrate that the haematotoxicity of hexachlorocyclohexane in the rats is enhanced by vitamin A-deficiency and its supplementation particularly in excess but not at hypervitaminotic level is protective against the toxicity.
Animals↗
Intravenous or inhaled pentamidine for treating Pneumocystis carinii pneumonia in AIDS. A randomized trial.
OBJECTIVE: To evaluate the efficacy and toxicity of aerosolized pentamidine and of reduced-dose intravenous pentamidine for the treatment of mild to moderate Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome (AIDS). DESIGN: Randomized open study with serial pulmonary function testing and measurement of pentamidine concentrations in plasma and bronchoalveolar lavage fluid. PATIENTS: Of 44 men and 1 woman with a mild to moderate first episode of P. carinii pneumonia (Pao2 greater than or equal to 7.3 kPa [55 mm Hg]), 23 received aerosolized pentamidine and 22, intravenous pentamidine. INTERVENTIONS: Pentamidine isethionate, 600 mg by inhalation using a Respirgard II nebulizer (Marquest Medical Products, Inc., Englewood, Colorado) or 3 mg/kg body weight intravenously, administered once daily for 2 to 3 weeks. MEASUREMENTS AND MAIN RESULTS: The planned 60-patient study was stopped after 45 patients had been enrolled. The rates (aerosolized compared with intravenous pentamidine) of initial failure, early recrudescence of symptoms, and relapse were 12% and 19% (difference, 7%; 99% confidence interval [CI], - 23% to 37%; P = 0.67), 35% and 0% (difference, 35%; CI, 13% to 58%; P = 0.02), and 24% and 0% (difference, 24%; CI, 4% to 49%; P = 0.03). The rates (aerosolized compared with intravenous pentamidine) of major toxicity were 0% (0 of 17 patients) and 10% (2 of 21 patients) (difference 10%; CI, -1% to 29%; P = 0.24). The mean (+/- SD) pentamidine concentration in bronchoalveolar lavage fluid for patients receiving aerosolized pentamidine was 96.6 +/- 65.1 ng/mL compared with 14.4 +/- 17.7 ng/mL for patients receiving intravenous treatment. Trough concentrations of pentamidine in plasma increased from 0 to 25.4 +/- 16.4, 56.5 +/- 26.1, and 61.1 +/- 56.0 ng/mL at the end of weeks 1, 2, and 3 of intravenous therapy, respectively. CONCLUSIONS: The data suggest that reduced-dose intravenous pentamidine was more effective than aerosolized pentamidine for treating mild to moderate P. carinii pneumonia. Systemic absorption during aerosolized therapy was minimal; daily doses of intravenous pentamidine resulted in increased accumulation of pentamidine in plasma.
Acquired Immunodeficiency Syndrome↗
Repetitive DNA sequence from the crayfish Procambarus clarkii.
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Animals↗
Omental pedicle grafting in the treatment of postcardiotomy sternotomy infection.
Postcardiotomy sternal infection occurred in 20 (2%) of 1007 patients undergoing cardiac surgery between September 1985 and December 1987, a 10-fold increase over the preceding 33 months (4 [0.24%] of 1627 patients). Cultures were sterile in 5 patients and yielded staphylococci in 12 and a variety of bowel organisms in 3. The cause for the increased occurrence of sternal wound infection is unclear after multivariate analysis, although infections have precipitously dropped subsequent to changing to cefuroxime sodium antibiotic prophylaxis. Treatment has evolved to appropriate antibiotics and early débridement of involved sternum and cartilage. Rewiring the sternum is not attempted. If gross purulence is not present, primary closure is accomplished using muscle flaps (2 patients) or omental pedicle grafts (17 patients). In the presence of gross purulence, the wound is packed open for 5 days and then closed in the above fashion. Two patients required skin grafts for primary closure. The omental pedicle flap is preferred due to simplicity and improved coverage of the sternal defect inferiorly. Nineteen patients healed primarily. A superficial wound infection was drained in 1 patient. Midline incisional hernias developed in 3 muscular patients. Omentum is now harvested through a left subcostal incision. Hospital stay was under 2 weeks in 13 patients. One death occurred due to multisystem failure prior to completion of wound closure. In our experience, early sternal débridement and omental pedicle grafting with primary closure is appropriate therapy for postcardiotomy sternotomy infections. The presence of gross purulence may require 5 days of open packing prior to omental grafting. No significant complications occurred, and mortality was low. A left subcostal incision for omental harvesting is utilized to avoid the occurrence of delayed incisional hernias.
Cardiac Surgical Procedures↗
Impairment of cerebral function following cardiac and other major surgery.
Patients undergoing routine coronary artery surgery (N = 76) were compared with those undergoing other major operations (N = 29) in a prospective multidisciplinary study designed to define the incidence of neurological and psychological sequelae. The preoperative state of the carotid and vertebral arteries was defined by digital subtraction angiography. Changes in clinical neurological examination, detailed neuropsychological testing, psychiatric assessment and cerebral blood flow were measured. All preoperative studies were repeated 8 days and 8 weeks after surgery. Clinical neurological examination was repeated in addition on the 1st postoperative day. New focal neurological signs were found in 8% of the cardiac patients and none of the comparison group 24 h after operation (P = 0.9). Global transient neurological dysfunction occurred in 59% of the coronary group in the 1st postoperative day compared with 21% in other forms of surgery (P = 0.0007) but this correlated with postoperative narcotic and sedative drugs. A deterioration in neuropsychological performance was detectable in 73% of coronary cases at 8 days which was more likely to occur with increasing age, longer bypass time and lower perfusion pressure, but similar neuropsychological changes also occurred in 50% of the comparison group. By 8 weeks, there was a significant improvement in the cardiac patients (37%, P less than 0.001) but not in the other group. Cerebral blood flow was reduced at 8 days in the coronary bypass patients but not in the comparison group suggesting that the mechanism of cerebral change may be different in the two groups.
Adult↗
Zidovudine-induced fever.
Zidovudine (3'-azido-3'deoxythymidine, AZT, Retrovir) is the first antiretroviral drug to demonstrate clinical efficacy for symptomatic human immunodeficiency virus infection. In a large, placebo-controlled trial, nausea and hematologic toxicity, but not fever, occurred more frequently in zidovudine- than in placebo-treated patients. However, in an open label study, fever severe enough to halt zidovudine administration occurred in 10% of 70 acquired immune deficiency syndrome (AIDS) patients receiving the drug. We now describe three AIDS patients with severe zidovudine-induced fever in whom other causes of fever were excluded. Zidovudine-induced fever was confirmed in each case by drug rechallenge. Using an enzyme immunoassay, we detected IgM antibodies directed against a zidovudine-serum protein conformational determinant in one of these three patients. Neither IgG nor IgM anti-zidovudine antibodies were present in sera from the other two patients with zidovudine fever, from four AIDS patients who discontinued zidovudine for reasons other than fever, or from five AIDS patients who never received zidovudine. Zidovudine may cause fever as a severe adverse effect in patients with AIDS. Either type III or type IV hypersensitivity may mediate this reaction.
Acquired Immunodeficiency Syndrome↗