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The Journal of Biological Chemistry Mar 1985Treatment of isolated rat adipocytes with adrenocorticotropin (ACTH) caused a 1.5-fold increase in phospholipid methyltransferase activity within 5 min. This effect of...
Treatment of isolated rat adipocytes with adrenocorticotropin (ACTH) caused a 1.5-fold increase in phospholipid methyltransferase activity within 5 min. This effect of ACTH was concentration-dependent with maximal activation at 2 milliunits/ml ACTH, and was reproduced by dibutyryl cyclic AMP. ACTH (2 milliunits/ml) caused an increase in the Vmax value of phospholipid methyltransferase without changing the Km for S-adenosyl-L-methionine. Insulin caused a concentration-dependent inhibition of both control and ACTH-stimulated phospholipid methyltransferase. Half-maximal inhibition by insulin was demonstrated with 5 microunits/ml insulin in control cells and with 25 microunits/ml insulin in ACTH-stimulated cells. The rapid and sensitive activation of adipocyte phospholipid methyltransferase by ACTH and inhibition by insulin are consistent with a role for this pathway in the hormonal response of the adipocyte.
Topics: Adipose Tissue; Adrenocorticotropic Hormone; Animals; Bucladesine; Chromatography, Thin Layer; Dose-Response Relationship, Drug; Insulin; Male; Methylation; Methyltransferases; Phosphatidyl-N-Methylethanolamine N-Methyltransferase; Phosphatidylcholines; Phosphatidylethanolamine N-Methyltransferase; Phosphatidylethanolamines; Phospholipids; Rats; Rats, Inbred Strains
PubMed: 2982871
DOI: No ID Found -
The Journal of Biological Chemistry Oct 1998Nitration of tyrosine residues of proteins has been suggested as a marker of peroxynitrite-mediated tissue injury in inflammatory conditions. The nitration reaction has...
Nitration of tyrosine residues of proteins has been suggested as a marker of peroxynitrite-mediated tissue injury in inflammatory conditions. The nitration reaction has been extensively studied in vitro by bolus addition of authentic peroxynitrite, an experimental approach hardly reflecting in vivo situations in which the occurrence of peroxynitrite is thought to result from continuous generation of .NO and O-2 at physiological pH. In the present study, we measured the nitration of free tyrosine by .NO and O-2 generated at well defined rates from the donor compound (Z)-1-[N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino]- dia zen-1-ium-1,2-diolate] (spermine NONOate) and the xanthine oxidase reaction, respectively. The results were compared with the established nitration reaction triggered by authentic peroxynitrite. Bolus addition of peroxynitrite (1 mM) to tyrosine (1 mM) at pH 7.4 yielded 36.77 +/- 1.67 microM 3-nitrotyrosine, corresponding to a recovery of about 4%. However, peroxynitrite formed from .NO and O-2, which were generated at equal rates ( approximately 5 microM x min-1) from 1 mM spermine NONOate, 28 milliunits/ml xanthine oxidase, and 1 mM hypoxanthine was much less efficient (0.67 +/- 0.01 microM; approximately 0.07% of total product flow). At O-2 fluxes exceeding the .NO release rates, 3-nitrotyrosine formation was below the detection limit of the high performance liquid chromatography method (<0.06 microM). Nitration was most efficient (approximately 0.3%) with the .NO donor alone, i.e. without concomitant generation of O-2. Nitration by .NO had a pH optimum of 8.2, increased progressively with increasing tyrosine concentrations (0.1-2 mM), and was not enhanced by NaHCO3 (up to 20 mM), indicating that it was mediated by .NO2 rather than peroxynitrite. Our results argue against peroxynitrite produced from .NO and O-2 as a mediator of tyrosine nitration in vivo.
Topics: Carbon Dioxide; Free Radicals; Hydrogen-Ion Concentration; Models, Chemical; Molsidomine; Nitrates; Nitrogen Oxides; Spermine; Tyrosine; Xanthine Oxidase
PubMed: 9765252
DOI: 10.1074/jbc.273.42.27280 -
Proceedings of the National Academy of... Aug 1999Hemophilia A is caused by a deficiency in coagulation factor VIII (FVIII) and predisposes to spontaneous bleeding that can be life-threatening or lead to chronic...
Hemophilia A is caused by a deficiency in coagulation factor VIII (FVIII) and predisposes to spontaneous bleeding that can be life-threatening or lead to chronic disabilities. It is well suited for gene therapy because a moderate increase in plasma FVIII concentration has therapeutic effects. Improved retroviral vectors expressing high levels of human FVIII were pseudotyped with the vesicular stomatitis virus G glycoprotein, were concentrated to high-titers (10(9)-10(10) colony-forming units/ml), and were injected intravenously into newborn, FVIII-deficient mice. High-levels (>/=200 milliunits/ml) of functional human FVIII production could be detected in 6 of the 13 animals, 4 of which expressed physiologic or higher levels (500-12,500 milliunits/ml). Five of the six expressers produced FVIII and survived an otherwise lethal tail-clipping, demonstrating phenotypic correction of the bleeding disorder. FVIII expression was sustained for >14 months. Gene transfer occurred into liver, spleen, and lungs with predominant FVIII mRNA expression in the liver. Six of the seven animals with transient or no detectable human FVIII developed FVIII inhibitors (7-350 Bethesda units/ml). These findings indicate that a genetic disease can be corrected by in vivo gene therapy using retroviral vectors.
Topics: Animals; Factor VIII; Gene Transfer Techniques; Genetic Therapy; Hemophilia A; Humans; Membrane Glycoproteins; Mice; Mice, Knockout; Organ Specificity; Phenotype; Polymerase Chain Reaction; RNA, Messenger; Retroviridae; Time Factors; Transcription, Genetic; Vesicular stomatitis Indiana virus; Viral Envelope Proteins
PubMed: 10468616
DOI: 10.1073/pnas.96.18.10379 -
FEBS Letters Mar 1995We have characterized the effect of angiotensin converting enzyme (ACE) inhibitors on the activity of CuZn-superoxide dismutase (CuZn-SOD), Mn-superoxide dismutase...
We have characterized the effect of angiotensin converting enzyme (ACE) inhibitors on the activity of CuZn-superoxide dismutase (CuZn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase, and selenium-dependent glutathione peroxidase (Se-GPx). CF1 mice (4-month-old females) were administered water containing enalapril (20 mg/l) or captopril (50 mg/l), during 4 to 11 weeks. After 11 weeks, enalapril treatment caused an increase in the activity of CuZn-SOD, Mn-SOD and Se-GPx, from 19 +/- 4 to 46 +/- 7, 2.1 +/- 0.2 to 3.8 +/- 0.2 units/mg protein and 27 +/- 3 to 54 +/- 3 milliunits/mg protein, respectively. After 11 weeks, captopril treatment increased the activities (P < 0.05) of CuZn-SOD, MnSOD and Se-GPx to 35 +/- 4, 2.9 +/- 0.2 units/mg protein, and 38 +/- 2 milliunits/mg protein, respectively. Catalase activity was not affected by the treatments. These results suggest that ACE inhibitors may protect cell components from oxidative damage by increasing the enzymatic antioxidant defenses.
Topics: Animals; Captopril; Catalase; Enalapril; Female; Glutathione Peroxidase; Liver; Mice; Superoxide Dismutase
PubMed: 7890034
DOI: 10.1016/0014-5793(95)00137-x -
The Journal of Biological Chemistry May 1996Human class 1 aldehyde dehydrogenase (hALDH-1) can oxidize aldophosphamide, a key aldehyde intermediate in the activation pathway of cyclophosphamide and other...
De novo expression of transfected human class 1 aldehyde dehydrogenase (ALDH) causes resistance to oxazaphosphorine anti-cancer alkylating agents in hamster V79 cell lines. Elevated class 1 ALDH activity is closely correlated with reduction in DNA interstrand cross-linking and lethality.
Human class 1 aldehyde dehydrogenase (hALDH-1) can oxidize aldophosphamide, a key aldehyde intermediate in the activation pathway of cyclophosphamide and other oxazaphosphorine (OAP) anti-cancer alkylating agents. Overexpression of class 1 ALDH (ALDH-1) has been observed in cells selected for survival in the presence of OAPs. We used transfection to induce de novo expression of human ALDH-1 in V79/SD1 Chinese hamster cells to clearly quantitate the role of hALDH-1 expression in OAP resistance. Messenger RNA levels correlated well with hALDH-1 protein levels and enzyme activities (1.5-13.6 milliunits/mg with propionaldehyde/NAD+ substrate, compared to < 1 milliunit/mg in controls) in individual clonal transfectant lines, and slot blot analysis confirmed the presence of the transfected cDNA. Expressed ALDH activity was closely correlated (r = 0.99) with resistance to mafosfamide, up to 21-fold relative to controls. Transfectants were cross-resistant to other OAPs but not to phosphoramide mustard, ifosfamide mustard, melphalan, or acrolein. Resistance was completely reversed by pretreatment with 25 microM diethylaminobenzaldehyde, a potent ALDH inhibitor. Alkaline elution studies showed that expression of ALDH-1 reduced the number of DNA cross-links commensurate with mafosfamide resistance, and this reduction in cross-links was fully reversed by the inhibitor. Thus, overexpression of human class 1 ALDH alone is sufficient to confer OAP-specific drug resistance.
Topics: Aldehyde Dehydrogenase; Animals; Antineoplastic Agents, Alkylating; Base Sequence; Cell Line; Cricetinae; Cricetulus; Cyclophosphamide; DNA; Drug Resistance; Enzyme Inhibitors; Humans; Isoenzymes; Molecular Sequence Data; Phosphoramide Mustards; Transfection
PubMed: 8662658
DOI: 10.1074/jbc.271.20.11884 -
European Journal of Biochemistry Jan 1988A casein kinase of type II has been highly purified from Xenopus laevis ovary. A new experimental protocol has been developed for the purification, consisting in four...
A casein kinase of type II has been highly purified from Xenopus laevis ovary. A new experimental protocol has been developed for the purification, consisting in four chromatographic steps: hydrophobic on tyrosine-agarose, ion exchange on DEAE-Sepharose, affinity on heparin-Sepharose and fast protein liquid on Mono Q. The purification was greater than 20,000, taking into account an inhibitor present in the starting material which masked the activity in the crude fraction. The overall yield was greater than 20%. Full-grown Xenopus oocytes contain 64 milliunits per oocyte corresponding to an intracellular concentration in the nanomolar range. The enzyme shares the following features with the mammalian casein kinase II: (a) comparable subunit composition (42-kDa doublet, 38 kDa and 26 kDa), (b) autophosphorylation of the 26-kDa subunit, (c) ability to use GTP as well as ATP as phosphate donor, (d) inability to use Mn2+ instead of Mg2+ to support the activity, (e) phosphorylation of both threonine and serine residues of casein, (f) inhibition by low doses of heparin. Biological effects of the highly purified enzyme have been investigated upon microinjection into Xenopus full-grown oocytes. At nanomolar concentrations (approximately 3 nM) the enzyme inhibited progesterone induction of meiotic cell division whereas it facilitates meiotic maturation at the level of maturation-promoting factor. These results suggest a role for the kinase in the phosphorylation cascade involved during the prophase/metaphase transition of meiotic cell division, both in the mechanism of the meiotic prophase arrest and in the activity of the cytoplasmic factor maturation-promoting factor. When microinjected into oocytes above 45 nM, the kinase provoked complex changes in the profile of the in ovo 32P-labelled proteins indicating that its targets could be other kinase/phosphatase regulatory proteins.
Topics: Animals; Casein Kinases; Female; Heparin; Meiosis; Oocytes; Oogenesis; Ovary; Phosphoproteins; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Substrate Specificity; Xenopus laevis
PubMed: 3422187
DOI: 10.1111/j.1432-1033.1988.tb13765.x -
The Journal of Biological Chemistry Nov 1998Hepatic lipase (HL) and scavenger receptor type B class I (SR-BI) have both been implicated in high density lipoprotein (HDL)-cholesteryl ester uptake in...
Hepatic lipase (HL) and scavenger receptor type B class I (SR-BI) have both been implicated in high density lipoprotein (HDL)-cholesteryl ester uptake in cholesterol-utilizing tissues. Inactivation of HL by gene-directed targeting in mice results in up-regulation of SR-BI expression in adrenal gland (Wang, N., Weng, W., Breslow, J. L., and Tall, A. R. (1996) J. Biol. Chem. 271, 21001-21004). The net effect on HDL-cholesteryl ester uptake is not known. We determined the impact of acute in vivo inhibition of rat adrenal HL activity by antibodies on SR-BI expression and on human and rat HDL-[3H]cholesteryl ether (CEth) uptake in the adrenal gland. Rat HDL was isolated from rats in which HL activity had been inhibited for 1 h. The rats were studied under basal conditions (not ACTH-treated) and after previous treatment with ACTH for 6 days (ACTH-treated). Intravenous injection of anti-HL resulted in 70% lowering of adrenal HL activity in both conditions which were maintained for at least 8 h. In not ACTH-treated rats, inhibition of adrenal HL increased adrenal SR-BI mRNA (5.2-fold) and mass (1. 6-fold) within 4 h. HL inhibition resulted in 41% and 14% more adrenal accumulation of human HDL-[3H]CEth during 4 and 24 h, respectively. The adrenal uptake of rat HDL-[3H]CEth increased by 68%, 4 h after the antibody injection. ACTH treatment increased total adrenal HL activity from 3.7 +/- 0.5 milliunits to 34.0 +/- 17. 2 milliunits, as well as adrenal SR-BI mRNA from 2.9 +/- 0.7 arbitrary units (A.U.) to 86.8 +/- 41.1 A.U. and SR-BI mass from 7.7 +/- 1.8 A.U. to 63.16 +/- 46.7 A.U. The human HDL-[3H]CEth uptake by adrenals was also significantly increased from 0.58 +/- 0.11% of injected dose to 7.24 +/- 1.58% of injected dose. Inhibition of adrenal HL activity did not result in further induction of SR-BI expression and did not affect human HDL-[3H]CEth uptake. These findings indicate that SR-BI expression may be influenced by changes in HL activity. HL activity is not needed for the SR-BI-mediated HDL-cholesteryl ester uptake by rat adrenal glands.
Topics: Adrenal Glands; Adrenocorticotropic Hormone; Animals; Biological Transport; CD36 Antigens; Cholesterol, HDL; DNA Primers; Ethers; Humans; Kinetics; Lipoprotein Lipase; Male; Membrane Proteins; Mice; Polymerase Chain Reaction; RNA, Messenger; Rats; Rats, Wistar; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; Scavenger Receptors, Class B; Time Factors; Transcription, Genetic; Up-Regulation
PubMed: 9822677
DOI: 10.1074/jbc.273.48.32038 -
Proceedings of the National Academy of... May 1980We have been investigating the extent to which separated thyroid follicles in suspension culture, free of endothelium and fibroblasts, have the properties of follicles...
We have been investigating the extent to which separated thyroid follicles in suspension culture, free of endothelium and fibroblasts, have the properties of follicles in vivo. To test whether thyrotropin (TSH) can cause thyroid epithelial cells to undergo mitosis, preparations of follicles suspended in Coon's modified F-12 medium with 0.5% calf serum were incubated with 10 milliunits of impure or pure TSH per ml. Three results were obtained: (i) TSH preparations stimulated the incorporation of [3H]thymidine into cell nuclei; (ii) mitotic figures were induced and they had the same characteristic ultrastructural features as those observed in vivo; and (iii) the cell number doubled in the course of 3 days of exposure to TSH. The results suggest that TSH is a mitogen for the principal thyroid epithelial celland that other substances found in the usual impure TSH preparations are not necessary for the mitogenic activity. It can act in the absence of nonfollicular cells. The initial multiplication rates are similar to those in vivo. The cells do not have to spread to divide in contrast to the requirement for spreading in the case of fibroblasts.
Topics: Animals; Autoradiography; Cell Division; Cell Survival; Cells, Cultured; DNA Replication; Epithelial Cells; Microscopy, Electron; Mitogens; Mitosis; Rats; Thyroid Gland; Thyrotropin
PubMed: 6930664
DOI: 10.1073/pnas.77.5.2743 -
Proceedings of the National Academy of... May 1976Occluding the main artery of one kidney in each of six dogs resulted in an average increase in the renin concentration of the peripheral blood from the original 0.040 to...
Occluding the main artery of one kidney in each of six dogs resulted in an average increase in the renin concentration of the peripheral blood from the original 0.040 to 0.198 milliunit/ml of serum. During the same time (3-4 days), the occlusion also resulted in a significant rise of directly measured mean systemic blood pressure from an average basal level of 104 to 139 mm Hg.
Topics: Animals; Blood Pressure; Disease Models, Animal; Dogs; Female; Hypertension, Renal; Ischemia; Kidney; Nephrectomy; Renin; Time Factors
PubMed: 1064043
DOI: 10.1073/pnas.73.5.1722 -
The Journal of Biological Chemistry May 1986The interaction between epinephrine and insulin in modulating in vivo glucose metabolism within individual tissues of the body has not previously been examined. This was...
The interaction between epinephrine and insulin in modulating in vivo glucose metabolism within individual tissues of the body has not previously been examined. This was investigated using the euglycemic hyperinsulinemic (120 milliunits/liter) clamp combined with administration of [3H]2-deoxyglucose and D-[U-14C]glucose. Epinephrine produced whole body insulin resistance due to increased hepatic glucose output and reduced peripheral glucose disposal. Despite elevated insulin levels liver glycogen content was reduced by 50% during epinephrine infusion (5 nM). However, this effect was transient, occurring predominantly during the initial 60 min of study. These effects were prevented during beta-adrenergic blockade with propranolol and potentiated during alpha 1-adrenergic blockade with prazosin. The most significant effect of epinephrine in peripheral tissues was increased glycogenolysis in both oxidative and glycolytic skeletal muscle. A significant reduction in insulin-mediated [3H]2-deoxyglucose uptake (30%) was evident in 5 of 9 muscles tested during epinephrine infusion. This effect was most pronounced in the more insulin-sensitive oxidative muscles. The latter effect was probably indirectly mediated via increased glycogenolysis--increased accumulation of metabolites--inhibition of hexokinase. In addition, it is evident that insulin-mediated glycogen synthesis occurred during epinephrine infusion. All effects of epinephrine on muscle glucose metabolism were prevented by propranolol but not prazosin. Similar effects to that observed in muscle were not evident in adipose tissue. It is concluded that epinephrine may override many of the actions of insulin in vivo, and most of these effects are mediated via the beta-adrenergic receptor. In the intact rat there may be a complex interaction between alpha- and beta-adrenergic effects in regulating hepatic glucose output.
Topics: Animals; Blood Glucose; Deoxyglucose; Drug Interactions; Epinephrine; Glycogen; Hyperinsulinism; Insulin; Male; Mathematics; Muscles; Prazosin; Propranolol; Rats; Rats, Inbred Strains
PubMed: 3516993
DOI: No ID Found