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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 Dec 2000To examine the impact of homozygous genetic disruption of insulin receptor substrate (IRS)-1 (IRS-1(-/-)) or IRS-2 (IRS-2(-/-)) on basal and insulin-stimulated...
To examine the impact of homozygous genetic disruption of insulin receptor substrate (IRS)-1 (IRS-1(-/-)) or IRS-2 (IRS-2(-/-)) on basal and insulin-stimulated carbohydrate and lipid metabolism in vivo, we infused 18-h fasted mice (wild-type (WT), IRS-1(-/-), and IRS-2(-/-)) with [3-(3)H]glucose and [(2)H(5)]glycerol and assessed rates of glucose and glycerol turnover under basal (0-90 min) and hyperinsulinemic-euglycemic clamp (90-210 min; 5 mm glucose, and 5 milliunits of insulin.kg(-)(1).min(-)(1)) conditions. Both IRS-1(-)(/-) and IRS-2(-)(/-) mice were insulin-resistant as reflected by markedly impaired insulin-stimulated whole-body glucose utilization compared with WT mice. Insulin resistance in the IRS-1(-)(/-) mice could be ascribed mainly to decreased insulin-stimulated peripheral glucose metabolism. In contrast, IRS-2(-)(/-) mice displayed multiple defects in insulin-mediated carbohydrate metabolism as reflected by (i) decreased peripheral glucose utilization, (ii) decreased suppression of endogenous glucose production, and (iii) decreased hepatic glycogen synthesis. Additionally, IRS-2(-)(/-) mice also showed marked insulin resistance in adipose tissue as reflected by reduced suppression of plasma free fatty acid concentrations and glycerol turnover during the hyperinsulinemic-euglycemic clamp. These data suggest important tissue-specific roles for IRS-1 and IRS-2 in mediating the effect of insulin on carbohydrate and lipid metabolism in vivo in mice. IRS-1 appears to have its major role in muscle, whereas IRS-2 appears to impact on liver, muscle, and adipose tissue.
Topics: Adipose Tissue; Animals; Carbohydrate Metabolism; Fatty Acids, Nonesterified; Food Deprivation; Gas Chromatography-Mass Spectrometry; Glucose; Glycerol; Insulin; Insulin Receptor Substrate Proteins; Intracellular Signaling Peptides and Proteins; Lipid Metabolism; Liver; Male; Mice; Muscles; Mutation; Phenotype; Phosphoproteins; Radioimmunoassay; Time Factors
PubMed: 10995761
DOI: 10.1074/jbc.M006490200 -
Journal of Pharmacy & Bioallied Sciences Jan 2014The purpose of this work was to develop a new oral drug delivery system intended for cattle and that enables delayed and pulsed release of an anthelmintic agent.
BACKGROUND
The purpose of this work was to develop a new oral drug delivery system intended for cattle and that enables delayed and pulsed release of an anthelmintic agent.
MATERIALS
This new tailored dosage form, also called reticulo-rumen device (RRD) has been evaluated on grazing calves by means of measurements of milliunits of tyrosine concentration, number of eggs per gram of feces, mean number of infective larvae on cattle pasture and increase in mean weight of cattle.
METHODS
The in vivo evaluation was carried out during two grazing seasons on different groups of dairy cattle. During the first grazing season, Group 1 was designated as an untreated control group. The remaining two were assigned to different treatments as follows: Group 2, early season suppression with a marketed intraruminal slow release bolus (Chronomintic(®), Virbac) administered immediately prior to turn-out and Group 3, mid-season suppression with a new RRD administered immediately prior to turn out. When the cattle were turned out at the start of the second grazing season, they were not given any anthelmintic treatment and were divided into two different groups, corresponding to the previous groups that received an anthelmintic treatment during the first grazing season, on that pasture that they had occupied as separate groups in the previous year. Furthermore, during the second season, samples of feces, blood and herbage were collected every month.
RESULTS AND CONCLUSION
During the first grazing season, the results indicated that the fecal egg counts and the number of infective larvae in herbage samples were slightly lower for the group receiving the new RRDs. Regular weighing of the cattle receiving the new RRDs revealed no significant difference with cattle receiving marketed RRDs. Conversely, during the second grazing season, the results for the mean weights of the cattle demonstrated that the weights of animals having been administered new RRDs during the first grazing season were significantly different (P < 0.05) from those in the second group treated with a Chronomintic(®) during the first grazing season. A difference in mean weight of 26 kg was observed between these two groups.
PubMed: 24459401
DOI: 10.4103/0975-7406.124311 -
Molecular Vision 2009Inflammation plays an important role in dry eye syndrome. In this study, inflammatory cytokine expression on the ocular surface in the Botulium toxin B (BTX-B) induced...
PURPOSE
Inflammation plays an important role in dry eye syndrome. In this study, inflammatory cytokine expression on the ocular surface in the Botulium toxin B (BTX-B) induced mouse dry eye model was investigated.
METHODS
CBA/J mice received an injection of saline or 20 milliunits (mU) of BTX-B into the lacrimal gland. Tear production and corneal fluorescein staining were evaluated in all groups before injection and at 3 time points after. The pro-inflammatory cytokines macrophage inhibitory factor (MIF), interleukin-1beta (IL-1 beta), tumor necrosis factor-alpha (TNF- alpha) and interleukin-6 (IL-6) in conjunctival and corneal epithelium were evaluated by real time quantitative PCR and immunohistochemistry.
RESULTS
BTX-B injected mice showed significantly decreased aqueous tear production and increased corneal fluorescein staining at the 1 week and 2 week time points compared with normal control and saline-injected mice. The BTX-B injected mice mRNA expression levels of TNF-alpha and IL-1beta from conjunctival and corneal epithelial cells increased significantly at two early time points comparing with that of normal and saline injected mice, but IL-1beta returned to normal levels at the 4 week time point. Saline injected mice showed no difference in mRNA expression of TNF-alpha, IL-1beta, MIF, and IL-6 on the ocular surface tissue at all time points. Immunohistochemistry confirmed these findings.
CONCLUSIONS
BTX-B induced mouse model showed decreased aqueous tear production, increased corneal fluorescein staining, and TNF-alpha and IL-1beta increased expression on the ocular surface within one month. The patterns seen appeared to mimic those in humans with non-Sjögren's syndrome keratoconjunctivitis sicca (NS-KCS).
Topics: Animals; Botulinum Toxins; Conjunctiva; Cornea; Cytokines; Disease Models, Animal; Dry Eye Syndromes; Female; Fluorescein; Fluorescent Antibody Technique; Gene Expression; Lacrimal Apparatus; Mice; Mice, Inbred CBA; Tears
PubMed: 19190733
DOI: No ID Found -
The Journal of Biological Chemistry Oct 2007A novel sialyltransferase, alpha/beta-galactoside alpha-2,3-sialyltransferase, was purified from the cell lysate of a luminous marine bacterium, Photobacterium...
A novel sialyltransferase, alpha/beta-galactoside alpha-2,3-sialyltransferase, was purified from the cell lysate of a luminous marine bacterium, Photobacterium phosphoreum JT-ISH-467, isolated from the Japanese common squid (Todarodes pacificus). The gene encoding the enzyme was cloned from the genomic library of the bacterium using probes derived from the NH(2)-terminal and internal amino acid sequences. An open reading frame of 409 amino acids was identified, and the sequence had 32% identity with that of beta-galactoside alpha-2,6-sialyltrasferase in Photobacterium damselae JT0160. DNA fragments that encoded the full-length protein and a protein that lacked the sequence between the 2nd and 24th residues at the NH(2) terminus were amplified by polymerase chain reactions and cloned into an expression vector. The full-length and truncated proteins were expressed in Escherichia coli, producing active enzymes of 0.25 and 305 milliunits, respectively, per milliliter of the medium in the lysate of E. coli. The truncated enzyme was much more soluble without detergent than the full-length enzyme. The enzyme catalyzed the transfer of N-acetylneuraminic acid from CMP-N-acetylneuraminic acid to disaccharides, such as lactose and N-acetyllactosamine, with low apparent K(m) and to monosaccharides, such as alpha-methyl-galactopyranoside and beta-methyl-galactopyranoside, with much lower apparent K(m). Thus, this sialyltransferase is unique and should be very useful for achieving high productivity in E. coli with a wide substrate range.
Topics: Animals; Cloning, Molecular; Decapodiformes; Disaccharides; Galactose; Gene Expression Regulation, Enzymologic; Kinetics; Models, Biological; Molecular Sequence Data; N-Acetylneuraminic Acid; Photobacterium; Protein Structure, Tertiary; Sialic Acids; Sialyltransferases; Substrate Specificity; beta-Galactoside alpha-2,3-Sialyltransferase
PubMed: 17702755
DOI: 10.1074/jbc.M701907200 -
Bioscience, Biotechnology, and... Mar 19991-Aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of ethylene in plants, has never been known to occur in microorganisms. We describe the synthesis of...
1-Aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of ethylene in plants, has never been known to occur in microorganisms. We describe the synthesis of ACC by Penicillium citrinum, purification of ACC synthase [EC 4.4.1.14] and ACC deaminase [EC 4.1.99.4], and their properties. Analyses of P. citrinum culture showed occurrence of ACC in the culture broth and in the cell extract. ACC synthase was purified from cells grown in a medium containing 0.05% L-methionine and ACC deaminase was done from cells incubated in a medium containing 1% 2-aminoisobutyrate. The purified ACC synthase, with a specific activity of 327 milliunit/mg protein, showed a single band of M(r) 48,000 in SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme by gel filtration was 96,000 Da. The ACC synthase had the Km for S-adenosyl-L-methionine of 1.74 mM and kcat of 0.56 s-1 per monomer. The purified ACC deaminase, with a specific activity of 4.7 unit/mg protein, showed one band in SDS-polyacrylamide gel electrophoresis of M(r) 41,000. The molecular mass of the native ACC deaminase was 68,000 Da by gel filtration. The enzyme had a Km for ACC of 4.8 mM and kcat of 3.52 s-1. The presence of 7 mM Cu2+ in alkaline buffer solution was effective for increasing the stability of the ACC deaminase in the process of purification.
Topics: Amino Acids; Amino Acids, Cyclic; Animals; Biotransformation; Carbon-Carbon Lyases; Chromatography, DEAE-Cellulose; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Ethylenes; Kinetics; Lyases; Molecular Weight; Organisms, Genetically Modified; Penicillium; S-Adenosylmethionine
PubMed: 10227140
DOI: 10.1271/bbb.63.542 -
The Journal of Biological Chemistry Oct 1988We recently described inositol polyphosphate 1-phosphatase, an enzyme which cleaves the 1-phosphate from inositol 1,4-bisphosphate (Ins(1,4)P2) and inositol... (Comparative Study)
Comparative Study
We recently described inositol polyphosphate 1-phosphatase, an enzyme which cleaves the 1-phosphate from inositol 1,4-bisphosphate (Ins(1,4)P2) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) (Inhorn, R. C., and Majerus, P. W. (1987) J. Biol. Chem. 262, 15946-15952). We have now purified the enzyme to homogeneity from calf brain. The enzyme hydrolyzes 50.3 mumol of Ins(1,4)P2/min/mg protein. The enzyme has an apparent mass of 44,000 daltons as determined both by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that it is monomeric. Lithium ions inhibit Ins(1,3,4)P3 hydrolysis uncompetitively with an apparent Ki of approximately 0.3 mM LiCl. Calcium inhibits hydrolysis of Ins(1,4)P2 and Ins(1,3,4)P3 equally, with approximately 40% inhibition occurring at 1 microM free Ca2+. Rabbit polyclonal antiserum against purified inositol polyphosphate 1-phosphatase was prepared which immunoprecipitates approximately 0.3 milliunits of activity/microliter serum (1 unit = 1 mumol of Ins(1,4)P2 hydrolyzed per min). This antiserum was used to determine the enzyme content in several bovine tissues, all of which had a similar intrinsic specific activity (i.e. approximately 0.3 milliunits/microliter antiserum). Tissues studied included brain, heart, kidney, liver, lung, parotid, spleen, testis, and thymus. Approximately 10-15% of the total inositol polyphosphate 1-phosphatase activity in calf brain homogenates remains in a particulate fraction; antiserum also binds 0.3 milliunits of membrane-associated activity/microliter antiserum. Thus, a single enzyme can account for Ins(1,4)P2 hydrolytic activity in the bovine tissues. Ins(1,3,4)P3 metabolism was also investigated in bovine tissue homogenates. Inositol polyphosphate 1-phosphatase accounts for greater than 80% of the hydrolytic activity in all tissues studied except brain, where inositol polyphosphate 4-phosphatase is the major enzyme that hydrolyzes Ins(1,3,4)P3. The apparent Km of inositol polyphosphate 1-phosphatase for Ins(1,3,4)P3 varies approximately 3-4-fold among the bovine tissues.
Topics: Animals; Brain; Cattle; Chlorides; Chromatography; Chromatography, Gel; Chromatography, Ion Exchange; Durapatite; Hydroxyapatites; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Lithium; Lithium Chloride; Organ Specificity; Phosphoric Monoester Hydrolases; Thymus Gland
PubMed: 2844776
DOI: No ID Found -
Biochemical Pharmacology Sep 1990Although the liver is recognized as a major site of glutathione (GSH) synthesis, it is thought to play only a minor role in GSH catabolism. This is primarily because in... (Comparative Study)
Comparative Study
Although the liver is recognized as a major site of glutathione (GSH) synthesis, it is thought to play only a minor role in GSH catabolism. This is primarily because in the rat, the most commonly used experimental animal, hepatic gamma-glutamyltransferase (gamma-GT) activity is very low, whereas kidney activity is quite high. gamma-GT is the only enzyme known to catalyze the initial step in GSH degradation. The present work compares gamma-GT and dipeptidase activities in liver, kidney, and gallbladder of six mammalian species to assess the importance of hepatobiliary catabolism of GSH, relative to renal degradation. Marked species differences were observed in gamma-GT activities, and in kidney to liver (K/L) ratios for both gamma-GT concentration (milliunits/mg protein) and whole organ activities (total activity per liver or two kidneys). The K/L concentration ratios for gamma-GT activities ranged from 875 in the rat to 15 in the guinea pig. Whole organ gamma-GT ratios were approximately 150 in mouse and rat, and only 2-5 in guinea pig. pig, and macaque. Human K/L ratios calculated from gamma-GT activities reported previously were similar to those of the guinea pig. Species differences were also observed in K/L ratios for dipeptidase activities, though these differences were not as large as those for gamma-GT, gamma-GT and dipeptidase activities were also measured in gallbladders of all species examined (except rat which does not have this organ), and were found to be comparable to those of liver. These results suggest that in species such as the guinea pig and perhaps humans, the liver and biliary tree play a prominent role in GSH turnover. Because of the low hepatic and high renal gamma-GT activities of the rat, and because it does not have a gallbladder, this species may not be the best model for studying the catabolism of GSH and GSH conjugates. Use of the rat model may underestimate the contribution of liver, and overestimate that of kidney, in these degradative processes.
Topics: Animals; Culture Techniques; Dipeptidases; Female; Gallbladder; Glutathione; Guinea Pigs; Kidney; Liver; Macaca fascicularis; Male; Mice; Mice, Inbred BALB C; Organ Size; Rabbits; Rats; Rats, Inbred Strains; Species Specificity; Swine; gamma-Glutamyltransferase
PubMed: 1975172
DOI: 10.1016/0006-2952(90)90503-d -
The Journal of Biological Chemistry Jun 1988Fructose-6-P,2-kinase:fructose-2,6-bisphosphatase has been purified to homogeneity from beef heart. The enzyme was bifunctional and the specific activities of the kinase...
Fructose-6-P,2-kinase:fructose-2,6-bisphosphatase has been purified to homogeneity from beef heart. The enzyme was bifunctional and the specific activities of the kinase and the phosphatase of the pure enzyme were 60 and 30 milliunits/mg, respectively. The molecular weight of the enzyme was 118,000, consisting of two subunits of 58,000. In some preparations of the enzyme a minor protein with a subunit Mr of 54,000 was present. This minor protein (54,000) was also bifunctional and showed the same immunoreactivity as the major protein. The specific activity of fructose-6-P,2-kinase of the minor component was three times higher than that of the major enzyme (58,000), but fructose-2,6-bisphosphatase activity was the same. These two forms have been separated by phosphocellulose chromatography. The tryptic peptide maps of these enzymes were very similar. The 58,000 enzyme was phosphorylated by cAMP-dependent protein kinase but the 54,000 enzyme was not. These results indicated that the minor 54,000 protein might be a proteolytically digested form of the 58,000 enzyme. The Km of the kinase for fructose-6-P and ATP was 70 microM and 260 microM, respectively for both the 58,000 and the 54,000 enzymes. Km for fructose-2,6-P2 and Ki for fructose-6-P of the phosphatase was approximately 40 and 11 microM, respectively. The enzyme was phosphorylated by fructose-2,6-P2 but the stoichiometry of the phosphate incorporation was 0.05 mol/mol subunit, while 0.4 mol/mol was incorporated in rat liver enzyme under the same conditions.
Topics: Animals; Cattle; Chromatography, Affinity; Chromatography, DEAE-Cellulose; Chromatography, Gel; Kinetics; Myocardium; Peptide Fragments; Phosphofructokinase-2; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Protein Kinases; Trypsin
PubMed: 2837472
DOI: No ID Found -
The Journal of Biological Chemistry Oct 1997Long term administration of leptin decreases caloric intake and fat mass and improves glucose tolerance. Here we examine whether leptin acutely regulates peripheral and...
Long term administration of leptin decreases caloric intake and fat mass and improves glucose tolerance. Here we examine whether leptin acutely regulates peripheral and hepatic insulin action. Recombinant mouse leptin (0.3 mg/kg.h, Leptin +) or vehicle (Leptin -) were administered for 6 h to 4-month-old rats (n = 20), and insulin (3 milliunits/kg.min) clamp studies were performed. During physiologic hyperinsulinemia (plasma insulin approximately 65 microunits/ml), the rates of whole body glucose uptake, glycolysis, and glycogen synthesis and the rates of 2-deoxyglucose uptake in individual tissues were similar in Leptin - and Leptin +. Post-absorptive hepatic glucose production (HGP) was similar in the two groups. However, leptin enhanced insulin's inhibition of HGP (4.1 +/- 0.7 and 6.2 +/- 0.7 mg/kg.min; p < 0.05). The decreased HGP in the Leptin + group was due to a marked suppression of hepatic glycogenolysis (0.7 +/- 0.1 versus 4.1 +/- 0.6 mg/kg.min, in Leptin + versus Leptin -, respectively; p < 0.001), whereas the % contribution of gluconeogenesis to HGP was markedly increased (82 +/- 3% versus 36 +/- 4% in Leptin + and Leptin -, respectively; p < 0.001). At the end of the 6-h leptin infusion, the hepatic abundance of glucokinase mRNA was decreased, whereas that of phosphoenolpyruvate carboxykinase mRNA was increased compared with Leptin -. We conclude that an acute increase in plasma leptin 1) enhances insulin's ability to inhibit HGP, 2) does not affect peripheral insulin action, and 3) induces a redistribution of intrahepatic glucose fluxes and changes in the gene expression of hepatic enzymes that closely resemble those of fasting.
Topics: Animals; Deoxyglucose; Glucokinase; Gluconeogenesis; Hyperinsulinism; Insulin; Leptin; Liver Glycogen; Male; Patch-Clamp Techniques; Phosphoenolpyruvate Carboxykinase (ATP); Proteins; RNA, Messenger; Rats; Rats, Sprague-Dawley
PubMed: 9346919
DOI: 10.1074/jbc.272.44.27758