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1.
Diabetes Obes Metab ; 14(7): 608-15, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22276636

ABSTRACT

AIMS: Amylinergic and melanocortinergic systems have each been implicated in energy balance regulation. We examined the interactive effects of both systems using gene knockout and pharmacological approaches. METHODS: Acute food consumption was measured in overnight fasted male wild-type (WT) and melanocortin-4 receptor (MC-4R) deficient rats and in male and female WT and amylin knockout mice (AmyKO). Changes in food intake, body weight and composition in male WT and MC-4R deficient rats and in male diet-induced obese (DIO) rats. Pharmacological treatments included either rat amylin, murine leptin and/or the MC-4R agonist, Ac-R[CEH-dF-RWC]-amide. RESULTS: Amylin (10 µg/kg, IP) decreased food intake in WT but not in MC-4R deficient rats (30 and 60 min post-injection). Ac-R[CEH-dF-RWC]-amide (100 µg/kg, IP) suppressed food intake similarly in male WT and AmyKO, but was ineffective in female AmyKO. Amylin (50 µg/kg/day for 28 days) and leptin (125 µg/kg/day) synergistically reduced food intake and body weight in WT and MC-4R deficient rats to a similar extent. Amylin (100 µg/kg) combined with Ac-R[CEH-dF-RWC]-amide (100 µg/kg, IP) decreased acute food intake over 3 h to a greater extent than either agent alone in fasted mice. In DIO rats, additive anorexigenic, weight- and fat-lowering effects were observed over 12 days with the combination of rat amylin (50 µg/kg/day) and Ac-R[CEH-dF-RWC]-amide (2.3 mg/kg, SC injected daily). CONCLUSIONS: Although amylin's acute anorexigenic effects are somewhat blunted in MC-4R deficiency and those of MC-4R agonism in amylin deficiency, these effects are surmountable with pharmacological administration lending therapeutic potential to combined amylin/melanocortin agonism for obesity.


Subject(s)
Anti-Obesity Agents/pharmacology , Body Weight , Eating , Islet Amyloid Polypeptide/deficiency , Islet Amyloid Polypeptide/pharmacology , Obesity/drug therapy , Receptor, Melanocortin, Type 4/deficiency , Animals , Disease Models, Animal , Drug Interactions , Energy Metabolism , Female , Gene Knockout Techniques , Islet Amyloid Polypeptide/administration & dosage , Male , Mice , Rats , Rats, Sprague-Dawley , Receptor, Melanocortin, Type 4/agonists
2.
Mitochondrion ; 5(1): 1-13, 2005 Feb.
Article in English | MEDLINE | ID: mdl-16060289

ABSTRACT

The adenine nucleotide translocase (ANT) is a key component in maintaining cellular energy homeostasis, and has also been implicated in formation of the mitochondrial permeability transition pore. Human ANT-3 was cloned from a human heart cDNA library and expressed as a histidine-tagged fusion protein in the mitochondria of the Trichoplusia ni. cell line. Overexpression resulted in a concomitant decrease in the endogenous ANT content, allowing for the characterization of binding of known ANT ligands to the human protein. Binding affinities for bongkrekic acid (BKA), ADP, and atractyloside (ATR) were measured in mitochondria from the human ANT-3 expressing cell line, and compared to similar preparations from bovine heart mitochondria by use of a novel radioiodinated derivative of ATR. Binding to ANT-3 by the high affinity inhibitors BKA and ATR, as well as the lower affinity natural ligand ADP, was similar to that measured in bovine heart mitochondria, and to that previously reported for mammalian heart mitochondria. Characterizations such as these of human ANT isoforms may lead to drug development for enhanced mitochondrial function and cellular viability.


Subject(s)
Adenine Nucleotide Translocator 3/genetics , Adenine Nucleotide Translocator 3/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Adenosine Diphosphate/metabolism , Animals , Atractyloside/analogs & derivatives , Atractyloside/chemistry , Atractyloside/metabolism , Bongkrekic Acid/metabolism , Cattle , Cell Line , Energy Metabolism , Gene Expression , Humans , In Vitro Techniques , Iodine Radioisotopes , Kinetics , Ligands , Mitochondria, Heart/metabolism , Moths , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
3.
Nucleic Acids Res ; 25(15): 3102-9, 1997 Aug 01.
Article in English | MEDLINE | ID: mdl-9224611

ABSTRACT

A sensitive and highly reproducible multiplexed primer extension assay is described for quantitative mutation analysis of heterogeneous DNA populations. Wild-type and mutant target DNA are simultaneously probed in competitive primer extension reactions using fluorophor-labeled primers and high fidelity, thermostable DNA polymerases in the presence of defined mixtures of deoxy- and dideoxynucleotides. Primers are differentially extended and the resulting products are distinguished by size and dye label. Wild-type:mutant DNA ratios are determined from the fluorescence intensities associated with electrophoretically resolved reaction products. Multiple nucleotide sites can be simultaneously interrogated with uniquely labeled primers of different lengths. The application of this quantitative technique is shown in the analysis of heteroplasmic point mutations in mitochondrial DNA that are associated with Alzheimer's disease.


Subject(s)
Alzheimer Disease/diagnosis , DNA Mutational Analysis/methods , DNA Primers , DNA, Mitochondrial , Fluoresceins/chemistry , Alzheimer Disease/genetics , Humans
4.
Adv Enzyme Regul ; 29: 141-57, 1989.
Article in English | MEDLINE | ID: mdl-2517377

ABSTRACT

Mercuration of 2'-deoxyuridine 5'-phosphate (dUMP) followed by alkylation with allylamine in the presence of K2PdCl4 afforded the 5-aminoallyl deoxynucleotide, which was isolated by sequential Dowex 50 H+ and DEAE-Sephadex chromatography. Further reaction of the product with the N-hydroxysuccinimide ester of methotrexate (MTX) in dry dimethyl sulfoxide gave an MTX-aminoallyl-dUMP covalent complex separable by DEAE-Sephadex chromatography. Reprecipitation with acid from basic solution offered further purification and the structure was confirmed by elemental analysis, NMR and absorbance spectra. The product was an inhibitor of rat liver dihydrofolate reductase (I50 approximately 250 nM, cf. MTX I50 approximately 60 nM) and Lactobacillus casei thymidylate synthase. With the latter enzyme, inhibition was competitive with both nucleotide and folate substrates (Ki = 2.6 and 3.5 microM, respectively) and partial enzyme-inhibitor binary complex could be detected by gel electrophoresis. Large fluorescence changes were observed on titration of the synthase with MTX-aminoallyl-dUMP and alterations in the UV difference spectra similar to those seen on titration of the enzyme with MTX were also noted. The compound was a poor growth inhibitor for cultured murine L1210 and human CCRF-CEM cell lines, which probably reflects low cellular uptake.


Subject(s)
Deoxyuracil Nucleotides/pharmacology , Methotrexate/analogs & derivatives , Thymidylate Synthase/antagonists & inhibitors , Animals , Biological Transport , Cell Division/drug effects , Deoxyuracil Nucleotides/chemical synthesis , Deoxyuracil Nucleotides/pharmacokinetics , Folic Acid Antagonists , Humans , Lacticaseibacillus casei/enzymology , Liver/enzymology , Methotrexate/chemical synthesis , Methotrexate/pharmacokinetics , Methotrexate/pharmacology , Rats , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , Tumor Cells, Cultured/drug effects
5.
Cancer Chemother Pharmacol ; 23(5): 283-90, 1989.
Article in English | MEDLINE | ID: mdl-2706732

ABSTRACT

The transport routes used by CCRF-CEM human lymphoblastoid cells for the influx and efflux of unconjugated pteridines were analyzed using [3H]6-hydroxymethylpterin as a model compound. Influx proceeds by a mechanism that exhibits a Km of 66.7 microM and a Vmax of 0.077 nmol/min per mg cellular protein. The process is somewhat sensitive to metabolic inhibitors, particularly uncouplers of oxidative phosphorylation, and is significantly affected by the presence of other pteridines in the extracellular medium. The results suggest that pterins with either no 6-substituent (pterin) or those with methyl, hydroxyl, or formyl groups in this position, which exhibit Ki values between 25 and 77 microM, may share the same pathway for uptake. 6-Carboxypterin exhibits low affinity for the system (Ki greater than 500 microM), as do 7-substituted and 6,7-di-substituted derivatives and compounds with larger groups at the 6-position, such as neopterin and biopterin (Ki = 250-300 microM). Efflux of [3H]6-hydroxymethylpterin occurs rapidly and can proceed by at least two routes. The first, comprising approximately 50% of total efflux, is inhibited by extracellular pterins and exhibits similar properties to the uptake system in both its pattern of sensitivity to metabolic inhibitors and its specificity for pteridine structure. The route by which the remaining efflux occurs is relatively insensitive to metabolic inhibition. Adenine significantly inhibits 6-hydroxymethylpterin influx and efflux (Ki = 10.6 microM for uptake) but does not appear to share the same transport system. Similarly, methotrexate and folic acid exhibit little affinity for the unconjugated pteridine transport routes.


Subject(s)
Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Pteridines/pharmacokinetics , Biological Transport , Carbon Radioisotopes , Cell Line , Chromatography, Gel , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Humans , Pteridines/analysis , Pteridines/antagonists & inhibitors , Pteridines/pharmacology , Pterins/analysis , Pterins/antagonists & inhibitors , Pterins/pharmacokinetics , Tritium , Tumor Cells, Cultured
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