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1.
J Biol Chem ; 275(11): 7826-31, 2000 Mar 17.
Article in English | MEDLINE | ID: mdl-10713097

ABSTRACT

The glucokinase regulatory protein (GKRP) inhibits glucokinase competitively with respect to glucose by forming a protein-protein complex with this enzyme. The physiological role of GKRP in controlling hepatic glucokinase activity was addressed using gene targeting to disrupt GKRP gene expression. Heterozygote and homozygote knockout mice have a substantial decrease in hepatic glucokinase expression and enzymatic activity as measured at saturating glucose concentrations when compared with wild-type mice, with no change in basal blood glucose levels. Interestingly, when assayed under conditions to promote the association between glucokinase and GKRP, liver glucokinase activity in wild-type and null mice displayed comparable glucose phosphorylation capacities at physiological glucose concentrations (5 mM). Thus, despite reduced hepatic glucokinase expression levels in the null mice, glucokinase activity in the liver homogenates was maintained at nearly normal levels due to the absence of the inhibitory effects of GKRP. However, following a glucose tolerance test, the homozygote knockout mice show impaired glucose clearance, indicating that they cannot recruit sufficient glucokinase due to the absence of a nuclear reserve. These data suggest both a regulatory and a stabilizing role for GKRP in maintaining adequate glucokinase in the liver. Furthermore, this study provides evidence for the important role GKRP plays in acutely regulating of hepatic glucose metabolism.


Subject(s)
Carrier Proteins , Glucokinase/antagonists & inhibitors , Glucose/metabolism , Liver/metabolism , Proteins/metabolism , Adaptor Proteins, Signal Transducing , Age Factors , Animals , Enzyme Inhibitors/metabolism , Gene Targeting , Glucose Tolerance Test , Heterozygote , Homeostasis , Homozygote , Insulin/blood , Intracellular Signaling Peptides and Proteins , Islets of Langerhans/metabolism , Mice , Mice, Knockout , Mutagenesis , Proteins/genetics
2.
J Interferon Cytokine Res ; 18(6): 357-60, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9660241

ABSTRACT

We have developed a solid-phase enzyme-linked immunoassay (EIA) for detecting antibodies to interferon-alpha2 (IFN-alpha2) in serum or plasma. In this assay, based on the sandwich principle, the capture antigen, IFN-alpha2, is covalently bound to the wells in 96-well plates. This novel procedure offers considerable advantages over the antigen binding by passive adsorption used in most previous EIA. Specific antibodies present in clinical specimens bind to the anchored antigen and are detected by adding peroxidase-labeled IFN-alpha2 and a peroxidase substrate mixture. The resultant color is a function of the concentration of antibody in the sample. The assay has proved to be convenient, precise, and reproducible and can detect as little as 1-5 ng/ml of specific antibody IgG.


Subject(s)
Antibodies/analysis , Immunoenzyme Techniques , Interferon-alpha/immunology , Immunoenzyme Techniques/instrumentation , Immunoglobulin G/analysis , Peroxidase/metabolism , Reproducibility of Results
3.
Cancer Chemother Pharmacol ; 38 Suppl: S16-21, 1996.
Article in English | MEDLINE | ID: mdl-8765410

ABSTRACT

Interleukin 12 (IL-12) is a heterodimeric cytokine with a number of biological effects that are consistent with its potential role as an antitumor agent. The antimetastatic and antitumor activities of IL-12 have been demonstrated in a number of murine tumor models. Both the inhibition of established experimental pulmonary or hepatic metastases and a reduction in spontaneous metastases have been achieved by treatment with murine IL-12. Systemic treatment of mice bearing subcutaneous tumors with IL-12 results in tumor growth inhibition, prolongation of survival, and, in some models, tumor regression. The antitumor effect of IL-12 in these models is dose-dependent and can be initiated against well-established tumors. Mice cured of their tumor by IL-12 treatment are specifically immune to rechallenge with the same tumor. A series of experiments have demonstrated that both T-cells and interferon-gamma (IFN-gamma) induction are necessary for the optimal antitumor effects of IL-12. However, the antitumor efficacy of IL-12 has not been observed after exogenous administration of murine IFN-gamma, suggesting that additional factors may be important for the antitumor effects of IL-12. In several tumor models, IL-12 is more active or has a larger therapeutic window than either IL-2 or IFN-alpha, two cytokines with demonstrated antitumor activity against human malignancies. Combining IL-12 with other cytokines or chemotherapeutic drugs can improve antitumor effects.


Subject(s)
Interleukin-12/therapeutic use , Neoplasms, Experimental/therapy , Animals , Drug Screening Assays, Antitumor , Humans , Interleukin-12/immunology , Interleukin-2/therapeutic use , Neoplasms, Experimental/immunology , Tumor Cells, Cultured/drug effects
4.
Res Commun Chem Pathol Pharmacol ; 64(3): 357-71, 1989 Jun.
Article in English | MEDLINE | ID: mdl-2551001

ABSTRACT

The regional distribution of calcineurin activity (measured using p-nitrophenyl-phosphate which detects the phospho-tyrosylphosphatase activity of calcineurin) shows that the striatum, hippocampus and cerebral cortex contains high calcineurin activity. Within the striatum, calcineurin activity does not appear to be present in dopaminergic terminals, since lesions of the nigro-striatal dopaminergic pathway (which reduce striatal dopamine levels by 97%) had no effect on calcineurin activity. On the other hand, kainic acid, which destroys neurons whose perikarya are in the striatum, reduced calcineurin activity by 86% indicating that calcineurin activity is localized in striatal intrinsic neurons. Calcineurin apparently does not exist in glia, since glial cells actually proliferate in kainic acid lesioned striatal tissues.


Subject(s)
Brain Chemistry/drug effects , Calcium/metabolism , Calmodulin-Binding Proteins/metabolism , Calmodulin/metabolism , Phosphoprotein Phosphatases/metabolism , Phosphoric Monoester Hydrolases/metabolism , 3,4-Dihydroxyphenylacetic Acid/metabolism , Animals , Calcineurin , Corpus Striatum/enzymology , Corpus Striatum/physiology , Dopamine/metabolism , Glutamate Decarboxylase , Homovanillic Acid/metabolism , Hydroxydopamines , Kainic Acid/pharmacology , Male , Oxidopamine , Rats , Rats, Inbred Strains , Substantia Nigra/drug effects , Sympathectomy, Chemical
5.
Article in English | MEDLINE | ID: mdl-2505797

ABSTRACT

Platelet MAO activity was measured in 79 Parkinson patients (56 males and 23 females) before and during L-deprenyl therapy. Baseline platelet MAO activity was higher in females than in males with no age dependent differences. During chronic L-deprenyl therapy, MAO activity was inhibited greater than 98%. Four hours after the oral administration of the first 5 mg dose of L-deprenyl, platelet MAO activity was inhibited by 86%. By 24 hours, greater than 98% inhibition was achieved and this degree of inhibition was maintained during continuous L-deprenyl administration. Following oral administration of 10 mg L-deprenyl once a day versus 5 mg L-deprenyl twice a day, the time course of platelet MAO inhibition was similar. Five days after the termination of chronic L-deprenyl therapy, platelet MAO activity was still inhibited by 96%. MAO activity returned to normal by 2 weeks after stopping L-deprenyl. Platelet MAO activity is a useful method of monitoring bioavailability, compliance, dose-response relationship and optimal dosage schedules for L-deprenyl in Parkinson patients.


Subject(s)
Blood Platelets/enzymology , Monoamine Oxidase/metabolism , Parkinson Disease/blood , Phenethylamines/therapeutic use , Selegiline/therapeutic use , Adult , Aged , Aged, 80 and over , Dose-Response Relationship, Drug , Humans , Middle Aged , Parkinson Disease/drug therapy , Sex Factors
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