Characterization of glucokinase regulatory protein-deficient mice.

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.

A second-generation enzyme immunoassay for detection of antibodies to IFN-alpha.

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.

Regional distribution of calcium/calmodulin-dependent phosphatase activity of calcineurin in rat brain.

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.

Platelet monoamine oxidase in Parkinson patients: effect of L-deprenyl therapy.

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.