Peptide bound hypohydroxyprolinuria in Handigodu Disease: a familial syndrome of spondylo epi(meta)physeal dysplasia.

Handigodu Disease (HD) is disorder of the osteoarticular system prevalent in few villages of two districts of the state Karnataka in southern India. 24 hrs urinary excretions of proline (Pro) and 4-hydroxyproline (Hyp) were analyzed by HPLC. Decreased peptide bound Hyp excretions (mumole/24 hrs) were found in patient group when compared with controls (Nonaffected; 113.02 +/- 67.96, Type-I; 36.22 +/- 20.76, Type-II; 45.74 +/- 14.95, Type-III; 40.46 +/- 22.68) and without significant difference in Pro excretions. Significant increased peptide bound Pro to Hyp ratio were found in patient group compared to control (Nonaffected n=63: 2.02 +/- 1.65, Type-I n=18: 3.144 +/- 1.42, Type-II n=28: 4.21 +/- 1.95, Type-III n=8: 8.60 +/- 6.55). 24 hrs urinary excretions of deoxypyridinoline (DPD) crosslinks were found without significant difference among affected and control, hence HD ruled out from general bone reduction. These results suggest hypohydroxyprolinuria may be because of reduced bone turnover or defective hydroxylation of prolyl residues during post translational modification of collagen biosynthesis.

Expression of p67 (Munc-18) in adult human brain and neuroectodermal tumors of human central nervous system.

p67 (Munc-18), is a neuron-specific protein of 67 kDa, known for its ability to bind with syntaxin and also to copurify with neuronal cdc2-like kinase. Earlier, in situ hybridization and immunocytochemical analysis of rat trigeminal ganglion and hippocampal cells demonstrated the specific localization of p67 in nerve cells and its rich distribution in axons. In the present study, we have looked for p67 expression in normal human brain and various neuroectodermal tumors. Immunohistochemical and Western immunoblot analysis of normal human brain tissue using antibodies against the N- and C-termini of p67 demonstrated the specific localization of this protein in postmitotic neurons but not in glia. Among neuroectodermal tumors, expression of p67 was observed in 100% of the tumors of neuronal origin studied, especially in the mature neuronal cell population of these tumors. Western immunoblot analysis of non-neuronal neuroectodermal tumors failed to reveal the expression of this protein in majority of cases. However, in gliomas and meningiomas, mild cytoplasmic immunohistochemical staining of neoplastic cells was noted in 64.7% and 25% of cases, respectively. Observed mild immunohistochemical staining of these tumors could be due to immunoreactivity to low molecular weight degraded products of p67, as seen on Western blot. The findings suggest that p67, by virtue of its ability to be expressed in postmitotic neurons of adult human brain and in tumors of neuronal origin, may serve as a molecular tool to understand the growth and differentiation of the nervous system in general.

A study of serum prolactin and plasma human growth hormone in male alcoholics.

Serum levels of prolactin (PRL) and Human Growth Hormone (HGH) were assayed in 38 male alcoholics and 24 male control subjects using radioimmunoassay (RIA) technique. Biochemical parameters of hepatic function and severity of withdrawal state were also assessed. Significantly elevated values of plasma HGH were found in alcoholics as a group. Nineteen percent and eight percent of the patient had elevated serum PRL and HGH levels respectively. Evidence of advanced liver disease was scant and withdrawal symptoms were by and large mild. The findings indicate a dysfunction in hypothalamic adenohypophyseal axis in a subgroup of alcoholics.

Utility of serum lysosomal enzyme assay in the detection of cerebral sphingolipidoses in patients with progressive neurologic dysfunction.

The cerebral sphingolipidoses which form part of a larger group of lysosomal disorders can be detected and conclusively confirmed by the demonstration of the relevent enzyme deficiency in easily available tissue samples like serum. We have assayed acid ß-galactosidase, ß-hexosaminidase and its isozymes hexosaminidase A and B, and arylsulfatase A in the serum of patients with progressive cerebral dysfunction and detected 18 patients with enzyme defects, thereby confirming the diagnosis of a specific type of cerebral lipidosis in these patients. The assay of serum lysosomal enzymes was of immense diagnostic use as it obviated the need for highly invasive techniques like a brain biopsy.

Disulfiram lowers Ca2+, Mg(2+)-ATPase activity of rat brain synaptosomes.

The chronic administration of disulfiram (DS) to rats resulted in significant decrease of synaptosomal Ca2+, Mg(2+)-ATPase activity. In vitro studies indicated that DS (ID50 = 20 microM) produced a dose-dependent inhibition of Ca2+, Mg(2+)-ATPase. However, diethyldithio-carbamate, a metabolite of DS, failed to modify Ca2+, Mg(2+)-ATPase activity, implying that the decrease in ATPase activity in DS administered rats was due to the effect of parent compound. The DS-mediated inhibition (48%) of ATPase activity was comparable with a similar degree of inhibition (49%) achieved by treating the synaptosomal membranes with N-ethylmaleimide (ID50 = 20 microM) in vitro. Furthermore, the inhibition by DS was neither altered by washing the membranes with EGTA nor reversed by treatment with sulfhydryl reagents such as GSH or dithiothreitol. About 74% and 68% decrease of synaptosomal Ca2+, Mg(2+)-ATPase specific activity was observed when treated with DS (30 microM) and EGTA (100 microM) respectively. The remaining 25-30% of total activity is suggested to be of Mg(2+)-dependent ATPase activity. This indicates that both these drugs may act on a common target, calmodulin component that represents 70-75% of total Ca2+, Mg(2+)-ATPase activity. Therefore, DS-mediated modulation of synaptosomal Ca2+, Mg(2+)-ATPase activity could affect its function of maintaining intracellular Ca2+ concentration. This could contribute to the deleterious effects on CNS.

Effect of disulfiram administration on rat brain glutathione metabolism.

Chronic administration of disulfiram (DS) to rats was found to affect glutathione (GSH) metabolism. Glutathione was measured in the rat brain following DS administration. Reduced glutathione was decreased significantly (1.52 +/- 0.3 mumol/g; p < 0.001), with a concomitant increase in oxidised glutathione (GSSG) content (0.12 +/- 0.013 mumol/g; p < 0.001) in the brain as a consequence of DS treatment. However, total glutathione (GSH + GSSG) content of the experimental group did not show any appreciable change. Similar changes were observed in the liver following chronic DS treatment. Brain glutathione reductase (GR) activity was found to be significantly depleted (100 +/- 0.16 mumol/min/mg protein), but glutathione peroxidase (GP) activity was not affected in rats chronically treated with DS. It is reported that the treatment with DS decreases the GSH content, with a concomitant increase in GSSG level, and perturbs the GSH/GSSG redox status, inducing an oxidative stress on the brain. Glutathione reductase implicated in maintaining GSH/GSSG homeostasis by replenishing GSH is also affected by DS potentiating the oxidative damage of the tissue. This effect of DS on glutathione metabolism in the brain would explain some of its known neurotoxic effects.

cdc2-like kinase from rat spinal cord specifically phosphorylates KSPXK motifs in neurofilament proteins: isolation and characterization.

A protein kinase that phosphorylates a specific KSP sequence [K(S/T)PXK], which is abundant in high molecular weight neurofilament (NF) proteins, was identified and isolated from rat spinal cord. Characterization of this enzyme activity revealed a close relationship with p34cdc2 kinase with respect to its molecular mass (32.5 kDa by SDS/PAGE) and substrate specificities. It could phosphorylate a synthetic peptide analog of the simian virus 40 large tumor antigen, reportedly a specific substrate for p34cdc2 kinase. Histone (H1) and peptide analogs of the KSP sequence present in the C-terminal end of rat and mouse neurofilament proteins were phosphorylated. This kinase did not phosphorylate alpha-casein and peptide substrates of other known second messenger-dependent or -independent kinases. Dephosphorylated rat NF protein NF-H was strongly phosphorylated by the purified enzyme; NF proteins NF-M and native NF-H, but not NF-L, were slightly phosphorylated. Studies on synthetic peptide analogs of KSP repeats with substitution of specific residues, known to be present in the C-terminal regions of NF-H, revealed a consensus sequence of X(S/T)PXK, characteristic of the p34cdc2 kinase substrate. On Western blots, the enzyme was immunoreactive with antibody against the C-terminal end of cdc2 kinase (mouse) and neuronal cdc2-like kinase from rat but not with an antibody against the conserved PSTAIRE region of the p34cdc2 kinase. The antibody against the C-terminal end of cdc2 kinase could immunoprecipitate (immunodeplete) the purified kinase activity. Since the adult nervous system is composed primarily of postmitotic cells, the present observations indicate a nonmitotic role for this cdc2-like kinase activity. The effective phosphorylation of NF-H by this kinase suggests a function in axonal structure.

Phosphatase activity against neurofilament proteins from bovine spinal cord: effect of aluminium and neuropsychoactive drugs.

Protein phosphatase activity associated with neurofilament (NF) rich (Triton X-100 insoluble) fraction was extracted and partially characterised by using known inhibitors of protein phosphatases such as vanadate and fluoride. Protein phosphatase activity was demonstrated with reference to the dephosphorylation of endogenous substrate, NF protein and exogenous protein substrates, casein and phosvitin. Phosphoamino acids and beta-glycerophosphate were found to be poor substrates. Further, new observations have been made regarding the in vitro inhibitory effect of aluminium and the differential effects of some of the neuropsychoactive drugs. The findings could possibly lead to studies explaining the biochemical basis of aluminium induced neurotoxicity as well as the side effects associated with the long term medication of neuropsychoactive drugs.

Alcoholic liver disease in a psychiatric hospital.

Liver biopsy was analysed for 41 alcoholics, satisfying RDC criteria. Alcoholic liver disease was diagnosed as per the guidelines from the WHO/ICMR Workshop. For the entire group mean consumption of alcohol was 183.lg. of ethanol for an average of 9.7 years. Only 5 persons (12.2%) had normal liver. Six persons (14.6%) had Fatty liver, 23 (56.1%) had alcoholic hepatitis, and 4(9.7%) had precirrhosis and cirrhosis. Biochemical analysis showed that alcoholics had elevated values of SGOT, SGPT and GGT as compared to control non - drinker population. Malnutrition did not appear to be a contaminating factor for the observed histopathological findings.

Occurrence of gamma-glutamyl transpeptidase activity in several mycobacteria including Mycobacterium leprae.

gamma-Glutamyl transpeptidase (gamma-GT) activity, which catalyzes the transfer of the "gamma-glutamyl" group of gamma-glutamyl compounds to several dipeptide and amino acid acceptors, was found to be present in several mycobacteria, including M. leprae, both in cell suspensions and in cell-free sonicates. Glycyl D-amino acids were active as acceptors, particularly glycyl-D-alanine and alpha, epsilon-diaminopimelic acid, among the amino acids. Two mycobacterial isolates obtained from biopsy material of lepromatous patients also exhibited similar enzyme activity. The need for further work to delineate the possible role of gamma-GT in mycobacterial metabolism is strongly indicated.