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.

Alterations in neurofilament protein(s) in human leprous nerves: morphology, immunohistochemistry and Western immunoblot correlative study.

Using a specific antibody (SMI 31), the state of phosphorylation of high and medium molecular weight neurofilaments (NF-H and NF-M) was studied in 22 leprous and four nonleprous human peripheral nerves by means of immunohistochemistry, sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot (WB). The results thus obtained were compared with morphological changes in the respective nerves studied through light and electron microscopy. Many of the leprous nerves showing minimal pathology revealed lack of or weak staining with SMI 31, denoting dephosphorylation. Remyelinated fibres stained intensely with SMI 31 antibody. The WB analysis of Triton X-100 insoluble cytoskeletal preparation showed absence of regular SMI 31 reactive bands corresponding to 200 and 150 kDa molecular weight (NF-H and NF-M, respectively) in 10 nerves. Three of the 10 nerves revealed presence of NF protein bands in SDS-PAGE but not in WB. Presence of additional protein band (following NF-M) was seen in four nerves. Two nerves revealed NF-H band but not NF-M band and one nerve showed trace positivity. In the remaining five nerves presence of all the three NF bands was seen. Thus, 77.3% (17/22) of human leprous nerves studied showed abnormal phosphorylation of NF protein(s). The ultrastructural study showed abnormal compaction and arraying of NF at the periphery of the axons in the fibres with altered axon to myelin thickness ratio (atrophied fibres) as well as at the Schmidt-Lantermann (S-L) cleft region. Such NF changes were more pronounced in the severely atrophied axons suggesting a direct correlation. The observed well-spaced NF in the remyelinated fibres under ultrastructural study was in keeping with both intense SMI 31 staining and presence of NF triplet bands seen in WBs in four of leprous nerves that showed a large number of regenerating fibres suggesting reversal of changes with regeneration. Findings in the present study suggest that atrophy, that is, the reduction in axonal calibre and paranodal demyelination, seen in leprous nerves may result from dephosphorylation of NF-H and NF-M proteins.

Alterations in neurofilament protein(s) in human leprous nerves: morphology, immunohistochemistry and Western immunoblot correlative study

Using a specific antibody (SMI 31), the state of phosphorylation of high and medium molecular weight neurofilaments (NF-H and NF-M) was studied in 22 leprous and four nonleprous human peripheral nerves by means of immunohistochemistry, sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot (WB). The results thus obtained were compared with morphological changes in the respective nerves studied through light and electron microscopy. Many of the leprous nerves showing minimal pathology revealed lack of or weak staining with SMI 31, denoting dephosphorylation. Remyelinated fibres stained intensely with SMI 31 antibody. The WB analysis of Triton X-100 insoluble cytoskeletal preparation showed absence of regular SMI 31 reactive bands corresponding to 200 and 150 kDa molecular weight (NF-H and NF-M, respectively) in 10 nerves. Three of the 10 nerves revealed presence of NF protein bands in SDS-PAGE but not in WB. Presence of additional protein band (following NF-M) was seen in four nerves. Two nerves revealed NF-H band but not NF-M band and one nerve showed trace positivity. In the remaining five nerves presence of all the three NF bands was seen. Thus, 77.3% (17/22) of human leprous nerves studied showed abnormal phosphorylation of NF protein(s). The ultrastructural study showed abnormal compaction and arraying of NF at the periphery of the axons in the fibres with altered axon to myelin thickness ratio (atrophied fibres) as well as at the Schmidt-Lantermann (S-L) cleft region. Such NF changes were more pronounced in the severely atrophied axons suggesting a direct correlation. The observed well-spaced NF in the remyelinated fibres under ultrastructural study was in keeping with both intense SMI 31 staining and presence of NF triplet bands seen in WBs in four of leprous nerves that showed a large number of regenerating fibres suggesting reversal of changes with regeneration. Findings in the present study suggest that atrophy, that is, the reduction in axonal calibre and paranodal demyelination, seen in leprous nerves may result from dephosphorylation of NF-H and NF-M proteins.

Cdk5 and MAPK are associated with complexes of cytoskeletal proteins in rat brain.

Neurofilament proteins, the major cytoskeletal components of large myelinated axons, are highly phosphorylated by second messenger-dependent and -independent kinases. These kinases, together with tubulins and other cytoskeletal proteins, have been shown to bind to neurofilament preparations. Cdk5 and Erk2, proline-directed kinases in neuronal tissues, phosphorylate the Lys-Ser-Pro (KSP) repeats in tail domains of NF-H, NF-M, and other axonal proteins such as tau and synapsin. In neurofilament and microtubule preparations from rat brain, we demonstrated by Western blot analysis that cdk5, a neuronal cyclin dependent kinase and Erk1/2 were associated with complexes of NF proteins, tubulins and tau. Using P13(suc1) affinity chromatography, a procedure known to bind cdc2-like kinases in proliferating cells with high affinity, we obtained a P13 complex from a rat brain extract exhibiting the same profiles of cdk5 and Erk2 bound to cytoskeletal proteins. The phosphorylation activities of these preparations and the effect of the cdk5 inhibitor, butyrolactone, were consistent with the presence of active kinases. Finally, during a column fractionation and purification of Erk kinases from rat brain extracts, fractions enriched in Erk kinase activity also exhibited co-elution of phosphorylated NF-H, tubulin, tau and cdk5. We suggest that in mammalian brain, different kinases, their regulators and phosphatases form multimeric complexes with cytoskeletal proteins and regulate multisite phosphorylation from synthesis in the cell body to transport and assembly in the axon.

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.

Trinucleotide repeats and neuropsychiatric disorders.

Expansions of trinucleotide repeats at the level of genomic DNA are increasingly recognized as a cause of a number of neuropsychiatric disorders. Triplet repeat disorders are commonly classified into two groups, those with moderate CAG expansions that result in a polyglutamine stretch in the gene products and those with very long expansions, usually non-CAG, that are not translated. The triplet repeat intergeneration and intra-generational instability, and genetic anticipation characterize disorders. Most of the diseases caused by expanded CAG repeat share common features, which include neurodegeneration, a dominant pattern of inheritance and widespread expression of the gene products with neuronal loss restricted to distinct subset of neurons. Neurodegenerative changes associated of CAG expansion disorders is explained in terms of intra and extra cellular aggregation of mutant gene products, the insoluble nature of the protein(s) being attributed to the presence of polyglutamine stretches, hence their neurotoxic effects. methods based on poymerase chain reaction have become handy in the diagnosis of these genetic disorders. Progress in transgenic animal models for these disorders will be critical for understanding the progress of these disorders and for testing new therapeutic strategies.

Maple syrup urine disease: An uncommon cause for neonatal metabolic distress.

Maple Syrup Urine Disease is an autosomal recessive disorder caused by a deficiency in the activity of the branched-chain α-ketoacid dehydrogenase complex. This rare disorder represents one of the causes of acute neonatal illness which results in devastating disturbances of neurological development. On investigation of 1750 infants with neurological impairment for inborn errors of amino acid metabolism, 4 neonates with classical maple syrup urine disease were detected. These otherwise normal neonates presented in the first week after birth with seizures, lethargy and refusal of feeds, hypoglycemia and metabolic acidosis. The plasma and urine concentrations of the branched-chain amino acids were increased and there was ketoaciduria. Two of these neonates expired before specific treatment could be instituted. Routine biochemical screening of neonates with acute illness could unearth many cases of this rare inherited metabolic disease.

Characterization of the phosphorylation sites of human high molecular weight neurofilament protein by electrospray ionization tandem mass spectrometry and database searching.

Hyperphosphorylated high molecular weight neurofilament protein (NF-H) exhibits extensive phosphorylation on lysine-serine-proline (KSP) repeats in the C-terminal domain of the molecule. Specific phosphorylation sites in human NF-H were identified by proteolytic digestion and analysis of the resulting digests by a combination of microbore liquid chromatography, electrospray ionization tandem (MS/MS) ion trap mass spectrometry, and database searching. The computer programs utilized (PEPSEARCH and SEQUEST) are capable of identifying peptides and phosphorylation sites from uninterpreted MS/MS spectra, and by use of these methods, 27 phosphopeptides and their phosphorylated residues were identified. On the basis of these phosphopeptides, 38 phosphorylation sites in human NF-H were characterized. These include 33 KSP, lysine-threonine-proline (KTP) or arginine-serine-proline (RSP) sites and four unphosphorylated sites, all of which occur in the KSP repeat domain (residues 502-823); and one threonine phosphorylation site observed in a KVPTPEK motif. Six KSP sites were not characterized because of the failure to isolate and identify corresponding phosphopeptides. Heterogeneity in serine and threonine phosphorylation was observed at three sites or deduced to occur at three sites on the basis of enzyme specificity. As a result of the phosphorylated motifs identified (KSPAKEE, KSPVKEE, KS/TPEKAK, KSPEKEE, KSPVKAE, KSPAEAK, KSPPEAK, KSPEAKT, KSPAEVK, and KVPTPEK), human NF-H tail domain is postulated to be a substrate of proline-directed kinases. The threonine-phosphorylated KVPTPEK motif suggested the existence of a novel proline-directed kinase.

Type Ib glycogenosis.

Type Ib glycogenosis is a rare glycogen storage disorder resulting from a defect in the enzyme, glucose-6-phosphatase microsomal translocase. We report a case of Type Ib glycogenosis in an 18 month-old male child who presented with a history of hypoglycemic seizures and recurrent infections and had a massive hepatomegaly, recurrent hypoglycemia, hyperuricemia, hypertriglyceridemia, neutropenia and fasting lactacidemia which decreased sharply on glucose administration.

Arginase deficiency.

Hyperargininemia due to arginase deficiency is a rare, inherited, urea cycle disorder. We report a case of arginase deficiency in a 5-year old boy presenting with mild hyperammonemia, hyperargininemia, and dibasic aminoaciduria.

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.

Inhibition of neuronal cyclin-dependent kinase-5 by staurosporine and purine analogs is independent of activation by Munc-18.

Neuronal cdk5 can phosphorylate certain lys-ser-pro (KSP) motifs of neurofilaments and tau protein in the nervous system. We have immunoprecipitated the cdk5 from rat brain using a polyclonal antibody raised against the C-terminus of cdk5. The immunoprecipitate has phosphorylated a KSPXK peptide analog of NF-H, as well as histone H1 and a bacterially expressed rat NF-H protein. The kinase activity was inhibited by staurosporine, isopentanyladenine and olomoucine in a dose dependent manner. Kinetic studies indicated Ki values of 39 nM, 38 microM and 8 microM, respectively for staurosporine, isopentanyladenine and olomoucine. The inhibition by staurosporine was non-competitive with respect to phosphoryl acceptor acceptor substrates. Western blot analysis of the immunoprecipitate showed both cdk5 and p67 (Munc-18), a putative regulator molecule of the kinase. Addition of p67 fusion protein enhanced the kinase activity of the immunoprecipitate by 60% above the basal activity. P67 elevated Ki values for both staurosporine and olomoucine. The degree of inhibition at high concentrations of these inhibitors was unaltered by exogenous p67 indicating a lack of competitive interactions with p67. The high affinity of staurosporine for cdk5 suggests that cdk5 may be one of the targets for the neurotropic effect of staurosporine.

GM2 gangliosidoses: a review of cases confirmed by beta-N-acetylhexosaminidase assay.

The inborn errors of GM2 ganglioside metabolism cause GM2 ganglioside to accumulate within the lysosomes of the nerve cells. The majority of the patients are infants with the Tay-Sachs form of the disease associated with a severe deficiency of beta-N-Acetylhexosaminidase A (hexosaminidase A). Both Hexosaminidase A and B are deficient in Sandhoff disease. The serum total hexosaminidase and the percentage of hexosaminidase A and B were estimated in 449 patients who presented with progressive mental-motor retardation. Three cases of Tay-Sachs disease and two cases of Sandhoff disease were detected. They presented with exaggerated startle response to acoustic stimuli, seizures, optic atrophy and retinal cherry red spots in addition to psychomotor retardation. One case of Sandhoff disease had hepatosplenomegaly and skeletal deformities.

Neuronal cyclin-dependent kinase-5 phosphorylation sites in neurofilament protein (NF-H) are dephosphorylated by protein phosphatase 2A.

Neurofilament (NF) protein [high molecular mass (NF-H)] is extensively phosphorylated in vivo. The phosphorylation occurs mainly in its characteristic KSP (Lys-Ser-Pro) repeat motifs. There are two major types of KSP motifs in the NF-H tail domain: KSPXKX and KSPXXX. Recent studies by two different laboratories have demonstrated the presence of a cdc2-like kinase [cyclin-dependent kinase-5 (cdk5)] in nervous tissue that selectively phosphorylates KSPXKX and XS/TXK motifs in NF-H and lysine-rich histone (H1). This article describes the identification of phosphatases dephosphorylating three different substrates: histone (H1), NF-H in a NF preparation, and a bacterially expressed C-terminal tail domain of NF-H, each containing KSPXKX repeats phosphorylated in vitro by cdk5. Among various phosphatases identified, protein phosphatase (PP) 2A from rabbit skeletal muscle appeared to be the most effective phosphatase in in vitro assays. Three phosphatase activity peaks--P1, P2, and P3--were partially purified from frozen rat spinal cord by ion exchange and size exclusion column chromatography and then characterized on the basis of biochemical, pharmacological, and immunochemical studies. One of the three peaks was identified as PP2A, whereas the others were mixtures of both PP2A and PP1. These three peaks could dephosphorylate cdk5-phosphorylated 32P-histone (H1), 32P-NF-H in the NF preparation, and 32P-NF-H tail fusion protein. These studies suggest the involvement of PP2A or a PP2A-like activity in the regulation of the phosphorylation state of KSPXKX motifs in NF-H.

Molecular characterization of a neuronal-specific protein that stimulates the activity of Cdk5.

Cyclin-dependent kinase, Cdk5, has been identified in neural tissue in connection with neurofilament and tau protein phosphorylation. This report describes the characterization of a 62-kDa protein that copurifies with Cdk5 from rat spinal cord homogenates. Dissociation of the protein from neural Cdk5 is concomitant with a reversible loss in kinase activity. Amino acid sequence information from tryptic peptide fragments was used to clone the complementary DNA from rat brain. A single full-length cDNA was characterized coding for a 67.5-kDa protein (p67). Exogenously expressed p67 stimulated Cdk5 kinase activity in vitro in a dose-dependent manner and when presented as an affinity matrix, selectively adsorbed Cdk5 from a cleared rat brain homogenate. In situ hybridization analysis of E18 rat embryos and adult rat brain demonstrated that p67 transcript expression is restricted to neural tissue. Immunohistochemical staining with an amino-terminal peptide-specific antibody further indicated that p67 is exclusively expressed in neurons. Localization in vivo and in cultured rat hippocampal neurons showed that p67 is highly enriched in axons. We propose that p67, by virtue of its regulation of Cdk5, participates in the dynamics of axonal architecture through the modulation of phosphorylation of cytoskeletal components.