Levels of gamma-aminobutyric acid in cerebrospinal fluid in various neurologic disorders.

Levels of gamma-aminobutyric acid (GABA) in CSF were measured by the ion exchange-fluorometric method in 136 patients who underwent evaluation for neurologic disorders. In 19 patients with no organic neurologic or mental disorders who acted as normal controls, the mean (+/-SD) GABA level in CSF was 239 +/- 76 picomoles/mL. Patients with acute hypoxic encephalopathy showed a mean GABA level in CSF higher than that of the controls, a difference that was statistically significant. In all the other disorders studied, the mean GABA level in CSF was either equal to or lower than that found in the controls. Statistically significant reductions of the GABA level in CSF were seen in patients with Huntington's disease, dementias, cerebellar cortical atrophy, multiple sclerosis, epilepsy, and Parkinson's disease.

Artifactual presence of beta-nerve growth factor in adult mouse brain.

Adult male mouse brain extracts were determined to contain 0.7 ng beta-nerve growth factor (NGF)/mg total protein in a one-site radioimmunoassay (RIA) and 3.6 ng NGF/mg protein in a competition radioreceptor assay using PC 12 cells. When brain extracts were immunoprecipitated with antiserum to NGF prior to use in a radioreceptor assay, no reduction in measured NGF content resulted, whereas immunoprecipitation of submaxillary gland extract reduced the NGF level by 83-100%. The immunoassay and radioreceptor assay were then modified by incubating antisera or PC 12 cells first with brain extract, and then, after washing, with 125I-labeled authentic mouse NGF. In the modified assays, no NGF was detected in the extracts, indicating that adult mouse brain does not contain NGF itself but does contain an NGF-binding component that causes false positive results in standard one-site RIAs and competition radioreceptor assays.

Stability of GABA levels in CSF under various conditions of storage.

Measurement of GABA in human lumbar CSF specimens stored under various conditions showed that the concentrations remained stable in untreated frozen specimens stored at -20 degrees C and at -70 degrees C. In untreated liquid specimens the GABA concentrations approximately doubled during 2 h at room temperature but did not change significantly during 10 min at room temperature or 2 h at 2-4 degrees C. The GABA level was stable at -70 degrees C in deproteinized specimens but doubled during 11 months of storage at -20 degrees C. The level was stable in liquid deproteinized samples for 49 h at room temperature but increased 1.3-fold and 2.0-fold in deproteinized specimens stored for 3 weeks at 2-4 degrees C and room temperature, respectively. Amino acid analyses of homocarnosine standards in 0.1 N HCl revealed a similar increase of GABA during storage at room temperature and at -20 degrees C, suggesting that at least part of the increase seen in CSF specimens might result from breakdown of GABA containing peptides. This instability of GABA level may account for some of the discrepancies among the reports of CSF GABA levels.

Electrophoretic analysis of low and high activity forms of catechol-O-methyltransferase in human erythrocytes.

Analysis of the catechol-O-methyltransferase (COMT) enzyme in human RBC lysates from 15 samples exhibiting inherited variations in level of activity and thermal stability was performed. Electrophoretic blotting and immune fixation was carried out following sodium dodecyl sulfate polyacrylamide gel electrophoresis or isoelectric focusing of lysate protein. These techniques did not reveal a major structural alteration of the protein that could account for the observed variation in enzyme activity or thermal stability. Future studies utilizing molecular genetic techniques should make it possible to determine the basis for inherited variations in human RBC COMT activity and thermal stability.

Molecular cloning and expression of catrocollastatin, a snake-venom protein from Crotalus atrox (western diamondback rattlesnake) which inhibits platelet adhesion to collagen.

A 50 kDa protein that inhibits platelet adhesion to collagen has been isolated from snake venom of Crotalus atrox (western diamondback rattlesnake) and has been named 'catrocollastatin'. The cDNA cloning of catrocollastatin has been accomplished. A full-length cDNA of 2310 bp with an open reading frame between nucleotides 51 and 1880 was obtained. The deduced amino acid sequence consists of 609 amino acids. The cDNA-predicted amino acid sequence is highly similar to that of haemorrhagic metalloproteinase jararhagin from Bothrops jararaca venom, HR1B from Trimeresurus flavoviridis, Ht-e from C. atrox and trigramin from T. gramineus. Like jararhagin and HR1B, catrocollastatin is a multidomain molecule composed of an N-terminal domain, a metalloproteinase domain, a disintegrin-like domain and a cysteine-rich C-terminal domain. In the disintegrin-like domain, the frequently seen RGD (Arg-Gly-Asp) sequence is replaced by SECD (Ser-Glu-Cys-Asp). This cDNA was expressed in Spodoptera frugiperda (fall armyworm) (Sf9) insect cells using a baculovirus expression system. Like native catrocollastatin, the expressed protein is capable of selectively blocking collagen-induced platelet aggregation. This is the first full-length clone of a high-molecular-mass haemorrhagin to be expressed.

Isolation of the mRNA encoding rat liver catechol-O-methyltransferase.

A highly specific, well characterized rabbit antiserum to purified rat liver catechol-O-methyltransferase (COMT; EC 2.1.1.6) and the procedure of polysome immunoadsorption have been used to isolate a messenger RNA which encodes a single polypeptide when translated in vitro. Western blotting and immune fixation have shown multiple active forms of the enzyme to exist; a major, soluble one with MW of 23,000 and pI of 5.2 and another, membrane-bound one with MW of 26,000 and a pI of 6.2 (1). When translated in vitro, the purified message synthesizes a protein of molecular weight (MW) 23,000 and pI 5.2, values in agreement with those for purified enzyme reported by other investigators (2,3). Only the soluble form is seen after in vitro translation; the other immunoreactive proteins possibly arise due to post-translational modifications which do not occur in the lysate; or perhaps another mRNA exists. Cloning of the COMT cDNA will resolve this issue and should be feasible in light of our data indicating that the mRNA isolated here represents 0.46% of total rat liver polyadenylated message.

Chromosomal mapping of the human catechol-O-methyltransferase gene to 22q11.1----q11.2.

Catechol-O-methyltransferase (COMT; EC 2.1.1.6) is a physiologically important enzyme in the metabolism of catecholamine neurotransmitters and catechol drugs. Using primers derived from the known rat cDNA sequence for COMT, we have used the polymerase chain reaction to produce an amplified DNA fragment corresponding to the complete coding region of the rat gene. With this fragment as a probe, we have hybridized DNAs from two panels consisting of human/rodent and human/hamster somatic cell hybrids carrying various translocations and deletions to refine the chromosomal location of human COMT. Southern blot analysis indicates that the human COMT gene is localized to 22q11.1----q11.2, a region to which several anonymous DNA sequences, but until now, no structural genes, have been assigned.

Eicosanoids in sickle cell disease: potential relevance of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid to the pathophysiology of vaso-occlusion.

The monohydroxyeicosanoid 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), which is derived from oxygenation of arachidonic acid by 12-lipoxygenase, is one of the major metabolites in platelets. In a recent study, we have showed that this eicosanoid stimulated basal sickle-red-cell-endothelial-cell adhesion. To understand the pathophysiologic significance of 12-HETE, we measured the levels of this eicosanoid in plasma and urine from children with sickle cell disease. We found that as compared with controls, plasma 12-HETE levels are increased in patients with sickle-cell disease in the steady state, and are increased further during vaso-occlusive crises. Urinary 12-HETE levels were also increased during the steady state. We also assessed plasma levels of soluble P-selectin (another potential marker for platelet activation), and found changes in the levels of this marker similar to those seen with plasma 12-HETE. In additional studies, we found that 12-HETE enhanced hypoxia-induced sickle-red-cell-endothelial adherence, and that this effect was mediated by potentiation of agonist-induced upregulation of the expression of the mRNA for vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells. Because 12-HETE appears to enhance both basal and agonist-induced sickle-red-cell adhesion, this metabolite could potentially play a role in the pathogenesis of the vaso-occlusive crisis (VOC) in sickle-cell disease.

Soluble and particulate forms of rat catechol-O-methyltransferase distinguished by gel electrophoresis and immune fixation.

Catechol-O-methyltransferase (COMT) was visualized in homogenates and subcellular fractions of rat tissues, including liver and brain, by gel electrophoresis, electrophoretic transfer of proteins to nitrocellulose (Western blotting), and immune fixation with antiserum to highly purified soluble rat liver COMT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of all tissue homogenates examined revealed three major immune-specific proteins with apparent molecular weights 23,000, 26,000, and 66,000 (23K, 26K and 66K). Centrifugation of homogenates at 100,000 X g for 60 min resulted in the enrichment of the 26K species protein in the pellet whereas the 23K and 66K proteins were the predominant forms in the supernatant. The 66K protein appeared in variable amounts depending on the tissue being examined and the length of transfer of protein and is assumed to be an "aggregate" of the smaller form(s). The 26K protein was essentially the only immunoreactive species seen in a purified preparation of rat liver outer mitochondrial membrane. Isoelectric focusing (IEF) under denaturing conditions and two-dimensional gel electrophoresis of brain and liver fractions showed that the 23K protein was resolved into three bands of pI 5.1, 5.2, and 5.3, whereas the 26K protein had a pI of 6.2. Analysis of COMT activity in slices from nondenaturing IEF gels indicated that the pI 5.1-5.3 species are biologically active; the pI 6.2 species could not be detected under these conditions. COMT activity was demonstrated, however, in outer mitochondrial membranes from rat liver, which contain predominantly the 26K, pI 6.2 immunoreactive species. The major form of COMT in all rat tissues examined is "soluble" with an apparent Mr of 23K and a pI of 5.2. The nature of the modifications giving rise to pI 5.1 and 5.3 forms of this enzyme are not clear, nor is the relationship between the 23K and 26K forms. Further studies are needed to elucidate the relationship of immunoreactive forms of COMT to each other, their intracellular location, and their functional significance.

Comparative mapping of 9 human chromosome 22q loci in the laboratory mouse.

We present a comparative map of genes on human chromosome 22q and homologous loci in the mouse genome. Gene order in humans was established using a panel of somatic cell hybrids. Genetic maps spanning homologous segments on three mouse chromosomes were generated using an interspecific backcross. The conserved linkage between human chromosome 22 and mouse chromosome 16 includes two closely linked loci, Comt and IgI-1. The second conserved linkage involves human chromosome 22 and mouse chromosome 11 and contains two genetically and physically linked loci, Lif and Nfh. Finally, conserved synteny involving mouse chromosome 15 and human chromosome 22 spans 30 cM and contains five loci (Acr, Bzrp, Dia-1, Il2rb and Pdgfb). Loci within this conserved synteny have been sublocalized to different portions of human chromosome 22. The order of genes on mouse chromosome 15 and human chromosome 22 provides further evidence for chromosomal rearrangements within the conserved synteny that have occurred since the divergence of lineages leading to mice and humans.