Abstract: RNA was extracted from five different rat brain regions during development, starting from embryonic day 15 (E15) until postnatal day 60 (P60). These RNA preparations were analyzed by both Northern and dot blot for their content of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), myelin proteolipid protein (PLP), and myelin basic protein (MBP)-specific transcripts. CNPase mRNA was readily detectable at E15 and PLP mRNA at P1 in all brain regions examined. In contrast, expression of MBP mRNA followed a caudorostral gradient. It was first observed at P1 in the mesencephalon and at P9–P11 in the olfactory bulb. Expression of these three transcripts displayed two types of developmental profiles. One was termed biphasic because the specific mRNA level increased regularly and then reached a plateau level. The other developmental profile was termed triphasic, because there was a gradual increase in the level of specific transcripts with a sudden appearance of a sharp peak followed by a decline to a plateau level. When the triphasic pattern was observed, the date of the peak appearance was probe-, but not region-, dependent. It was P15 for CNPase, P18 for MBP, and P21 for PLP. As these peaks occurred at a time during development when myelination was the most active, we postulate the existence of a transient external signal, perhaps neuronal, which would be responsible for this increased amount of myelin-related transcripts.

Abstract: We investigated regulation of the dynamic state of enkephalin and endorphin brain stores during morphine tolerance and dependence using cDNA hybridization and radioimmunoassay of the biologically active peptide(s) and their respective peptide precursors. Rats were made tolerant to morphine with the subcutaneous implantation of three morphine pellets (75 mg each) for a period of five days. Hypothalamic proopiomelanocortin (POMC) mRNA, POMC, and corticotropin-like intermediate lobe peptide content were decreased by 50% in morphine-dependent rats. However, beta-endorphin content remained unchanged. Enkephalin and proenkephalin mRNA content in various brain structures failed to change. A single injection of naltrexone (2 mg/kg) 1 hour before decapitation did not reverse the decrease in POMC mRNA and POMC content elicited by morphine. However, a slower, spontaneous withdrawal caused by removal of the pellets did reverse (after two days) the down-regulation of the hypothalamic POMC system. A single injection of morphine (10 mg/kg) failed to affect any parameter used to assess the dynamic state of opioid peptides.

Abstract: Microtubule-associated protein 2 (MAP-2), a cytoskeletal protein of 280 kilodalton that is highly enriched in dendrites and neuronal perikarya, is subject to both cyclic AMP-, calcium/calmodulin-, and calcium/phospholipid-regulated phosphorylation when incubated with [γ-32P]ATP in vitro. We have analyzed the different sites in MAP-2 phosphorylated by these three kinases in fresh or boiled cytosol from different regions of the rat brain, in particular the olfactory bulb, where only one form (MAP-2B) is present, and the cerebral cortex, where both forms (MAP-2A and MAP-2B) are equally enriched. Cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II phosphorylated four common phosphorylation sites, as well as a number of distinct sites that were unique to each enzyme. Calcium/phospholipid-dependent protein kinase phosphorylated a minimum of 15 sites, only one of which appeared to be shared with the other protein kinases. Only serine residues were phosphorylated by cyclic AMP-dependent and calcium/phospholipid-dependent protein kinases, while both serine and threonine residues were phosphorylated by calcium/calmodulin-dependent protein kinase II. No differences were observed in the peptide maps of phospho-MAP-2 prepared from different brain regions. These results emphasize the complexity of the phosphorylation systems that may regulate the function of MAP-2 in situ.

Abstract: In situ hybridization was used to study the macroscopic distribution and regulatory control of proenkephalin mRNA and prodynorphin mRNA in rat striatum. While proenkephalin mRNA was widely distributed throughout the striatum, levels of prodynorphin mRNA were highest in the medial and ventral portions of the striatum. Furthermore, in contrast to the results for proenkephalin mRNA, the levels of prodynorphin mRNA appeared higher in the nucleus accumbens than in the striatum. The mesostriatal dopaminergic pathway was destroyed by discrete, unilateral injection of 6-hydroxydopamine (6-OHDA) into either the substantia nigra or the neighboring ventral tegmental area (VTA). Lesions of the substantia nigra caused a dramatic ipsilateral increase in the hybridization signal for proenkephalin mRNA, but no change was observed in the hybridization signal for prodynorphin mRNA. Similar effects were seen with VTA lesions. Since destruction of the mesostriatal dopamine system elevates the levels of proenkephalin mRNA, but not of prodynorphin mRNA, in the striatal target neurons, it appears that the mesostriatal pathway exerts a tonic and selective suppression of striatal proenkephalin gene expression at the mRNA level.

Abstract: Previous studies have shown that insulin and IGF-I bind to their respective receptors and stimulate autophosphorylation of the receptor β subunits in detergent extracts of neuronal and glial cells. In the present study, intact neuronal and glial cells in primary culture have been utilized to characterize insulin- and IGF-I-stimulated phosphorylation of their receptors. Following [32P]orthophosphate labeling and stimulation by insulin or IGF-I, the cells were solubilized and the phosphorylated receptors were partially purified on wheat germ agglutinin-agarose columns, and immunoprecipitated using anti-phosphotyrosine or anti-insulin receptor antibodies. Insulin stimulated the phosphorylation of its receptor β subunit (95 kD phosphoprotein) in a dose-dependent manner, within at least 20 seconds in both neuronal and glial cells. Additionally, a 102-kD phosphoprotein was observed in insulin-stimulated neuronal cells. Maximal stimulation of receptor phosphorylation occurred at 1 minute for the glial cells, and 10 minutes for the neuronal cells. IGF-I stimulated the phosphorylation of two phosphoproteins in intact neuronal and glial cells; a 95-kD protein and a 102-kD protein, in a dose-dependent manner. These observations demonstrate that both insulin and IGF-I stimulate the phosphorylation of the β subunits of their respective receptors in brain cells in a similar fashion to their effects on receptors from nonneural tissues.

Abstract: We have characterized and localized phorbol ester binding sites in human autopsied brains, using [3H]phorbol 12,13-dibutyrate ([3H]PDBu). When the tissue was homogenized in the absence of Ca2+ chelator (10 mM EGTA/2 mM EDTA), Scatchard analysis of the specific [3H]PDBu bindings to both particulate and soluble fractions yielded a single class of high-affinity binding site (K d = 7.1 and 7.4 nM:B max = 45.4 and 3.1 pmol/mg protein, respectively). The particulate fraction retained the majority of [3H]PDBu binding (98% of total binding activity), while the soluble fraction was almost devoid of binding activity (2%). In the presence of Ca2+ chelator, more of the activity was found in the soluble fraction (30%). The binding of [3H]PDBu was potently inhibited by active phorbol esters and related diterpenes withK i of nanomolar concentration but not by inactive ones. Diolein (OAG), a synthetic diacylglycerol, and polymixin B, an inhibitor of protein kinase C (PKC), inhibited the binding moderately (K i = 5.8 and 1.3 µM, respectively). H-7, an inhibitor of PKC and cyclic nucleotides-dependent kinase, did not compete with [3H]PDBu for the binding sites (K i > 100,000 nM). The regional distribution of specific [3H]PDBu binding in the human brain was rather uneven and resembled that of [3H]PDBu autoradiograms and PKC-like immunoreactivities in the rat brain. The binding capacities were generally in the order: rhinencephalon > basal ganglia > cerebral cortex > diencephalon > cerebellum > mesencephalon. Age-related loss of binding sites was observed in the prefrontal cortex of the subjects 33–81 years of age. In Parkinson’s disease, the phorbol ester binding showed a significant reduction in the substantia nigra, caudate putamen, and pallidum, whereas it was unchanged in the prefrontal cortex and caudate nucleus of schizophrenics, when compared with the relevant controls.

Abstract: Bombesin/gastrin-releasing peptide receptors were characterized in human glioblastoma cell lines. [125I]Gastrin-releasing peptide or ([125I]Tyr4)bombesin bound with high affinity to these cell lines. Binding to cell line U-118 was time dependent, reversible, and specific. ([125I]Tyr4)Bombesin bound with high affinity (Kd = 1.6 nM) to a single class of sites (Bmax = 30,000/cell). The C-terminal of bombesin- or gastrin-releasing peptide was essential for high-affinity binding. Bombesin- or gastrin-releasing peptide elevated the cytosolic Ca2+ levels in a dose-dependent manner. Because gastrin-releasing peptide, but not gastrin-releasing peptide, increased the cytosolic Ca2+ levels, the C-terminal but not the N-terminal of GRP is essential for biological activity. These data indicate that biologically active bombesin receptors are present in human glioblastoma cell lines.

Abstract: In addition to their well-known involvement in neuromuscular junctions and in brain cholinergic synapses, cholinergic mechanisms have been implicated in the growth and maturation of oocytes in various species. Functional acetylcholine receptors were electrophysiologically demonstrated in amphibian and mammalian oocyte membranes, and activity of the acetylcholine-hydrolyzing enzyme, acetylcholinesterase (AChE), was biochemically measured in the exceptionally big oocytes of the frogXenopus laevis. However, biochemical methods could not reveal whether AChE was produced within the oocytes themselves or in the surrounding follicle cells. Furthermore, this issue is particularly important for understanding growth and fertilization processes in the much smaller human oocytes, in which the sensitivity of AChE biochemical measurements is far too low to be employed. To resolve this question, a molecular biology approach was combined with biochemical measurements on ovarian extracts and sections. To directly determine whether the human cholinesterase (ChE) genes are transcriptionally active in oocytes, and, if so, at what stages in their development, the presence of ChE mRNA was pursued. For this purpose frozen ovarian sections were subjected to in situ hybridization using35S-labeled human ChE cDNA. Highly pronounced hybridization signals were localized within oocytes in primordial, preantral, and antral follicles, but not in other ovarian cell types, demonstrating that within the human ovary ChE mRNA is selectively synthesized in viable oocytes at different developmental stages. Sucrose gradient centrifugation followed by [3H]acetylcholine hydrolysis measurements revealed in the ovarian extracts the presence of low levels of soluble AChE dimers, sensitive to the specific AChE inhibitor BW284C51 but resistant to the BuChE inhibitor iso-OMPA. In view of the low numbers of oocytes out of total cells in the ovary, these findings strongly suggest that AChE is a prominent protein in human oocytes throughout their development and further support the hypothesis that cholinergic mechanisms may be involved in oocyte growth and maturation in humans.

Abstract: Messenger RNA (mRNA) coding for vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI-27) were localized in cortical and thalamic neurons with synthetic DNA probes complementary to the PHI-27 and VIP exon coding sequence of the rat VIP precursor gene. Hybridization signal with these probes was found widely distributed in the thalamus, neocortex, and pyriform cortex, and the distribution of hybridization signal for each probe was identical. Furthermore, the distribution of each message was correlated closely with peptide distribution demonstrated immunohistochemically. Labeling of individual neurons with in situ hybridization histochemistry was characterized by a dense accumulation of silver grains in the cytoplasm of these cells with little or no label in the nucleus. Labeled neurons in the thalamus were observed in the ventrolateral, ventromedial, ventrobasal, and lateral reticular nuclei. In the neocortex, the distribution of labeled neurons was concentrated in layers II and III with scattered cells also apparent in deeper cortical layers. Hybridization signal was limited to nonpyramidal neurons in both the neo- and pyriform cortex. The coextensive distribution of immunoreactivity and mRNA coding regions for VIP and PHI-27 establishes that these peptides are synthesized from the same precursor mRNA in the same thalamic and cortical cell groups. Although the physiological role of these peptides in thalamocortical function remains unknown, these data provide an anatomical substrate which suggests that VIP and PHI-27 may be cotransmitters in thalamic and cortical neurons.

Abstract: The expression of MAP2 during rat brain development was studied by using specific antibodies and cDNA probes. MAP2 cDNAs were isolated from a rat brain λgt11 library, and their identity was confirmed by the reactivity of their fusion proteins with several independent monoclonal antibodies that recognize MAP2. Northern blot analyses of the RNA prepared from whole brains, cerebral cortex, hypothalamus, brain stem, olfactory bulbs, and cerebellum showed that the levels of MAP2 mRNA increase during the initial phase of development, reach a maximum between postnatal weeks 2 and 3, and then decrease in the adult. The time course and the kinetics of this change varied between different brain regions and appeared to reflect the pattern of morphological changes in these regions RNA blots were also analyzed with β-tubulin and β-actin cDNA probes to ensure the quality and the quantity of the RNA. The levels of MAP2 mRNA and protein showed similar changes during the initial part of brain development and suggested a transcriptional control. However, while MAP2 protein levels remained high throughout development, MAP2 mRNA levels decreased in adulthood. We suggest that the increased stability of the MAP2 molecule may be a contributing factor in the developmental regulation of steady-state levels of MAP2. *** DIRECT SUPPORT *** A07GO001 00014

Abstract: The tyrosine kinase substrate p36 (calpactin heavy chain) is a calcium-dependent membrane- and cytoskeletal-binding protein. Using an affinity-purified antiserum raised against the p36 heavy chain from bovine adrenal medulla, we have examined the cellular distribution of p36 in developing and adult cerebellum. From immunoblotting, the level of p36 in cerebellum was found to decline during development. In dissociated cell cultures of P4 cerebellum, all cell types were labeled by anti-p36. In vibratome sections from cerebella of P10 rats, anti-p36 stained Purkinje cell bodies strongly and all other cell types less strongly, with the exception of cells in the external germinal layer, which were unstained. By 18 days postnatally p36 was present at higher levels in Bergmann glia and astrocytes of the white matter. In sections of adult cerebella, anti-p36 staining was restricted to Bergmann glial processes and to the processes of a subclass of astrocytes in the granular layer and the white matter. At no developmental stage was anti-p36 staining detectable in axons or dendrites in vibratome sections. These results suggest that p36 plays a role in some aspect of cellular differentiation common to all cerebellar cell types and may have additional functions in astrocytes of the adult cerebellum.

Abstract: Cyclic GMP-dependent protein kinase displays an uneven distribution in brain, being highly concentrated only in cerebellar Purkinje cells. Using DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr 32,000) as exogenous substrate, and performing assays in the absence or presence of the protein inhibitor of cyclic AMP-dependent protein kinase, we have now identified both cyclic AMP-dependent and cyclic GMP-dependent protein kinase activities in the rat neostriatum and substantia nigra. Quinolinic acid-induced degeneration of neostriatal neurons and the straitonigral fibers emanating from neostriatal neurons decreased the activities of both cyclic nucleotide-dependent enzymes by 70-85% in the neostriatum, while cyclic GMP-dependent protein kinase was decreased by 44% and cyclic AMP-dependent protein kinase was decreased by 18% in the substantia nigra. In the basal ganglia, cyclic GMP-dependent protein kinase therefore appears enriched in striatonigral neurons, while cyclic AMP-dependent protein kinase is present both in striatonigral neurons and in other cells. The results indicate that cyclic GMP-regulated protein phosphorylation may play a role in the function of distinct basal ganglion neurons.

Abstract: The myelin-deficient (MD) rat does not express the major protein of CNS myelin, proteolipid protein (PLP). Here we further analyze whether this defect is reflected at the level of mRNA and whether the expression of other myelin proteins is affected in oligodendrocytes in vivo and in vitro. Both myelin basic protein (MBP) and PLP message levels were reduced in MD rats to 10–20% of the normal littermate controls, while the level of expression of an astrocyte-specific gene, glial fibrillary acidic protein (GFAP), was normal. Although MBP and PLP mRNAs were equally depressed, only MBP was detected with immunolabeling of corpus callosum, while PLP was absent in oligodendrocytes both in vivo and in vitro. A reduced number of MD rat oligodendrocytes express MBP in vitro compared to controls. The MD rat optic nerve contains normal numbers of 0–2A progenitors, but they tend to differentiate into GC-positive oligodendrocytes faster than oligodendrocytes from control littermates. In conclusion, the absence of PLP and reduced levels of MBP in the MD rats point to similarities with the jimpy mouse lesion. Moreover, the defect influences the expression of other myelin proteins and the oligodendrocyte developmental pathway.

Abstract: Recently, a subset of fucosicosis patients was identified in which the single EcoRI site in the open reading frame of the human cDNA encoding α-l-fucosidase was obliterated. We have employed the polymerase chain reaction technique to amplify α-l-fucosidase DNA from the five patients known to carry the EcoRI abnormality as well as four patients and two additional fucosidosis patients who do not carry the EcoRI abnormality. Sequence analysis of the amplified DNA has determined that the EcoRI site was destroyed by a C-T transition in the last position of the EcoRI site. This single base change results in the generation of a stop codon 120 base pairs upstream of the normal stop codon. In addition, we have determined that EcoRI cleavage of amplified DNA may be a useful diagnostic tool in the diagnosis of heterozygotes and in prenatal diagnosis of fetuses at risk for this disease.

Abstract: Mixed oligonucleotide primers complementary to the translation product of the sphingolipid activator protein (SAP)-2 were used to generate a 144-base pair (bp) complementary DNA (cDNA). This cDNA probe was used to isolate a 2,649-nucleotide-long cDNA that was sequenced and found to contain coding sequences for two known activators of lysosomal enzymes, namely, the sphingolipid activator protein (SAP)-1 and SAP-2. The cDNA contains an open reading frame of 1,482 nucleotides and 1,167 nucleotides of 3'-nontranslated region, followed by a stretch of 24 residues of adenylic acid. At 20 nucleotides upstream from the poly(A) tail there is a consensus AATAAA polyadenylation signal that is preceded by another potential polyadenylation signal. The cDNA, designed SAP-1/SAP-2 cDNA, hybridizes with two human mRNA species of approximately 3 kb in length, which most probably arise from polyadenylation at different sites. There are higher amounts of steady-state RNA levels of the SAP-1/SAP-2 mRNA in skin fibroblasts in comparison to B cells. The steady-state SAP-1/SAP-2 mRNA levels in Gaucher B cells are higher than in their normal counterparts. There is one human SAP-1/SAP-2 gene that has been cloned and is localized on two approximately 5 kb BamHI fragments.

Abstract: The myelin-associated glycoprotein (MAG) and the brain 1B236 protein are 100-kDa glycoproteins containing 30% carbohydrate that exist in two developmentally regulated forms and are specific to the nervous system. Recent cDNA cloning experiments in several laboratories using primarily immunological means of identification have determined the complete primary sequence of a rat brain glycoprotein that seems to correspond to both MAG and 1B236, suggesting that these proteins are identical. However, MAG was previously considered to be an oligodendrocyte/myelin specific component in the CNS at all ages, whereas 1B236 was thought to be primarily a neuronal component in adult rats but synthesized by oligodendrocytes at the time of active myelination. The composite term 1B236/MAG was proposed to describe the molecule identified by the cDNAs. In order to explore further the relationship between MAG and 1B236, as well as their developmentally regulated forms, experiments were carried out on rat samples utilizing synthetic peptides corresponding to sequences throughout the 1B236 molecule, antisera raised to synthetic peptides in the C-terminus of 1B236 that distinguish between the two developmentally regulated forms, and well-characterized polyclonal and monoclonal antibodies raised to purified MAG. Epitope mapping demonstrated that reactive sites were distributed throughout the extracellular and intracellular domains of 1B236/MAG. Only antibodies reacting with the smaller of the two forms of 1B236/MAG detected the glycoprotein in the peripheral nervous system. Both anti-MAG and anti-1B236 antibodies revealed a drastic reduction of the level of 1B236/MAG in 25-day-old myelin-deficient rats and in adult quaking mice, and both types of antibodies revealed a slight shift of 1B236/MAG toward higher apparent Mr in quaking mice as had previously been reported for MAG. The results indicate that MAG and 1B236 are almost certainly identical since they cannot be distinguished immunologically by the reagents available and that quantitatively most of the glycoprotein is associated with oligodendrocytes and myelin rather than neurons at all ages.

Abstract: Expression of mRNAs for the muscarinic acetylcholine receptor subtypes in various brain regions and in the heart of male rats was examined during postnatal development. The mRNAs for the four subtypes displayed different developmental patterns in the different regions, depending both on the cell type composition and on the age of the brain region examined.

Abstract: Bovine chromogranin A (CGA) was purified by three steps of column chromatography to a single-band purity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antibody against this preparation was purified by a CGA-coupled Sepharose column, and F(ab')2 and Fab' of the antibody IgG were prepared by enzymatic digestion. An enzyme immunoassay (EIA) system was developed with the F(ab')2 immobilized on polystyrene balls and the Fab' labeled with beta-D-galactosidase. The EIA was able to detect 1 pg of CGA and was three to four orders more sensitive compared with any radioimmunoassay systems hitherto reported. Several neural acidic proteins (dopamine beta-hydroxylase, neuron specific enolase, S-100a protein, and brain-type creatine kinase) showed no cross-reaction. Using this EIA, CGA was detected in all regions of bovine central nervous system. CGA concentrations were within a narrow range, being lowest in the cerebellar white matter and highest in the putamen (17.3 and 78.1 ng/mg protein, respectively). The concentrations were extremely low compared to the concentration in the adrenal medulla (205,000 ng/mg protein). The highly sensitive EIA system should be useful for studies of materials containing very small amounts of CGA.

Abstract: Protein phosphorylation systems regulated by cyclic adenosine 3',5'-monophosphate (cyclic AMP), or calcium in conjunction with calmodulin or phospholipid/diacylglycerol, have been studied by phosphorylation in vitro of particulate and soluble fractions from human postmortem brain samples. One-dimensional or two-dimensional gel electrophoretic protein separations were used for analysis. Protein phosphorylation catalyzed by cyclic AMP-dependent protein kinase was found to be highly active in both particulate and soluble preparations throughout the human CNS, with groups of both widely distributed and region-specific substrates being observed in different brain nuclei. Dopamine-innervated parts of the basal ganglia and cerebral cortex contained the phosphoproteins previously observed in rodent basal ganglia. In contrast, calcium/phospholipid-dependent and calcium/calmodulin-dependent protein phosphorylation systems were less prominent in human postmortem brain than in rodent brain, and only a few widely distributed substrates for these protein kinases were found. Protein staining indicated that postmortem proteolysis, particularly of high-molecular-mass proteins, was prominent in deeply located, subcortical regions in the human brain. Our results indicate that it is feasible to use human postmortem brain samples, when obtained under carefully controlled conditions, for qualitative studies on brain protein phosphorylation. Such studies should be of value in studies on human neurological and/or psychiatric disorders.

Abstract: Dopamine beta-hydroxylase exists as soluble and membrane-bound forms in secretory vesicles. The soluble form of the enzyme contains identical subunits of 72 kDa and the membrane-bound form contains two non-identical subunits of 72 kDa and 75 kDa. The difference in the banding pattern on sodium dodecyl sulfate-poly-acrylamide gel electrophoresis suggests the presence of either an extra peptide, or membrane-binding segment, or differential glycosylation of 75-kDa subunits of the membranous form. Soluble and membranous forms of the enzyme were deglycosylated with endoglycosidases to elucidate the contribution of the carbohydrate moieties to the banding pattern on a sodium dodecyl sulfate-poly-acrylamide gel. The deglycosylated species of both forms appeared to be identical and showed a decrease in apparent molecular weights to 66 kDa. These results indicate that the banding pattern of soluble and membranous dopamine beta-hydroxylase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis may not be due to a membrane-binding anchor but rather to carbohydrate moieties.

Abstract: By employing a highly sensitive immunoassay method, concentration of the α subunit of GTP-binding protein, Go (Goα), recently shown to be localized mainly in nervous tissues and neuroendocrine cells, was determined in cerebrospinal fluids (CSF) of 192 patients with various neurological disorders and 50 control subjects The results were compared with CSF levels of neuronspecific enolase (NSE) and S-100b protein (S-100b) in the same samples Normal levels of Goα were 519 ± 217 pg/ml The levels of Goα, as well as NSE and S-100b, in CSF were enhanced in some patients with acute conditions, eg, meningitis (48%), encephalitis (100%), and cerebral infarct (56%) In these disorders, cases with enhanced Goα levels were more frequent than those with enhanced NSE or S-100b Three patients with encephalitis whose Goα levels were more than 1000 pg/ml all died; the remaining two patients with encephalitis and slightly elevated Goα levels had a good prognosis Concentration of Goα in CSF correlated well with that of NSE but poorly with that of S-100b However, cervical spondylosis and demyelinating diseases, CSF levels of Goα were generally lower than those of NSE or S-100b These results suggest that Goα in CSF is a useful marker for monitoring patients with acute neuronal damage Since these three proteins are distributed differently in the central nervous system, simultaneous determination of Goα, NSE, and S-100b levels in CSF might provide valuable information about the pathophysiology of neurological disorders

Abstract: Huntington’s disease is generally considered to be a late-onset neurodegenerative disorder, which follows a protracted course of deteriorating motor control and cognitive impairment. However, in a minority of cases, the onset of symptoms occurs early in life. A preponderance of the juvenile-onset HD victims have inherited the genetic defect from their fathers. This variation in age of onset, based on the sex of the affected parent, has suggested that maternally inherited genes may influence expression of the disorder. We describe a portion of a large Venezuelan HD pedigree in which both the mother and father of three juvenile-onset HD patients share a common maternal lineage. Scanning of mtDNA from members of this family with 43 restriction endonucleases failed to reveal any differences in the mitochondrial genotype that could account for the difference in age of onset between the affected father and his progeny. Members of a related family with an affected father but no juvenile-onset progeny also appeared to share the same mitochondrial genotype. In addition, the mitochondrial gene products from lymphoblast cell lines of these family members were analyzed on polyacrylamide gels after incubation of cells with [35S]methionine, but no detectable alterations were seen. Taken together, these data suggest that the maternally inherited mitochondrial genome does not play a crucial role in determining in age of onset in HD.

Abstract: Protein methyltransferases, rich in most mammalian brains, were studied in human cerebrospinal fluid (CSF). Among several well-characterized groups of methyltransferases, protein methylase I (S-adenosyl-methionine:protein-arginineN-methyltransferase, EC 2.1.1.23) was found in significant amounts in human CSF samples. Both myelin basic protein (MBP)-specific and histone-specific protein methylase I activities were observed, the latter being generally higher in most CSF.S-Adenosyl-l-homocysteine, a potent product inhibitor for the methyltransferase, inhibited approximately 90% of MBP-specific protein methylase I activity at a concentration of 1 mM. The optimum pH of the MBP-specific protein methylase I was found to be around 7.2. Identity of exogenously added MBP as the methylated substrate for CSF enzyme was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An amino acid analysis of the [methyl-3H]protein hydrolysate showed two major radioactive peaks cochromatographing with monomethyl- and dimethyl (symmetric)-arginine. Human CSF contained relatively high endogenous protein methylase I activity (activity measured without added substrate protein): The endogenous substrate can be immunoprecipitated by antibody raised against calf brain MBP. Finally, CSF from several neurological patients were analyzed for protein methylase I, and the results are presented.

Abstract: The malignant transformation of congenital nevocellular nevi, both large and small, is controversial and presents problems in management. The size of the lesion is taken to indicate potential malignant transformation, but this is an arbitrary scale. A more reliable biological indicator is needed to help predict the lesions at risk. Following the localization of neuron-specific enolase to most cells of the diffuse neuroendocrine system and their neoplasms (including benign and malignant melanocytic lesions), it has been suggested that its level is related to tumor activity. In a prospective trial, the presence of neuron-specific enolase immunoreactivity, its concentration, and gene expression in nevus cells were studied in 31 congenital melanocytic nevi of various sizes (1.5 cm to bathing trunk) using immunocytochemistry, biochemical assay, and in situ hybridization. Twenty-five of the 31 congenital nevi were immunoreactive to neuron-specific enolase antiserum, with stronger immunostaining in the larger lesions. There is an apparent linear relationship between the size of the nevi and the level of neuron-specific enolase (expressed as nanograms per milligram protein). Neuron-specific enolase mRNA was highly expresed in most of large congenital nevi (>15 cm in diameter), as revealed by autoradiography following in situ hybridization. Our results show that neuron-specific enolase and its mRNA are expressed to a greater extent in large congenital nevi compared with smaller lesions. This might prove to be a useful indicator of those lesions at risk of malignant transformation.

Abstract: Soluble calmodulin-stimulated protein kinase II has been purified from 2-day and adult chicken forebrain At both ages the holoenzyme eluted from a Superose-6B column with an apparent molecular weight of approximately 700,000 daltons and contained three subunits. The subunits were found to be the counterparts of the alpha, beta, and beta subunits of the enzyme purified from adult rat brain in that they had one-dimensional phosphopeptide maps that were indistinguishable from those of the corresponding subunit in the rat enzyme and they migrated in SDS-polyacrylamide gels with the same apparent molecular weights. However, the doublet formed by the beta subunit was much more clearly resolved in the chicken enzyme and the beta subunit, which was much more abundant in the adult chicken than in the adult rat, was also found to be a doublet. The ratio of the concentrations of the alpha and beta subunits changed during development. By autoradiography following autophosphorylation, the alpha:beta ratios of the 2-day and adult enzymes were 0.89±0.07 and 1.92±0.26, reapectively; by silver staining the alpha:beta ratios were 0.95±0.11 and 1.85±0.17, respectively. The concentration of the beta subunit was equal to that of the beta subunit at both ages. Autophosphorylation produced a decrease in the electrophoretic mobility of the alpha and beta subunits in SDS-polyacrylamide gels and a marked decrease in the calcium dependence of the substrate phosphorylation activity of the enzyme at both ages. The purified enzyme from chicken brain appeared to be more stable under standard in vitro assay conditions than the rat enzyme, and this was particularly so for the enzyme from 2-day forebrain.

Abstract: The participation of gonadal steroid hormones in regulation of the vasoactive intestinal peptide (VIP) gene expression in the hypothalamus was studied using a quantitative densitometric hybridization assay. In the female rat the levels of VIP mRNA were found to be significantly decreased following ovariectomy (4.41±0.7 arbitrary units of absorbance vs. 8.52±0.18). This decrease was largely reversed after three days of treatment with estradiol dibenzoate. In contrast to the female rats, no significant change in VIP mRNA levels was observed in the male rats, following orchidectomy. These results suggest a sexual dimorphism with regard to the steroid regulation of hypothalamic VIP gene expression in the rat.

Abstract: [3H]Inositol 1,4,5-trisphosphate [( 3H]Ins-(1,4,5)P3) binding studies were done on the human brain obtained at autopsy. The specific [3H]Ins(1,3,4,5)P3 binding sites in the cerebral and cerebellar cortices consisted of a single component with a high affinity (Kd = 11.3 and 16.5 nM, Bmax = 0.8 and 6.4 pmol/mg protein, respectively). The binding of [3H]Ins(1,4,5)P3 was potently inhibited by Ins(1,4,5)P3, in a nanomolar concentration, while other inositol phosphates and inositol were either much less potent or did not inhibit binding. The binding sites for [3H]Ins(1,4,5)P3 were discretely localized and were in the order: cerebellum much greater than basal ganglia, cerebral cortex greater than rhinencephalon greater than diencephalon, mesencephalon. There was an age-related loss of [3H]Ins(1,4,5)P3 binding in the frontal cortex. In the brains of patients with Parkinson's disease, [3H]Ins(1,4,5)P3 binding sites were reduced by about 50% in the caudate nucleus, putamen, and pallidum, while there were no differences in the frontal cortex, as compared to findings in the age-matched controls. Our findings suggest that [3H]Ins(1,4,5)P3 binding sites are closely linked to neural elements in the human brain.