bluuebunny/crossref_metadata_2025 huggingface dataset:
58,000 Dalton Intermediate Filament Proteins of Neuronal and Nonneuronal Origin in the Goldfish Visual Pathway
Authors: Quitschke Wolfgang , Schechter Nisson
Published: October 2006

Abstract: A group of proteins in the goldfish optic nerve with a molecular weight of 58K daltons was analyzed by two‐dimensional gel electrophoresis. Results show that the proteins are differentially phosphorylated and found exclusively in a cytoskeletal‐enriched fraction. The proteins from this fraction can be reconstituted into typical intermediate filament structures, as shown by electron microscopy. Two components which are of neuronal origin are transported within the slow phase of transport. The 58K proteins are the most abundant proteins in the optic nerve, and they are distinct from actin and tubulin. It was concluded that they are intermediate filament proteins. Cytoskeletal preparations of rat spinal cord, rat optic nerve, and goldfish optic nerve were compared by one‐dimensional gel electrophoresis. The rat spinal cord contains glial fibrillary acidic protein (GFAP), and the rat optic nerve contains vimentin and GFAP, in addition to the neurofilament triplet. A typical mammalian neurofilament triplet is not detected in the goldfish optic nerve, while the major cytoskeletal constituent is a 58K band which coelectrophoreses with vimentin in the rat optic nerve by one‐dimensional gel electrophoresis.
Amino Acids in Plasma and CSF and Monoamine Metabolites in CSF: Interrelationship in Healthy Subjects
Authors: Hagenfeldt L. , Bjerkenstedt L. , Edman G. , Sedvall G. , Wiesel F.‐A.
Published: October 2006

Abstract: Plasma and cerebrospinal fluid (CSF) concentrations of amino acids were measured in 65 healthy volunteers (50 men and 15 women). The CSF levels of the monoamine metabolites homovanillic acid (HVA), 3‐methoxy‐4‐hydroxyphenylethylene glycol (MOPEG), and 5‐hydroxyindoleacetic acid (5‐HIAA) were also determined. Sex differences were observed in both plasma and CSF amino acid levels as well as in the relationship between these concentrations. No significant correlations were observed between the CSF levels of HVA and 5‐HIAA, and the concentrations of their precursor amino acids in either plasma or CSF. The MOPEG level in CSF correlated positively with the plasma concentrations of several amino acids.
Release of Aspartate and Glutamate Caused by Chloride Reduction in Synaptosomal Incubation Media
Authors: Hardy J. A. , Boakes R. J. , Thomas D. J. E. , Kidd A. M. , Edwardson J. A. , Virmani M. , Turner J. , Dodd P. R.
Published: October 2006

Abstract: Release of the amino acid neurotransmitter candidates aspartate and glutamate from synaptosomes can be stimulated by reduction of [Cl−] in the incubation medium by replacement with the impermeant anions pro‐pionate and isethionate. Replacement by the permeant anion Br− is without effect on amino acid release. This release offers a biochemical method of studying the Cl−ion channel in CNS membrane. Key Words: Synaptosomes — Chloride — Transmitters — Release — Amino acid—Permeability. Hardy J. A. et al. Release of aspartate and glutamate caused by chloride reduction in synaptosomal incubation media.
Dialytrode Technology and Local Profiles of Amino Acids in the Awake Cat Brain
Authors: Delgado J. M. R. , Lerma J. , del Río R. Martín , Solís J. M.
Published: October 2006

Abstract: Methods to investigate the in vivo effects and release of neuroactive substances include cortical cups, push‐pull cannulae, chemitrodes, and dialytrodes. Critical evaluation of these procedures is necessary in order to interpret related results and to select the most suitable devices for further studies. Recent improvements in the dialytrode include structural modifications and the use of a small, permeable membrane constructed of thin polyester. The dialytrode system and its diffusion rates have been characterized with in vitro studies. In vivo long‐term experiments in awake cats have been conducted to test injection rates, diffusion of [14C]urea, temporal variability, pressure factors, and other experimental variables. Using dialytrodes we have measured the normal profile of amino acids present in different cerebral structures and their possible correlations.
Changes in the Expression of a Neuronal Surface Protein During Development of Cerebellar Neurones In Vivo and in Culture
Authors: Meier Eddi , Regan Ciaran M. , Balazs Robert
Published: October 2006

Abstract: The expression of the neurone‐specific D2 protein changes both quantitatively and qualitatively during development in vivo and in cultures of cerebellar nerve cells. The total D2 content per unit protein shows a twofold increase in vivo from birth to postnatal day 6, after which it declines progressively to about 50% of the maximal value. This increase can be accounted for by an immature form of the protein anodic D2 being preferentially expressed at the early stages of cerebellar development. After postnatal day 9 this form gradually switches to a mature form cathodic D2. This switch can be mimicked by neuraminidase treatment, suggesting a developmental loss of sialic acid from the D2 protein. In freshly isolated cells the total D2 content per unit protein is only 30% of that in the corresponding intact tissue from 8‐day‐old cerebella, but it increases rapidly during the first 8 days of culture to levels similar to those of the equivalent age in vivo. The switch from anodic D2 to cathodic D2 also occurs at a faster rate in culture, probably reflecting the culture conditions that favour differentiation. The changes in the expression of D2 during development of cerebellar nerve cells in culture suggest that anodic D2 is preferentially expressed on nerve cells that are proliferating, migrating, or in the initial stages of differentiation, whereas cathodic D2 is associated with differentiated neurones. The transition between the two forms appears to occur during the formation of interneuronal contacts.
Resistance to Disruption of Multilamellar Fragments of Central Nervous System Myelin
Authors: Sedzik Jan , Toews Arrel D. , Blaurock Allen E. , Morell Pierre
Published: October 2006

Abstract: Single‐bilayer vesicles of myelin are desirable for studying myelin development and metabolism. Accordingly, our interest was drawn to a procedure for ves‐iculating myelin (Steck et al., Biochim. Biophys. Acta509, 397–408, 1978). We used X‐ray diffraction analysis to examine these putative vesicle preparations because much larger amounts of material can be surveyed by this method than by electron microscopy. The sharpness (width) of the rings in the X‐ray diffraction pattern varies inversely with the number of bilayers per multilayer structure. We therefore expected to see the diffuse diffraction pattern characteristic of single bilayers. Diffraction patterns were recorded from isolated rat brain myelin before and after the vesiculation procedure. Both patterns showed sharp rings, indicating numerous multilayered structures. Average values ranging from 7 to 10 bilayers per multilayer were calculated in both cases. This procedure did produce a small fraction of single‐bilayer structures, which were isolated by differential centnfu gation; however, these accounted for only about 1% of the total myelin present. The diffraction pattern of this material showed the diffuse band typical of single‐bilayer structures, and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis indicated it had the same protein composition as in normal myelin. Similar results were also obtained using either fresh or frozen bovine brain myelin. Variations of the published vesiculation procedure (incubation in 0.1 M NaCl or in buffers containing glycerol; disruption by sonication or use of a Tissumizer) also were not effective in breaking down the multilamellar fragments into thinner structures. We conclude that the multilamellar fragments of isolated CNS myelin resist disruption into single‐bilayer structures.
Decrease in N‐Acetyl‐l‐Aspartic Acid in Brain of Myodystrophic Mice
Authors: Marcucci Franca , Colombo Laura , De Ponte Gloria , Mussini Emilio
Published: October 2006

Abstract: Brain tissue from myodystrophic mice (male 129/ReJ‐dy and female 129 B6F1/J‐dy) was examined to determine whether CNS abnormalities accompany the known muscular defects. Brain N‐acetyl‐l‐aspartate, aspartate, and glutamate were significantly lower than in brain from control mice. These amino acids are only slightly reduced in brains of female dystrophic mice.
Blood‐Brain Transfer of Galactose in Experimental Galactosemia, with Special Reference to the Competitive Interaction Between Galactose and Glucose
Authors: Gjedde Albert
Published: October 2006

The interaction between glucose and galactose during transport across the cerebral capillary endothehum was studied in anesthetized rats. Although galactose is present in the diet of suckling mammals and is a potential substrate for brain metabolism in adult mammals, its effect on glucose transport in adult rats is unknown. A kinetic model was formulated to analyze the effect of chronically elevated galactose levels on glucose transport in adult rats. The analysis indicated that galactose and glucose compete for the same transport mechanism in the cerebral capillary endothelium. The Tmax of glucose and galactose were both about 380 μmol 100 g‐1 min‐1 and the Kt of galactose (30 mM) was about three times that of glucose (10 mM). During prolonged galactosemia in adult rats, neither the Tmax, nor the Kt of either competitor changed substantially when compared with rats subjected to acute galactosemia. At 10 mM galactose in plasma in rats with acute galactosemia, the inhibition of glucose transport, simulated a 25% reduction of plasma glucose, and in rats with chronic galactosemia a 20% reduction. This moderate effect is in contrast to the effect of galactose in suckling rats in which 10 mM galactose in plasma reduced the glucose transport to a level corresponding to a 50% reduction of the plasma glucose concentration.
Tubulin‐Associated Calmodulin‐Dependent Kinase: Evidence for an Endogenous Complex of Tubulin with a Calcium‐Calmodulin‐Dependent Kinase
Authors: Goldenring James R. , Casanova James E. , DeLorenzo Robert J.
Published: October 2006

A Ca2+‐calmodulin kinase that phosphorylates tubulin and microtubule‐associated proteins as major substrates has been purified and characterized from brain cytoplasm. It is important to determine if cytoskeletal proteins are major natural substrates for this kinase system. This report demonstrates that a significant fraction of brain cytosolic calmodulin‐dependent kinase activity exists in tight association with tubulin in the form of a stable complex. The tubulin‐calmodulin kinase complex displayed an apparent molecular weight on gel filtration of approximately 1.8 × 106 daltons. The specific activity of tubulin kinase in the complex was enriched over 20‐fold in comparison with brain cytosol. Although purified tubulin alone did not adhere to a calmodulin column, the tubulin associated with the calmodulin kinase complex did bind specifically to the calmodulin affinity resin. The kinase activity was shown to be tightly associated in complex with tubulin by (1) copurification, (2) isolation on gel filtration chromatography, (3) isolation on ion‐exchange chromatography, and (4) binding to calmodulin. The kinase complexed with tubulin was identical to the previously purified kinase as judged by several criteria including (1) subunit molecular weights, (2) isoelectric points, (3) autophosphorylation characteristics, (4) calmodulin binding properties, (5) kinetic parameters of tubulin phosphorylation, (6) phosphoamino acid phosphorylation sites on α‐and β‐tubulin, and (7) identical subunit 125I‐tryptic peptide maps. The results indicate that a significant fraction of this previously purified calmodulin kinase is endogenously associated with tubulin in brain cytoplasm and may play a role in mediating some of the effects of calcium on neuronal function.
Parvalbumin, a Neuronal Protein in Brain Cell Cultures
Authors: Pfyffer Gaby E. , Bologa Liane , Herschkowitz Norbert , Heizmann Claus W.
Published: October 2006

Abstract: Dissociated brain cell cultures were derived from 14‐day‐old embryonic as well as from newborn mice. The cells were grown in a medium containing 10% fetal calf serum. Indirect immunofluorescence was performed using antisera directed against the Ca + ‐binding protein parvalbumin (M., 12,000). In embryonic cultures a large proportion of cells was intensely stained by anti‐ parvalbumin. In double‐labelling experiments involving the simultaneous application of antisera against parvalbumin and the neuron‐specific enolase, the enolase‐con‐ taining cells were also parvalbumin‐positive and both antisera revealed identical intracellular staining patterns. Conversely, almost no parvalbumin‐ and enolase‐positive cells were present in cultures derived from newbornmice. However, in these cultures many cells were immunoreactive toward the myelin basic protein, an accepted marker for oligodendrocytes. The presence of parvalbumin within the embryonic brain cell cultures was confirmed by analyses of the culture extracts (4 mM EDTA, pH 7.5) by HPLC on reverse‐phase supports, two‐dimensional polyacrylamide gel electrophoresis, and immunoblotting. The present study suggests that in mouse brain cell cultures, parvalbumin is localized in neurons.
Monoclonal Antibodies to Gel‐Excised Glial Filament Protein and Their Reactivities with Other Intermediate Filament Proteins
Authors: Lee V. M.‐Y. , Page C. D. , Wu H.‐L. , Schlaepfer W. W.
Published: October 2006

Abstract: A series of 14 monoclonal antibodies (MAs) has been obtained from a single rat‐mouse fusion using gel‐excised bovine glial filament (GF) proteins as immunogens. These MAs were characterized by two separate immunochemical assays and by two different immunohistochemical methods. Nine MAs demonstrated specificity for GF proteins. One MA also recognized an epitope shared by intermediate filaments (IF) of the vimentin class (VF). Using the enzyme‐linked immunosorbent assay, four of the MAs recognized 200,000, 150,000, and 51,000 dalton proteins, suggesting that these MAs were specific for GF proteins (the 51,000 dalton protein) and neurofilament (NF) proteins (the two high‐molecular‐weight proteins). However, in both of the immunohistochemical assay systems, these MAs stained neurons and their processes but not astroglial cells. These observations strongly suggest that the 51,000–dalton protein recognized by these four MAs was not derived from GF proteins but instead represents derivatives of NF protein subunits comigrating in gels with GF proteins. These data provide additional information concerning the unique and shared antigenic determinants of the three classes of IF (NF, GF, and VF) of the CNS. In addition, they draw attention to the fact that proteins of certain IF may undergo degradation and comigrate in gels with the proteins of unrelated IF. This emphasizes the need for the use of independent immunochemical and immunohistochemical assays in the characterization of the specificity of MAs.
Effects of Dichloroacetate on Brain Pyruvate Dehydrogenase
Authors: Abemayor Elliot , Kovachich Gyula B. , Haugaard Niels
Published: October 2006

Abstract: The action of dichloroacetate (DCA) on pyruvate dehydrogenase (PDH) activity of rat brain has been studied in vitro and in vivo. In a crude brain mitochondrial fraction, DCA inhibits PDH kinase and in rat brain slices this compound increases PDH activity and stimulates glucose oxidation. In the whole animal, intraperitoneal injection of DCA causes activation of brain PDH, indicating that this inhibitor crosses the blood‐brain barrier. The same treatment with DCA also produced a large increase in heart PDH activity. Further studies of the effects of DCA on the CNS should lead to results of considerable importance.
Isolation and Characterization of an Unsaturated Fatty Acid‐Binding Protein from Developing Chick Neural Retina
Authors: Lee L. , Wiggert B.
Published: October 2006

Abstract: An unsaturated fatty acid‐binding protein has been isolated from the cytosol fraction of developing chick neural retina. It has a molecular weight of approximately 14,800 and specifically binds not only added radiolabeled arachidonic and oleic acids but has also been found to bind unsaturated fatty acids endogenously. This protein was detected in chick neural retina at all stages examined, from 8 to 16 days of development. It is also present in chick heart, brain, and retinal pigmented epithelium‐choroid as well as in adult bovine neural retina. It is distinct from both cellular retinol‐binding protein and cellular retinoic acid‐binding protein on the bases of binding specificity and isoelectric point.
Characterization of Human Brain Phenolsulphotransferase
Authors: Rein G. , Glover Vivette , Sandier M.
Published: October 2006

Abstract: Both human phenolsulphotransferase M (for monoamines) and P (for phenol) were detected in eight out of twelve brains examined postmortem. Activity values were low compared with those in other human tissues and in brains from other species. The activity of both forms was unevenly distributed in different brain regions in a pattern different from that of the mono amines. From a study of substrate specificity, Km values, and inhibitor sensitivity, the two forms of the human brain enzyme did not appear to differ from their counterparts in platelet.
Insulin Binding in Four Regions of the Developing Rat Brain
Authors: Kappy Michael , Sellinger Scott , Raizada Mohan
Published: October 2006

Abstract: Specific insulin binding has been demonstrated in partially purified membranes prepared from four regions of the developing rat brain. Insulin binding to brain membranes demonstrated kinetics and hormonal specificity that were quite similar to those reported for traditional insulin target tissues (e.g., liver and adipose tissue), and binding was significantly correlated with receptor concentration. Binding in the olfactory bulbs, cerebrum, cerebellum, and hypothalamus all reached highest values at 15 days of postnatal life, with the olfactory bulbs generally showing the greatest binding at all ages studied. A temporal relationship was found between insulin binding to brain membranes in the postnatal rat and plasma membrane protein synthesis, especially in the cerebellum and olfactory bulbs.
Triazolam, an Anomalous Benzodiazepine Receptor Ligand: In Vitro Characterization of Alprazolam and Triazolam Binding
Authors: Clow Angela , Glover Vivette , Sandler Merton
Published: October 2006

Abstract: Both alprazolam and triazolam displaced clonazepam (but not Ro 5–4864) from rat brain membranes with high affinity, showing them to act at central but not peripheral benzodiazepine receptors. At 0°C, 10 μMγ‐aminobutyric acid (GABA) increased the ability of alprazolam, but not of triazolam, to displace ethyl‐β‐carboline‐3‐carboxylate (β‐CCE) and Ro 15–1788 from these receptors. At 37°C, GABA increased the affinity of the receptors for both drugs, with a +GABA/–GABA ratio of 1.5 for each in promoting Ro 15–1788 binding displacement. As both triazolam and alprazolam act as anxiolytics in vivo, the results at 37°C would be compatible with the hypothesis that GABA causes an increase in affinity of drugs that act in this way, but the results at 0°C would not be compatible. At 37°C, alprazolam had a higher IC50 for the benzodiazepine receptor than at 0°C, whereas triazolam showed the reverse effect. The relative IC50 values in vitro at 37°C correlated better with the potency in vivo than those obtained at 0°C. At 0°C, both drugs showed Hill plots with slopes of 0.9–1 with β‐CCE and Ro 15–1788. At 37°C, the slopes with triazolam were much reduced, indicating that the drug may have a selective action on a subclass of central benzodiazepine receptors. In the studies reported here, alprazolam behaved like other benzodiazepines, whereas triazolam showed several anomalous properties. It would be of interest if these properties could be related either to the drug's use as a hypnotic or to the side effects it sometimes induces.
Inhibition of the Electrically Evoked Release of [3H]Acetylcholine in Rat Striatal Slices: An Experimental Model for Drugs that Enhance Dopaminergic Neurotransmission
Authors: Baud P. , Arbilla S. , Langer S. Z.
Published: October 2006

Abstract: The activation by endogenous dopamine of the inhibitory 3,4‐dihydroxyphenylethylamine (dopamine) receptors modulating the electrically evoked release of [3H]acetylcholine ([3H]ACh) and [3H]dopamine in rat striatal slices is a function of the concentration of dopamine accumulated in the synaptic cleft during electrical stimulation. When the release of 3H‐neurotransmitters was elicited with a 2‐min period of stimulation at a frequency of 1 Hz, neither dopamine autoreceptors nor dopamine receptors modulating [3H]ACh were activated by endogenously released dopamine. On the other hand, exposure to (S)‐sulpiride facilitated the release of [3H]dopamine and [3H]ACh elicited when the 2‐min stimulation was carried out at a frequency of 3 Hz but this effect was not observed at a lower frequency of stimulation (1 Hz). In the presence of amphetamine the dopamine receptors modulating the electrically evoked release of [3H]ACh can be activated by endogenous dopamine even at the lower frequency of stimulation (1 Hz). Similar effects can be obtained if the neuronal uptake of dopamine is inhibited by cocaine or nomifensine. The inhibition by amphetamine of the release of [3H]ACh elicited by electrical stimulation at 1 Hz involves dopamine receptors and can be fully antagonized by clozapine, haloperidol, chlorpromazine, or pimozide. The stereoselectivity of this antagonism can be demonstrated with the optical enantiomers of sulpiride and butaclamol. This inhibitory effect of amphetamine on cholinergic neurotransmission appears to be the result of the stimulation of dopamine receptors of the D2 subtype, as they were resistant to blockade by the preferential D1 receptor antagonist SCH 23390. Inhibition of dopamine synthesis with α‐methyl‐p‐tyrosine antagonized the effects of amphetamine on [3H]ACh release. The dopamine receptor‐mediated inhibition of the release of [3H]ACh elicited at low frequencies of stimulation from rat striatal slices is a suitable model to test drugs that enhance dopaminergic neurotransmission. In addition, the antagonism of the inhibition of cholinergic neurotransmission by amphetamine in striatal slices represents a useful test for drugs with potential usefulness in clinical situations in which dopaminergic neurotransmission is exacerbated.
Regulation of Myelination: Schwann Cell Transition from a Myelin‐Maintaining State to a Quiescent State After Permanent Nerve Transection
Authors: Poduslo Joseph F. , Dyck Peter J. , Berg Carole T.
Published: October 2006

Abstract: Permanent nerve transection of the adult rat sciatic nerve forces Schwann cells in the distal nerve segment from a myelin‐maintaining to a quiescent state. This transition was followed by serial morphometric evaluation of the percentage fascicular area having myelin (myelin percent of area) in transverse sections of the distal nerve segment and revealed a rapid decline from a normal value of 36.6% to 3.2% by 14 days for the sciatic nerve to < 1.0% throughout the remaining time course (up to 105 days). No evidence of axonal reentry into the distal nerve segment or new myelin formation was observed at times under 70 days. In some of the distal nerve segments at 70, 90, and 105 days, new myelinated fibers were observed that usually consisted of only a few myelinated fibers at the periphery and in the worst case amounted to 1.6% (myelin percent of area). Radioactive precursor incorporation of [3H]mannose into endoneurial slices at 4 and 7 days after transection revealed two species of the major myelin glycoprotein, P0, with Mr of 28,500 and 27,700. By 14 days after nerve transection, only the 27,700 Mr species remained. Incorporation of [3H]mannose into the 27,700 Mr species increased progressively to 35 days after transection and then began to decline at 70 and 105 days. Alterations in the oligosaccharide structure of this down‐regulated myelin glycoprotein accounted for the progressive increase in mannose incorporation. Lectin affinity chromatography of pronase‐digested P0 glycopeptides on concanavalin A‐Sepharose revealed that the 28,500 Mr species of P0 had the complex‐type oligosaccharide as the predominant oligosaccharide structure (92%). In contrast, the high mannose‐type oligosaccharide was the predominate structure for the 27,700 Mr form, which increased to 70% of the total radioactivity by 35 days after nerve transection. Since the biosynthesis of the complex‐type oligosaccharide chains on glycoproteins involves high mannose‐type intermediates, the mechanism of down‐regulation in the biosynthesis of this major myelin glycoprotein, therefore, results in a biosynthetic switch from the complex‐type oligosaccharide structure as an end product to the predominantly high mannose‐type oligosaccharide structure as a biosynthetic intermediate. This biosynthetic switch occurs gradually between 7 and 14 days after nerve transection and likely reflects a decreased rate of processing through the Golgi apparatus. It remains to be determined if the high mannose‐type oligosaccharide chain on P0 can undergo additional processing steps in this permanent nerve transection model.
Dopamine‐β‐Hydroxylase: Structural Comparisons of Membrane‐Bound Versus Soluble Forms from Adrenal Medulla and Pheochromocytoma
Authors: Sokoloff Roger L. , Frigon Ronald P. , O'Connor Daniel T.
Published: October 2006

Abstract: Dopamine‐β‐hydroxylase (DBH) in membrane‐bound (mDBH) and water‐soluble (sDBH) forms was isolated from chromaffin granules of bovine adrenal medullae and a human pheochromocytoma tumor. sDBH was purified by concanavalin A‐agarose column chromatography followed by DEAE‐Sepharose column chromatography. The final bovine preparation had a specific activity of 16.27 IU/mg; the human preparation had a specific activity of 9.16 IU/mg. mDBH was isolated in enzymatically inactive form by preparative polyacrylamide gel electrophoresis. The proteins were subjected to amino acid analysis, as well as digestion with trypsin, followed by separation of the resulting peptides by twodimensional TLC/electrophoresis. No intraspecies differences between sDBH and mDBH were found from comparisons of amino acid composition or peptide maps. Thus the basis of the difference between sDBH and mDBH cannot easily be explained by differences in primary structure, within the resolution of these techniques.
An Immunoblot Study of Neurofilament Degradation In Situ and During Calcium‐Activated Proteolysis
Authors: Schlaepfer W. W. , Lee C. , Lee V. M.‐Y. , Zimmerman U.‐J. P.
Published: October 2006

Abstract: The degradation of neurofilament (NF) proteins was examined by immunoblot methods to identify, characterize, and monitor the appearance of immunoreactive breakdown products during the loss of NF triplet proteins. Individual NF proteins and their breakdown products were identified using polyclonal and monoclonal antibodies to NF proteins. NF degradation was compared during calcium‐activated proteolysis of isolated rat NF, during an experimental influx of calcium into excised rat spinal nerve roots, and during NF breakdown in transected rat peripheral nerve. These different experimental conditions produced similar patterns of NF fragmentation, including the transient appearance of NF immunobands between Mr 150,000–200,000 and 110,000–120,000 as well as the appearance and accumulation of NF immunobands between Mr 45,000 and 65,000. Most immunoreactive NF fragments remained Triton‐insoluble. Low levels of the same immunoreactive fragments were present in control neural tissues, suggesting that calciumactivated proteolysis may be operative in the turnover and/or processing of NF proteins in vivo. Very similar patterns of NF degradation during experimental calcium influxes into different CNS and PNS tissues are indicative of the widespread distribution of calcium‐activated NF protease in neural tissues.
Temporal Profiles of Proteins Responsive to Transient Ischemia
Authors: Dienel Gerald A. , Cruz Nancy F. , Rosenfeld Stephen J.
Published: October 2006

Abstract: The responses of long and short half‐lived proteins to ischemia were measured in rat brain during 6 days of recovery from 30 min of transient forebrain ischemia produced by four‐vessel occlusion. At the end of the ischemic interval, the neocortical activities of four vulnerable enzymes [ornithine (ODC) and S‐adenosylmethionine (SAMDC) decarboxylases, and RNA polymerases I and II] were unchanged, but within 30 min of reperfusion, their activities dropped by 25–50%. The loss of substance P in the striatum and substantia nigra was slower, reaching about 50% by 12 h. On the other hand, the activities of 5 long half‐lived enzymes did not change in the neocortex at 5 and 15 h of reperfusion and regional protein concentrations were essentially unaffected over 6 days survival. The rate and extent of normalization of the amounts or activities of the vulnerable proteins varied. RNA polymerase II and ODC activities were restored within 4 h, and ODC showed a biphasic increase in activity, with peaks at 10 h and 2–3 days. RNA polymerase I and SAMDC activities were restored by 18 h and 5 days, respectively, whereas substance P concentrations did not completely recover, even at 6–15 days. The greater the regional reduction of blood flow during ischemia, the larger the net change (gain or loss) of SAMDC or ODC activity and the longer the time required to normalize the activities of these enzymes. The average rate of proteolysis, assessed by measuring the rate of clearance of 14C from protein prelabeled with [14C]bicarbonate, was abnormal during the first 2 days of reperfusion. Postischemic changes in both protein synthesis and degradation could affect the amounts of some of the proteins responsive to transient ischemia.
Desipramine Binding: Relationship to Central and Sympathetic Noradrenergic Activity
Authors: Swann Alan C. , Duman Ron , Hewitt Lance
Published: October 2006

Abstract: We examined the effects of treatments affecting norepinephrine release on the number of norepinephrine reuptake recognition sites as reflected by desipramine binding. To do this, we used manipulations having similar presynaptic but contrasting postsynaptic effects. Presynaptic inhibition by 6‐hydroxydopamine lesion or by clonidine, and postsynaptic receptor stimulation by isoproterenol, reduced desipramine binding. Presynaptic stimulation by d‐amphetamine and postsynaptic receptor blockade by prazosin increased desipramine binding. Similar effects and binding properties were seen in cerebral cortex, heart, and soleus muscle. After unilateral noradrenergic lesions, reduction in desipramine binding correlated with reduction in norepinephrine uptake. These results show that norepinephrine reuptake appears to be regulated by transmitter release regardless of effects on postsynaptic transmission, and that this regulation is analogous in the central and sympathetic nervous systems.
Calcium‐ and Calmodulin‐Dependent Phosphorylation of Diphosphoinositide in Acetylcholine Receptor‐Rich Membranes from Electroplax of Narke japonica
Authors: Hayashi Fumio , Amakawa Taisaku
Published: October 2006

Abstract: The phosphoryiation of phosphoinositides in the acetylcholine receptor (AChR)‐rich membranes from the electroplax of the electric fish Narke japonica has been examined. When the AChR‐rich membranes were incubated with [γ‐32P]ATP, 32P was incorporated into only two inositol phospholipids, i.e., tri‐ and diphosphoinositide (TPI and DPI). Even after the alkali treatment of the membrane, AChR‐rich membranes still showed a considerable DPI kinase activity upon addition of exogenous DPI. It is likely that the 32P‐incorporation into these lipids was realized by the membrane‐bound DPI kinase and phosphatidyl inositol (PI) kinase. Such a membrane‐bound DPI kinase was activated by Ca2+ (>10−‐6M), whereas the PI kinase appeared to be inhibited by Ca2+. The effect of Ca2+ on the DPI phosphorylation was further enhanced by the addition of ubiquitous Ca2+‐depen‐dent regulator protein calmodulin. Calmodulin antagonists such as chlorpromazine (CPZ), trifluoperazine (TFP), and N‐(6‐aminohexyl)‐5‐chloro‐l‐naphthalenesul‐fonamide (W‐7) inhibited the phosphorylation of DPI in the AChR‐rich membranes. It is suggested that the small pool of TPI in the plasma membrane is replenished by such Ca2+– and calmodulin‐dependent DPI kinase responding to the change in the intracellular Ca2+ level.
An Ex Vivo Method for Evaluating Prostaglandin Synthetase Activity in Cortical Slices of Mouse Brain
Authors: Shohami E. , Gross J.
Published: October 2006

Abstract: The release of prostaglandin E2 (PGE2) from cortical slices of mice into incubation medium is followed for 3 h and compared to PGE2 levels in the corresponding slice. Immediately after decapitation, the rate of PGE2 released into the incubation medium is elevated and a steady low rate of spontaneous release is gained within 1–2 h of incubation. PGE2 synthesis and release is blocked in a dose‐dependent manner by either indometh‐acin (3 × 1(10−6‐ 3 × 10−4M) or flufenamic acid (2.6 × 10−6M) either when added in vitro or administered in vivo. Full recovery of PGE2 synthesis is reached after 3 h incubation of slices following in vivo administration, of indomethacin. In vivo administration of flufenamic acid results in prolonged inhibition of PGE2 released in vitro. The inhibition of PGE2 released by indomethacin is also correlated with the slice PGE2 content. Administration of lipopolysaccharide (LPS), a known activator of phospho‐lipase A2, results in a fivefold increase in PGE2 and a twofold increase in 6‐keto‐PGFlQ released into the medium. The release of thromboxane B2 is not affected by LPS.
A Comparison of the Evolutionary Distribution of the Two Neuroendocrine Markers, Neurone‐Specific Enolase and Protein Gene Product 9.5
Authors: Jackson Peter , Thomson Virginia M. , Thompson R. J.
Published: October 2006

Abstract: One‐ and two‐dimensional polyacrylamide gel electrophoresis followed by immunoblotting has been used to examine the phylogenetic distribution of the two neuronal and neuroendocrine proteins, neurone‐specific enolase and protein gene product 9.5, in animal brains. A new immunoblotting procedure was used in which complex two‐dimensional patterns of brain proteins were transferred to nitrocellulose paper simultaneously with the Coomassie Blue stain. This produced a copy of the blue spot pattern against which brown protein spots reacting in a specific antibody‐immunoperoxidase procedure could be identified unequivocally. Extracts of human, bovine, sheep, rabbit, rat, guinea‐pig, chicken, trout, and frog brains were examined. Proteins cross‐reacting with antisera to the human forms of both proteins could be demonstrated in all species examined. This suggests that proteins corresponding to neurone‐specific enolase and protein gene product 9.5 could have evolved at least 400 million years ago and have been highly conserved throughout evolution.
Effect of Hyperammonemia and Methionine Sulfoximine on the Kinetic Parameters of Blood‐Brain Transport of Leucine and Phenylalanine
Authors: Jonung Torbjorn , Rigotti Paolo , James J. Howard , Brackett Kim , Fischer Josef E.
Published: October 2006

Abstract: The activity of the blood‐brain neutral amino acid transport system is increased in rats infused with ammonium salts or rendered hyperammonemic by a portacaval anastomosis. This effect may be due to a direct action of ammonia or to some metabolic consequence of high ammonia levels, such as increased brain glutamine synthesis. To test these possibilities we evaluated the kinetic parameters of blood‐brain transport of leucine and phenylalanine in control rats, in rats after continuous 24 h infusion of ammonium salts (NH4+= 2.5 mmol. kg−1 h−1), and in rats treated with methionine sulfoximine, an inhibitor of glutamine synthetase, before infusion of ammonium salts. In ammonia‐infused rats without methionine sulfoximine treatment, the KD and Vmax of phenylalanine transport were increased, respectively, about 170% and 80% compared to controls, whereas the Km and Vmax of leucine transport were increased, respectively, about 100% and 200%. Electron microscopy demonstrated marked swelling of astrocytic processes around brain capillaries of ammonia‐infused rats; however, capillary permeability to horseradish peroxidase apparently was not increased by ammonia infusion. Administration of methionine sulfoximine before ammonia infusion inhibited glutamine synthesis and prevented the changes in transport of leucine and phenylalanine, but apparently did not reverse the perivascular swelling. These results suggest that the ammonia‐induced increase in the activity of transport of large neutral amino acids across the blood‐brain barrier requires glutamine synthesis in brain, and is not a direct effect of ammonia.
Enzymatic Regulation of Glycoprotein Synthesis in Peripheral Nervous System Myelin
Authors: Smith Marion Edmonds , Somera Florentino P. , Sims Terry J.
Published: October 2006

Abstract: The enzyme UDP‐N‐acetylglucosamine: dolichyl phosphate, N‐acetylglucosamine‐1‐phosphate transferase initiates the synthesis of the oligosaccharide chain of complex‐type glycoproteins. In view of the high content of glycoprotein in peripheral nerve myelin, the properties of this enzyme, its changes with age, and the effect of the specific inhibitor tunicamycin were investigated. The enzyme activity in rat peripheral nerve homogenate was completely dependent on the presence of exogenous dolichyl phosphate as well as Mg2+ and a detergent (Triton X‐100) and was also greatly stimulated by a high salt concentration (0.4 M KCl) and AMP. The highest specific activity was present in the postmitochondrial membranes. The specific activity in postmitochondrial membranes in the presence of exogenous dolichyl phosphate reached a maximum at 17 days and remained relatively high throughout development, up to 2 years of age, but the activity was much lower when dolichyl phosphate was not added. This indicates that the enzyme level does not decrease with age, but that the content of the lipid cofactor may limit glycoprotein synthesis in vivo. Tunicamycin (5 μg) was injected intraneurally into 24‐day‐old rat sciatic nerve, and the enzyme was assayed from 1 to 24 days after injection. The specific activity of the transferase remained at low levels (5–40% of the level in control nerve) in most injected nerves assayed throughout this postinjection period. A protein previously identified as the unglycosylated P0 protein was synthesized in vitro by the tunicamycin‐injected nerve and could be demonstrated to be incorporated into myelin in large amounts at 2 days and in small amounts at 6 days after injection. At 11 days, the protein was synthesized but not incorporated into myelin, and morphological examination showed evidence of myelin disintegration at 14 and 30 days after tunicamycin injection. These results indicate an important role for the enzyme UDP‐N‐acetylglucosamine:dolichyl phosphate, N‐acetylglucosamine‐1‐phosphate transferase in the synthesis of P0 in peripheral nerve myelin. The activity of this enzyme may be a limiting factor in the maintenance of the nerve.
Regulation of Rat Pineal α1‐Adrenoceptors
Authors: Sugden David , Klein David C.
Published: October 2006

Some aspects of the physiological regulation of the pineal α1‐adrenoceptor have been studied using the selective, high‐affinity ligand [125I] iodo‐2‐[β‐(4‐hydroxyphenyl)ethylaminomethyl]tetralone ([125I]HEAT). Pineal glands taken from rats housed in a diurnal lighting cycle showed no circadian rhythm in the number of specific [125I]HEAT binding sites, although a characteristic rhythm in pineal melatonin was seen. It was established that the pineal α1‐adrenoceptor is under neural control because interruption of neural stimulation of the pineal by bilateral superior cervical ganglionectomy (SCGX) or by exposing rats to constant light for 3 weeks doubled receptor density but did not change affinity for [125I]HEAT. Administration of various α1‐adrenoceptor agonists either acutely (i.p. injection) or chronically (s.c. infusion) did not alter the number of specific [125I]HEAT binding sites. Together these results indicate that the pineal α1‐adrenoceptor, like the pineal β‐adrenoceptor, is regulated by sympathetic nerve activity, probably through the physiological release of the neurotransmitter norepinephrine. However the absence of a circadian rhythm in α1‐adrenoceptor number and lack of down‐regulation by adrenergic agonists imply different mechanisms of regulation.
Properties and Partial Purification of Choline Acetyltransferase from the Nematode Caenorhabditis elegans
Authors: Rand James B. , Russell Richard L.
Published: October 2006

We have stabilized and studied choline acetyltransferase from the nematode Caenorhabditis elegans. The enzyme is soluble, and two discrete forms were resolved by gel filtration. The larger of these two forms (MW ∼ 154,000) was somewhat unstable and in the presence of 0.5 M NaI was converted to a form indistinguishable from the “native”small form (MW ˜ 71,000). We have purified the small form of the enzyme >3,300‐fold by a combination of gel filtration, ion‐exchange chromatography, and nucleotide affinity chromatography. The purified preparation has a measured specific activity of 3.74 μmol/min/mg protein, and is free of acetylcholinesterase and acetyl‐CoA hydrolase activities. The Vmax of the purified enzyme is stimulated by NaCl, with half‐maximal stimulation at 80 mM NaCl. The Km for each substrate is also affected by salt, but in different manners from each other and the Vmax; the kinetic parameter Vmax/Km thus changes significantly as a function of the salt concentration.
Neurotoxicity of L‐Glutamate and DL‐Threo‐3‐Hydroxyaspartate in the Rat Striatum
Authors: McBean Gethin J. , Roberts Peter J.
Published: October 2006

Destruction of the glutamatergic corticostriatal pathway potentiates the neurotoxic action of 1 μmol L‐glutamate injected into the rat striatum, whereas the toxic effects of 10 nmol kainate are markedly attenuated. Injection of 170 nmol of the glutamate uptake inhibitor, DL‐threo‐3‐hydroxyaspartate, into the intact striatum also causes neuronal degeneration, which is accompanied by a reduction in markers for cholinergic and GABAergic neurones. Prior removal of the corticostriatal pathway destroys the ability of DL‐threo‐3‐hydroxyaspartate to cause lesions in the striatum. These results indicate that removal, or blockade, of uptake sites for glutamate increase the vulnerability of striatal neurones to the toxic effects of synaptically released glutamate.
Short Communication Occupation of Dopamine Receptors by N‐n‐Propylnorapomorphine or Spiperone and Acetylcholine Levels in the Rat Striatum
Authors: Korf Jakob , Sebens Jantiena B. , Flentge Frans , van der Werf Jan F.
Published: October 2006

In an attempt to quantify the interactions between dopaminergic and cholinergic processes, the consequences of complete or partial activation (with N‐n‐propylnorapomorphine) or blockade (with spiperone) of dopamine receptors for the acetylcholine levels in the rat striatum were studied. The number of specific striatal binding sites (receptors) of spiperone was nearly three times that of N‐n‐propylnorapomorphine (76 and 26 pmol g−1 wet weight, respectively). The agonist produced a significant increase in the striatal levels of acetylcholine, but there was no simple relationship between receptor binding and these levels. A linear negative correlation was found between the striatal levels of acetylcholine and specific spiperone binding, showing that further receptor blockade induces a decrease in acetylcholine levels, which is independent of the receptors already occupied by the antagonist. The results of this study are evidence that one striatal dopamine receptor regulates the metabolism of at least 400 molecules of acetylcholine.
Characterization of “High‐Affinity”[3H]Ouabain Binding in the Rat Central Nervous System
Authors: Hauger R. , Luu H. M. D. , Meyer D. K. , Goodwin F. K. , Paul S. M.
Published: October 2006

The characteristics of [3H]ouabain binding were examined in various areas of rat brain. In the striatum, Scatchard analysis revealed a single class of “high‐affinity” binding sites with an apparent binding affinity (KD) of 10.4 ± 0.9 nM and an estimated binding capacity (Bmax) of 7.6 ± 1.9 pmol/mg protein. Similar monophasic Scatchard plots were found in the brainstem, cerebellum, hypothalamus, and frontal cerebral cortex. [3H]Ouabain binding to rat brain was sodium‐ and ATP‐dependent and strongly inhibited by potassium. Proscillariden A was the most potent cardiac glycoside tested in inhibiting specific [3H]ouabain binding to brain membranes, and the rank order of inhibitory potencies for a series of cardiac glycosides was similar to that previously reported for inhibition of heart Na,K‐ATPase. To assess whether the high‐affinity binding sites for [3H]ouabain were localized to neuronal or nonneuronal membranes, the effect of discrete kainic acid lesions on striatal [3H]ouabain binding was examined. Kainic acid lesions of the striatum reduced [3H]ouabain binding to striatal homogenates by 79.6 ± 1.6%. This suggests that the “high‐affinity”[3H]ouabain binding sites measured in our experiments are localized to neuronal elements. Thus, the high‐affinity binding of [3H]ouabain to brain membranes may selectively label a neuronal form or conformation of Na,K‐ATPase.
Amino Acids in Rat Striatal Dialysates: Methodological Aspects and Changes After Electroconvulsive Shock
Authors: Korf Jakob , Venema Kor
Published: October 2006

Abstract: Extracellular levels of amino acids were estimated in dialysates of the rat striatum that were collected 1,2, and/or more than 5 days after surgery, before (resting release) and during exposure to high K concentrations (50 mM) or electroconvulsive shocks. The resting release of several amino acids (Glu, Asn, Thr, Tau, Tyr, Gly, and Ala) was higher 9 days as compared to 1 day after surgery. In the 1‐day preparation the resting release correlated highly with that observed with push‐pull cannulas. The correlation with the tissue content of the amino acids was high only when they were divided into two groups (putative transmitters and metabolic intermediates). High K exposure produced increased output of Ala, ethanolamine (Eam). Asp, Glu. Tau, and Gly and a decrease in the egress of Gln 1 or 2 days after surgery. The effects on Asp and Glu had disappeared, and that on Gln reversed after 4–9 days. Electrically induced convulsions produced increased output of Ala, Gln, and Eam 1 or 2 days and 2 weeks after implantation of the probe. Changes were seen not only during but also (and some cases even more prominent) after the seizure. This study shows the usefulness of dialysis to monitor extracellular transmitter amino acids in the striatum of conscious rats (also bilateral dialysis was possible) for only a limited time after implantation of the probe. The dialysis method is suitable for longer time, when metabolic changes in amino acids are to be followed. In addition to transmitter release, glycolysis can be monitored by the measurement of Ala in the dialysate.