bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of Phosphorylation of a 30‐kD Polyribosomal Protein (pp30) by ACTH and Spermine: Comparison with Modulation of Brain Protein Synthesis
Authors: Schrama L. H. , Frankena H. , Edwards P. M. , Schotman P.
Published: October 2006

Gel electrophoretic separation of proteins phosphorylated in a postmitochondrial supernatant fraction of brain in the presence of spermine or adrenocorticotropin (ACTH) indicated modulation in only one region (30 kD) of the gel. The 30‐kD (pp30) protein together with enzyme activity catalyzing its phosphorylation and sensitivity of the phosphorylation to spermine and ACTH were retained in a free polyribosomal fraction of this extract. ACTH(11–24) inhibited phosphorylation at all the spermine or Mg2+ concentrations tested. Structure‐activity studies revealed that the inhibitory activity within ACTH(l–24) resides in the sequences ACTH(11–24), (5–18,17Lys,18Lys)‐NH2, (15–24), (7–16)‐NH2, and (1–16)‐NH2 and can also be found in certain polylysine fragments. Phosphorylation under conditions suitable for measuring protein synthesis revealed only one phosphoprotein (pp30), sensitive to both ACTH(15–24) and spermine. The possibility of a relationship between modulation of pp30 phosphorylation and modulation of brain cell‐free protein synthesis is discussed in relation to the effects of ACTH, spermine, and Mg2+ .
Photoaffinity Labeling of Different Benzodiazepine Receptors at Physiological Temperature
Authors: Eichinger A. , Sieghart W.
Published: October 2006

Irreversible labeling of benzodiazepine receptors in membranes from cerebellum or hippocampus was compared at 0°C or 37°C using [3H]flunitrazepam as a photoaffinity ligand. [3H]Flunitrazepam reproducibly and irreversibly labeled mainly one protein (P51) in cerebellum and at least two proteins (P51 and P55) in hippocampus at both temperatures. Differential inhibition at 37°C of irreversible [3H]flunitrazepam binding to the individual proteins by several selective benzodiazepine receptor ligands supports the hypothesis that P51 and P55 are associated with different benzodiazepine receptors.
Free Fatty Acid Content and Release Kinetics as Manifestations of Cerebral Lateralization in Mouse Brain
Authors: Pediconi María F. , Rodríguez de Turco Elena B.
Published: October 2006

Abstract: Free fatty acid (FFA) content was analyzed in mouse cerebral hemispheres and cerebellum under basal and postdecapitative ischemic conditions. Total FFA content immediately after decapitation (2 s) was about two‐fold higher in the left hemisphere than in the right. Marked dissimilarities between hemispheres were also apparent when FFA levels were measured during short periods of ischemia. Whereas in the right side a significant FFA release took place as early as 10 s, no accumulation was detected in the left in the 2–20 s interval. The highest rates of total fatty acid release occurred in the 20–30 s interval in both hemispheres and decreased afterwards (3 min). Individual FFA, especially stearate and arachidonate, differed in their rates of production, the right cerebral hemisphere being more active in releasing arachidonic acid. In cerebellum, FFA levels were lower and accumulation was slower than in cerebrum in both intervals. When subjected to 3 min ischemia, the same difference in FFA levels between right and left hemispheres (50%) was observed in heads kept at 20 or 30°C. The differences between hemispheres are interpreted as manifestations of an inherent lateralization in the regulation of acylation‐deacylation reactions of complex lipids.
Changes in Regional Neurotransmitter Amino Acid Levels in Rat Brain During Seizures Induced by l‐Allylglycine, Bicuculline, and Kainic Acid
Authors: Chapman Astrid G. , Westerberg Eva , Premachandra Maithri , Meldrum Brian S.
Published: October 2006

Abstract: Changes in amino acid concentrations were studied in the cortex, cerebellum, and hippocampus of the rat brain, after 20 min of seizure activity induced by kainic acid, 47 μmol/kg i.v.;l‐allylglycine, 2.4 mmol/kg i.v.; or bicuculline, 3.27 μmol/kg i.v. in paralysed, mechanically ventilated animals. Metabolic changes associated with kainic acid seizures predominate in the hippocampus, where there are decreases in aspartate (‐26%), glutamate (‐ 45%), taurine (‐20%), and glutamine (‐32%) concentrations and an increase in ‐γ‐aminobutyric acid (GABA) concentration (+26%). l‐Allylglycine seizures are associated with generalized decreases in GABA concentrations (‐32 to ‐54%), increases in glutamine concentrations (+ 10 to +53%), and a decrease in cortical aspartate concentration (‐14%). Bicuculline seizures, in fasted rats, are associated with marked increases in the levels of hippocampal GABA (+106%) and taurine (+ 40%). In the cerebellum, there are increases in glutamine (+ 50%) and taurine concentrations (+ 36%). These changes can be explained partially in terms of known biochemical and neurophysiological mechanisms, but uncertainties remain, particularly concerning the cer‐ ebellar changes and the effects of kainic acid on dicar‐ boxylic amino acid metabolism.
Picrotoxinin and Phorbol‐12‐Myristate‐13‐Acetate Stimulate the Secretion of Multiple Forms of Somatostatin from Cultured Rat Brain Cells
Authors: Peterfreund Robert A. , Vale Wylie W.
Published: October 2006

Abstract: The molecular forms of somatostatin (SRIF) secreted by cultured fetal rat brain cells were resolved using reverse phase high performance liquid chromatography followed by radioimmunoassay. Multiple forms of SRIF‐like immunoactivity were detected in media from cells treated with either picrotoxinin, phorbol‐12‐myris‐ tate‐13‐acetate, or high potassium. For stimulated cells, elevated levels of an SRIF‐28‐like molecule, an SRIF‐14‐like molecule, and a hydrophobic SRIF‐like molecule were observed compared to basal conditions. All three forms of SRIF‐like molecules were also detected in acid extracts of whole cells. The data are consistent with the possibility that secretion of multiple SRIFs, including SRIF‐28, may be regulated by multiple effectors and mechanisms.
Effects of Monensin and Colchicine on Myelin Galactolipids
Authors: Townsend Laurace E. , Benjamins Joyce A. , Skoff Robert P.
Published: October 2006

Abstract: Monensin and colchicine have been used in a variety of systems to disrupt functioning of the Golgi apparatus and transport of Golgi‐derived vesicles to the plasma membrane. In this study the effects of monensin and colchicine on the synthesis of cerebroside and sulfatide and their appearance in myelin were examined to determine whether these myelin components are processed through the Golgi apparatus. Brain slices from rats 17 days old were incubated with [3H]galactose and [35S]sulfate to label cerebroside and sulfatide. Myelin was isolated on sucrose density gradients. Fractions highly enriched in cerebroside and sulfatide were prepared from homogenates and myelin fractions by lipid extraction, alkaline methanolysis, and in some cases TLC. Monensin at 0.1 μM had no significant effect on synthesis of these galactolipids as measured by incorporation of [3H]galactose into cerebroside or [35S]sulfate into sulfatide in homogenates. However, appearance of [35S]sulfatide in the myelin fraction was reduced to 49% of control, while appearance of [3H]cerebroside was not significantly reduced. Colchicine from 1 mM to 0.1 μM had effects similar to monensin, that is, appearance of [35S]sulfatide in myelin was depressed, but again [3H]cerebroside was not affected. Incorporation of [35S]sulfate into sulfatide in homogenate was 93% of control, while appearance of [35S]sulfatide in the myelin fraction was depressed to 58% of control. The inhibition of appearance of sulfatide in myelin by colchicine and monensin is consistent with the view that sulfation of cerebroside occurs in the Golgi and that sulfatide is transported via Golgi‐derived vesicles to the forming myelin membrane. Further, synthesis of cerebroside and its appearance in myelin are not inhibited by colchicine or monensin, indicating that cerebroside destined for myelin is not processed through the Golgi apparatus.
Diazotization and Thiocyanate Differentiate Agonists from Antagonists for the High‐ and Low‐Affinity Receptors of γ‐Aminobutyric Acid
Authors: Maksay Gabor , Ticku Maharaj K.
Published: October 2006

Abstract: The differentiation of high‐ and low‐affinitypostsynaptic y‐aminobutyric acid (GABA) receptors wasexamined in a washed cortical membrane preparation of the rat. The selective elimination of the high‐ and low‐affinity GABA sites by the chaotropic anion thiocyanateand diazotization by p‐diazobenzenesulfonic acid (DSA), respectively, offered two model systems for the sepa‐rate sites. The [3H]GABA displacing potencies of some GABA agonists [GABA, 4,5,6,7‐tetrahydro‐isoxa‐zole[4,5c]pyridine‐3‐01 (THIP), and muscimol] and antagonists [bicuculline methiodide (BCM), 3‐α‐hydroxy‐16‐imino‐5β;‐l7‐aza‐androstan‐ll‐one (R‐5 135), and d‐tubo‐curarine] and their slope factors were examined in thesemodel systems and in control membranes. The displacingpotency of the agonists was increased in the DSA‐pre‐treated membranes and decreased in the presence of thiocyanate. The displacing potency of the antagonists wasshifted in an opposite manner. The chaotropic effect ofthiocyanate was reversible and not additive with the inhibitory effect of diazotization on the specific binding of GABA. Inhibition of specific GABA binding by pyri‐doxal‐5‐phosphate (PLP) could not be protected by GABA antagonists (BCM and R‐5135) but only by agonists. The results can be interpreted in the frame work of a dual (agonist‐antagonist) receptor model, postulating a hydrophobic accessory site at the low‐affinity GABA re‐ceptor. The effect of thiocyanate on the GABA receptor may result in the exposure of the hydrophobic accessory sites.
Phosphate Derivatives of Thiamine and Na+ Channel in Conducting Membranes
Authors: Schoffeniels Ernest , Dandrifosse Guy , Bettendorff L.
Published: October 2006

Abstract: The results show that thiamine derivatives are copurified with the specific proteins forming the Na+ channel in conducting membranes. Therefore, thiamine derivatives could well play a specific role in the molecular aspects of bioelectrogenesis, an interpretation that could help explain the neurological symptoms observed in human pathology as well as in animals experimentally rendered deficient in vitamin Bl.
Effects of Calcium, Strontium, and Barium Ions on Phosphorylation of Hippocampal Proteins In Vitro
Authors: Hoch Daniel B. , Wilson John Eric
Published: October 2006

Abstract: Calcium ion alone or in the presence of added calmodulin stimulated in vitro transfer of 32P from [γ32P]ATP into several proteins of mitochondrial and synaptosomal particulate fractions from rat brain. Strontium ion was capable of substituting for calcium ion in this stimulation, but barium ion lacked this capacity. These results bring into question the hypothesis that calciumdependent protein phosphorylation of synaptic proteins is intrinsic to neurotransmitter release during neurotransmission, but they do not rule out that possibility.
γ‐Aminobutyric Acid Turnover in Rat Striatum: Effects of Glutamate and Kainic Acid
Authors: Giorgi Osvaldo , Meek James L.
Published: October 2006

Abstract: The turnover rate of γ‐aminobutyric acid (GABA) in the rat striatum was estimated by measuring its accumulation after inhibition of GABA‐transaminase (GABA‐T) with gabaculine. Intrastriatal injections of 100 μg gabaculine induced a rapid and complete inhibition of GABA‐T. GABA accumulation was linear with time for at least 60 min (estimated turnover rate = 25 nmol/mg protein/h). The accumulation of GABA after gabaculine administration in animals that had been treated with kainic acid (5 nmol intrastriatally, 7 days) was only 40% of the control value, indicating that a major fraction of the net increase in GABA content induced by gabaculine originates in kainic acid‐sensitive neurons. Intrastriatal injection of a mixture of kainic acid (5 nmol) and gabaculine caused a net increase in striatal GABA content significantly greater than that observed in controls, suggesting that neuronal death induced by kainic acid is preceded by a period of increased neuronal activity. Glutamic acid, the putative neurotransmitter for the excitatory corticostriatal pathway, also produced a significant increase in striatal GABA accumulation when injected together with gabaculine. This effect was blocked by the administration of the glutamate receptor antagonist glutamic acid diethyl ester. The interactions between GABAergic neurons and other neurotransmitters present in the striatum were also analyzed.
Metabolism of 15NH3 in Organotypic Cerebellar Explants and Cultured Astrocytes: Studies with Gas Chromatography‐Mass Spectrometry
Authors: Yudkoff Marc , Nissim Itzhak , Kim Seung U. , Pleasure David , Segal Stanton
Published: October 2006

Abstract: Gas chromatography‐mass spectrometry was used to study the metabolism of 15NH3 in organotypic cerebellar explants and cultured astrocyte monolayers. A steady‐state level of 15NH3 was present by 1 min in both systems. Steady‐state labeling in l‐[amide‐15N] glutamine, l‐[15N]alanine, l‐[15N]glutamate, and l‐[15N]aspartate was attained by 1 min after 15NH3 addition in the organotypic cerebellar explants and by approximately 5 min in the cultured astrocytes. No measurable 15N labeling was noted in either glycine or serine in either system.
Localization and Characterization of Tubulin‐Like Proteins Associated with Brain Mitochondria: The Presence of a Membrane‐Specific Isoform
Authors: Hargreaves Alan J. , Avila Jesús
Published: October 2006

Abstract: A mitochondrial fraction, purified from pig brain, was found to contain associated polypeptides with the same electrophoretic migration and isoelectric points as the α‐ and β‐tubulin subunits present in brain microtubules. When analyzed by Western blotting these polypeptides reacted specifically with purified tubulin antibodies. The tubulin‐like proteins were then visualized in mitochondrial membranes by protein A‐gold complexes after the incubation of purified mitochondria with tubulin antibodies. When membrane and microtubule proteins were compared by isoelectric focussing and two‐dimensional gel electrophoresis, differences were observed in the patterns of tubulin isoforms. An additional polypeptide, with the electrophoretic migration of β‐tubulin but the isoelectric point of α‐tubulin, was found to be enriched in the mitochondrial fraction. This peptide had several Staphylococcus aureus V8 protease peptides in common with α‐tubulin and may result from a posttranslational modification of that subunit.
Quantitative Autoradiography of [3H]Indalpine Binding Sites in the Rat Brain: II. Regional Distribution
Authors: Savaki H. , Malgouris C. , Bénavidès J. , Laplace C. , Uzan A. , Guérémy C. , Le Fur G.
Published: October 2006

Abstract: The localization of binding sites for [3H]indalpine to sections of rat brain was studied by a quantitative autoradiographic technique. Binding sites for this specific neuronal 5‐hydroxytryptamine (5‐HT) uptake inhibitor are concentrated in areas rich in 5‐HT neuronal cell bodies, fibers, and synaptic terminals. One of the most interesting features of this regional distribution is the very high density of these sites found in the dorsal raphe, substantia nigra, ventral tegmental area, and locus ceruleus. Components of the visual system also show pronounced labelling with [3H]indalpine. The finding that limbic structures are strongly labelled by this drug may be related to the antidepressant activity of indalpine. The anatomical distribution of binding sites demonstrated is Consistent with the specific labelling of 5‐HT neurons by [3H]indalpine and confirms previous studies carried out with another 5‐HT uptake inhibitor, [3H]imipramine.
Solubilization of an Adenosine Uptake Site in Brain
Authors: Verma Ajay , Houston Michelle , Marangos Paul J.
Published: October 2006

Abstract: Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine ([3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 ± 0.02 nM and 133 ± 18 fmol/mg protein, respectively. with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep > hexobendine > dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine ([3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high‐affinity site with a KD of 0.32 ± 0.06 nM and a Bmax of 105 ± 30 fmol/mg protein and a loweraffinity site with a KD of 5.50 ± 0.52 nM and Bmax of 300 ± 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake ([3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.
Biogenic Aldehydes in Brain: Characteristics of a Reaction Between Rat Brain Tissue and Indole‐3‐Acetaldehyde
Authors: Nilsson Göran E. , Tottmar Olof
Published: October 2006

Abstract: When indole‐3‐acetaldehyde was incubated with rat brain tissue, an aldehyde dehydrogenase‐independent irreversible disappearance of the aldehyde was found. This was accompanied by an increase in absorbance at 240–400 nm, with a peak at 310 nm. The results suggested that this change in absorbance was caused by a membrane‐bound nonenzymatic reaction between indole‐3‐acetaldehyde and phospholipids. A similar reaction occurred between indole‐3‐acetaldehyde and pure preparations of phosphatidylethanolamine and phosphatidylserine, but not phosphatidylcholine. Indole‐3‐acetaldehyde levels also decreased slightly when incubated with albumin but absorbance at 310 nm was unaltered. It is suggested that nonenzymatic reactions between indole‐3‐acetaldehyde (or other biogenic aldehydes) and membrane components might occur in vivo, and could be involved in the effects of drugs such as ethanol and barbiturates.
Methionine‐Induced Changes in Glutamate, Aspartate, Glutamine, and γ‐Aminobutyrate Levels in Brain Tissue
Authors: Wood J. D. , Kurylo E. , Geddes J. W.
Published: October 2006

Abstract: Intramuscular administration of methionine to mice resulted in changes in the levels of aspartate, glutamate, glutamine, and γ‐aminobutyrate in both nerve endings (synaptosomes) and “non‐nerve‐ending” tissue in the brain. However, the amino acid changes in the two locations differed considerably, not only in the time to onset of the changes, but also in the direction of the changes and in their duration. The results provide additional support for a glutamate‐glutamine cycle between neurons and glia, and suggest that the decreases in amino acid levels in the nerve endings are due to an insufficient supply of glutamine from glia or other cellular structures, possibly compounded by an impairment in the uptake of glutamine into the nerve terminals. The primary cause of the glutamine deficiency is unknown because methionine did not affect the enzymes of glutamate and glutamine metabolism. Treatment of mice with methionine also resulted in an anticonvulsant action, but no correlation was observed between the latter phenomenon and the glutamate content of nerve endings.
Axonal Transport of Glycoconjugates in the Rat Visual System
Authors: Gammon C. M. , Goodrum J. F. , Toews A. D. , Okabe A. , Morell P.
Published: October 2006

Abstract: Long‐Evans rats at 45 days of age were injected intraocularly with 25 μCi of [3H]glucosamine. Incorporation of radioactivity into retinal gangliosides, glycoproteins, and glycosaminoglycans (GAGs) was determined at various times after injection. Portions of all three classes of radioactive macromolecules were committed to rapid axonal transport in the retinal ganglion cells. With respect to gangliosides about 60% of those synthesized in the retina were retained in that structure, 30% were committed to transport to regions containing the nerve terminal structures (lateral geniculate body and superior colliculus), and about 10% were deposited in stationary structures of the axons (optic nerve and tract). With the exception of ganglioside GD3 the molecular species distribution of gangliosides synthesized in the retina matched that committed to transport. In contrast to gangliosides a smaller fraction of newly synthesized retinal glycoprotein (less than 12% of that synthesized in the retina) was committed to rapid transport to nerve ending regions and only about 0.5% was retained in the nerve and tract. The molecular‐weight distribution of glycoproteins committed to transport differed quantitatively from that of the retina. With respect to GAGs an even smaller portion (1‐2%) of that synthesized in the retina was committed to rapid transport; of this portion almost all was recovered in nerve terminal‐containing structures. A constant proportion of each retinal GAG species was transported to the superior colliculus. We suggest that most of the retinal gangliosides are synthesized in neurons and preferentially in ganglion cells (possibly a function of the large surface membrane area supported by these cells). Subcellular fractionation experiments indicated that transported gangliosides, glycoproteins, and GAGs may be preferentially distributed into different subcellular compartments.
Sites in Myelin Basic Protein that React with Monoclonal Antibodies
Authors: Hruby Sarka , Alvord Ellsworth C. , Martenson Russell E. , Deibler Gladys E. , Hickey William F. , Gonatas Nicholas K.
Published: October 2006

Abstract: The epitopes (antigenic sites) for seven monoclonal antibodies (MAbs) evoked in rats or mice by guinea pig or monkey myelin basic protein (BP) have been located in four different sequences of the BPs extracted from various species. Six of the MAbs were evoked by guinea pig BP. (1) One epitope, possibly a pair, is included within residues 1–14 of all BPs tested and reacts with two rat IgG MAbs. (2) A definite pair of over‐lapping epitopes includes the central Phe91‐Phe92 sequence. One epitope is contained entirely within sequence 90–99 and reacts with a rat IgG MAb. The substitution of Ser in chicken BP for Thr97 destroys this epitope. The other epitope appears to include residues on the amino side of Phe44 and even of His32 and suggests some tertiary structure in BP. This epitope reacts with a mouse IgM MAb that does not recognize the chicken substitution. (3) The third epitope lies within residues 114–121, specifically including Trp118, and reacts with a rat IgG MAb. A cross‐reacting epitope probably includes residues 44–45 in certain species (guinea pig and bovine but not rabbit). (4) Another pair of epitopes is located within residues 131–140 but is severely species‐restricted. This region in guinea pig BP evoked a species‐specific mouse IgM MAb. The same region in monkey BP evoked the seventh MAb, a mouse IgG, which reacts with human, chimpanzee, monkey, bovine, and rat‐18.5 kDa BPs and to a lesser extent rabbit BP but not with guinea pig, pig, or chicken BPs. Some tertiary structure in guinea pig BP is also suggested by the reactivities with the IgM MAb. All of the MAbs react with myelin in histologic preparations, but the optimum method of preparation of the tissue varies with each.
Phylogenetic Survey of Proteins Related to Synapsin I and Biochemical Analysis of Four Such Proteins from Fish Brain
Authors: Goelz Susan E. , Nestler Eric J. , Greengard Paul
Published: October 2006

Abstract: A phylogenetic survey of proteins immunologically related to Synapsin I, a major synaptic vesicle‐associated phosphoprotein in mammals was carried out. Proteins antigenically related to Synapsin I were found by use of radioimmunoassay and other radioimmunochemical techniques in the nervous systems of several vertebrate and invertebrate species, which included birds, reptiles, amphibians, fish, echinoderms, arthropods, and mollusks. Four proteins present in fish brain, antigenically related to Synapsin I, were further studied and found to resemble mammalian Synapsin I in several respects. Like Synapsin I, the fish proteins were present in high amounts in nervous tissue, were enriched in synaptosomal fractions of brain where they were substrates for endogenous protein kinases, were acid extractable, and were sensitive to digestion by collagenase. In addition, two‐dimensional peptide‐mapping analysis revealed some homology between major phosphopeptide fragments of Synapsin I and the fish proteins. The results indicate that proteins related to Synapsin I are widespread in the animal kingdom.
Accumulation, Acetylation, and Releasability of Diethylhomocholine from a Sympathetic Ganglion
Authors: Welner S. A. , Collier B.
Published: October 2006

Abstract: Superior cervical ganglia of the cat perfused with [14C]diethylhomocholine ([14C]DEHCh) synthesized acetyldiethylhomocholine (ADEHCh), but rather little of this ester was released by subsequent preganglionic nerve stimulation. Stimulation evoked the release of an appreciable amount of unchanged DEHCh when ganglia had been exposed to the analogue in the absence of choline (Ch), but did not do so when exposed to both Ch and DEHCh. The release of DEHCh was Ca2+ dependent, and was not the result of the release and subsequent hydrolysis of ADEHCh. This is the first clear demonstration of the release of an unacetylated compound from mammalian tissue; therefore, the characteristics of the transmitter release mechanism are further defined. The effect of preganglionic nerve stimulation on the uptake and acetylation of DEHCh was also measured. Stimulated ganglia accumulated ∼4 times more labeled analogue and synthesized 7.5 times more ADEHCh than did rested ganglia. Stimulated ganglia perfused with 2‐(4‐phenylpi‐peridino)cyclohexanol, a compound considered to inhibit acetylcholine (ACh) release by inhibiting its transport into synaptic vesicles, accumulated 3.4 times as much and acetylated 6 times as much DEHCh as did rested ganglia. When the concentration of Mg2+ in the perfusion medium was increased to block ACh release, accumulation of the labelled analogue was enhanced by stimulation, but its acetylation was increased much less than during perfusion with normal medium. It is concluded that the synthesis of ADEHCh is subject to the same regulation as is ACh synthesis and that the activation of ester synthesis during activity can be dissociated from ester release. The relationship between stimulation‐induced increases of DEHCh accumulation and ADEHCh synthesis suggests that some other factor associated with nerve terminal impulse invasion, such as increased Ca2+ influx, is necessary for the manifestation of increased synthesis of the acetyl compound.
Differential Degradation of Different Benzodiazepine Binding Proteins by Incubation of Membranes from Cerebellum or Hippocampus with Trypsin
Authors: Eichinger A. , Sieghart W.
Published: October 2006

Abstract: When rat brain membranes were incubated with [3H]flunitrazepam in the presence of UV light, predominantly one protein (P5I) was irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. On digestion of membranes with increasing concentrations of trypsin up to 40% of radioactivity irreversibly bound to proteins was removed from the membranes. In addition, P51 was nearly completely degraded to a peptide with apparent molecular weight 39,000 and this peptide was further degraded to a peptide with apparent molecular weight 25,000. In contrast, protein P55 was only partially degraded by trypsin and yielded two proteolytic peptides with apparent molecular weights 42,000 and 45,000 which seemed to be rather stable against further attack by trypsin. Membranes treated with trypsin still had the capacity to bind [3H]‐flunitrazepam reversibly with an affinity similar to that of membranes not previously treated with trypsin. When these membranes were irradiated with UV light, the same proteolytic peptides were detected as in membranes first photolabeled and then digested with trypsin. These results suggest a close association between reversible and irreversible benzodiazepine binding sites and indicate that membrane‐associated proteins P51 and P55 are differentially protected against degradation by trypsin.
Properties of N‐Acetylhistamine Deacetylase in Mammalian Brain
Authors: Hegstrand Linda R. , Kalinke Tom H.
Published: October 2006

Abstract: Properties of N‐acetylhistamine deacetylase in rat brain were studied, utilizing a sensitive coupled radioenzymatic assay. The Km for N‐acetylhistamine for this deacetylase was 660 μM and its Vmax was 330 pmol/ h/mg protein. N‐Acetylhistamine deacetylase activity increased 80% in the presence of 1 mM Mn2+. The Km of Mn2+ was 40 μM. The enzyme is primarily a soluble enzyme with a relatively uniform regional distribution, unlike the distribution for histamine and histidine decarboxylase. Neonatal activity of this enzyme in rat brain is higher than in adult brain. α‐Fluoromethylhistidine does not affect the activity of N‐acetylhistamine, indicating that deacetylation probably does not play a regulatory role in the synthesis of brain histamine.
Studies on the Oxidation of the Dopaminergic Neurotoxin 1‐Methyl‐4‐Phenyl‐1,2,5,6‐Tetrahydropyridine by Monoamine Oxidase B
Authors: Heikkila Richard E. , Manzino Lawrence , Cabbat Felicitas S. , Duvoisin Roger C.
Published: October 2006

Abstract: 1‐Methyl‐4‐phenyl‐ 1,2,5,6 ‐tetrahydropyridine (MPTP) is a chemical that, after injection into experimental animals, including mice and monkeys, causes a degeneration of the nigrostriatal pathway. We carried out experiments designed to study the in vitro oxidation of MPTP by mouse brain mitochondrial preparations. MPTP was actively oxidized by the mitochondrial preparations, with Km and Vmax values very similar to those of benzyl amine, a typical substrate for MAO‐B. MPTP was oxidized considerably better than many of its analogs, even those with relatively minor structural changes. Several monoamine oxidase inhibitors (MAOI) were potent inhibitors of MPTP oxidation, and there was a highly significant correlation between the capacity of the MAOI tested to inhibit MPTP oxidation and benzylamine oxidation. There was no correlation between the capacity of the MAOI to inhibit MPTP oxidation and their capacity to inhibit the oxidation of tryptamine, a substrate for MAO‐A. In other experiments, MPTP was an excellent substrate for pure MAO‐B, prepared from bovine liver. All of these data. combined with the fact that MAO‐B inhibitors can protect against MPTP‐induced dopami nergic neurotoxicity in vivo. point to an important role for MAO‐B in MPTP metabolism in vivo.
Identification and Topography of Substrates for Protein Carboxyl Methyltransferase in Synaptic Membrane and Myelin‐Enriched Fractions of Bovine and Rat Brain
Authors: Johnson Brett A. , Aswad Dana W.
Published: October 2006

Abstract: The major components of crude brain synaptosomes (synaptic membranes, mitochondria, and myelin) have been separated and analyzed by polyacrylamide gel electrophoresis for the presence of proteins that serve as substrates for protein carboxyl methyltransferase. Of the three fractions, synaptic membranes contain the largest number of individual methyl acceptors (at least seven), while mitochondria contain no well‐defined methyl acceptors. Undisrupted myelin contains a single major methyl acceptor with a very low apparent molecular weight. The patterns of protein methylation in synaptic membranes prepared from cerebral cortex, hippocampus, striatum, thalamus, and tectum showed marked differences; however, these differences could largely be explained by differential degrees of myelin contamination in synaptic membranes from the different regions. The effect of trypsin pretreatment on the carboxyl methylation of intact and lysed synaptosomes was studied to estimate the sidedness of the major methylation sites on synaptic membranes. One of the methyl acceptors (Mr 48K) appears to be facing the intracellular surface of the synaptosome, but most sites appear to be outward facing.
The Presence of Aldehyde‐Reacted Proteins in Normal and Multiple Sclerosis White Matter
Authors: Epand Richard M. , Dell Karen , Tonogai Wayne T. , Moscarello Mario A.
Published: October 2006

Abstract: The incorporation of tritium from NaB3H4 into the major protein components of myelin and the presence of weak fluorescence emission bands at wavelengths of –440 and 500.nm from sodium dodecyl sulfate‐solubilized, delipidated white matter are indicative of the presence of the products of aldehyde reactions with proteins. The incorporation of tritium from NaB3H4 into myelin proteins was confirmed by reaction with purified components of myelin basic protein or with lipophilin, a purified fraction of proteolipid protein. From the extent of tritium incorporation into the purified proteins, it is estimated that ‐0.2 mol of tritium is incorporated/mol of myelin basic protein and ‐0.4 mol of tritium/mol of proteolipid protein. There is ‐50% greater incorporation of tritium into a more degraded, less positively charged form of the basic protein. The incorporation of tritium into normal and multiple sclerosis white matter was compared. There is a small but statistically significant difference in the percentage of the total counts incorporated into the major protein fractions for the two groups, with the multiple sclerosis samples showing a higher percentage of the counts in the Wolfgram protein and a lower percentage in the myelin basic protein compared with the normal samples.
Comparative Ontogenesis of Brain Tryptamine, Serotonin, and Tryptophan
Authors: Artigas F. , Suñol C. , Tusell J. M. , Martínez E. , Gelpí E.
Published: October 2006

The pattern of ontogenetic development of tryptophan (TP), tryptamine (T), indole‐3‐acetic acid (IAA), 5‐hydroxytryptamine (5‐HT; serotonin), and 5‐hydroxyindole‐3‐acetic acid (5‐HIAA) in the brains of rats aged 1–45 days is presented. Analysis of the five components in each brain allows the calculation of the acid/amine and amine/amino acid ratios. These metabolic indexes are a useful tool to study and compare the metabolic origins and fates of both amines. The ontogenetic patterns of TP, T, and IAA are very similar, especially during the first week postpartum. The highest and lowest levels found for T were 2.2 ng/g and 0.1 ng/g at the 1st and 5th day, respectively. The temporal relationship between the T/TP and IAA/T ratios suggests the existence of mechanisms protecting T against monoamine oxidase (MAO) which develop in parallel to synaptogenesis. Significant correlations were found between TP and IAA during the whole period studied and between TP and T during the first week after birth. The 5‐HT peak found during the first postpartum week could be due to a non‐neuronal pool of 5‐HT protected against MAO and possibly contained in mast cells. Preliminary determinations on leptomeningeal membranes suggest the existence of such a pool.
Protein Turnover in Brain of the Rat Fetus
Authors: Johnson John D.
Published: October 2006

Protein synthesis in vivo was studied in whole brain of rat fetuses using continuous intravenous infusion of L‐[U‐14C]tyrosine into unrestrained pregnant rats at 19 and 21 days gestation. Protein degradation (KD) was calculated by subtracting fractional growth rate of brain protein (KG) from the fractional synthesis rate (Ks). Ks was high at both gestational ages (0.42 ± 0.03 days−1 at day 19, 0.47 ± 0.029 days−1 at 21 days), comparable to values previously reported for newborn rat cerebral hemispheres, and threefold higher than is seen in adult animals. KD was similar at both 19 and 21 days gestation (0.19‐0.24) and lower than that reported in neonatal rat brain using similar techniques. Protein accretion during the most rapid phase of brain growth (fetus) is accomplished by similar rates of protein synthesis, but decreased rates of degradation when compared with a slower growth phase (newborn). KD in the brain of the rapidly growing fetus is slightly higher than in adult cerebral hemispheres.
Regional Mitochondrial Respiratory Activity in Huntington's Disease Brain
Authors: Brennan William A. , Bird Edward D. , Aprille June R.
Published: October 2006

This study investigated mitochondrial respiratory activity in Huntington's disease (HD) brain. Mitochondrial membranes from caudate and cortex of HD and non‐HD autopsied brains were assayed for succinate oxidation, cytochrome oxidase activity, and cytochromes b, cc1, and aa3. There was a significant decrease in HD caudate mitochondrial respiration, cytochrome oxidase activity, and cytochrome aa3, whereas cytochromes b and cc1 were normal. These findings are consistent with the hypothesis that mitochondrial dysfunction may contribute to the localized hypometabolism and progressive atrophy of the HD caudate.
[3H]Nitrendipine Binding to Calcium Channels in Bovine and Rat Pituitary
Authors: Titeler Milt , De Souza Errol B. , Kuhar Michael J.
Published: October 2006

[3H]Nitrendipine was used to label sites in homogenates of bovine anterior and neurointermediate lobes of the pituitary gland. The amount of specific binding in the anterior lobe was 1.82 < 0.30 pmol/g wet weight of tissue and the KD was 1.44 < 0.02 x 10−10M. Preliminary experiments indicated a similar amount of binding in bovine neurointermediate lobe. In competition studies nimodipine and nisoldipine (two potent voltage‐sensitive calcium channel blockers) displayed IC50 values of 1.6 and 6.8 x 10−10M, respectively. Verapamil and the verapamil‐like calcium channel blockers D‐600 and tiapamil competed in a complex manner for the [3H]nitrendipine specific binding to bovine anterior pituitary homogenates. Autoradiographical studies demonstrated specific [3H]nitrendipine binding sites distributed approximately equally in the anterior and posterior lobes, but not in the intermediate lobe of the rat pituitary. In general the properties of [3H]nitrendipine binding in the pituitary tissue resemble strongly the properties of [3H]nitrendipine binding in the brain which is believed to be to voltage‐sensitive calcium channels. These results provide support for the hypothesis that calcium channels are involved in pituitary hormone secretion and that drugs that interact with calcium channels may modulate the secretory process directly at the level of the pituitary.
Uptake and Release of γ‐Aminobutyric Acid in the Guinea Pig Cochlear Nucleus After Axotomy of Cochlear and Centrifugal Fibers
Authors: Potashner S. J. , Lindberg N. , Morest D. K.
Published: October 2006

Abstract:This study attempts to determine if γ‐aminobutyric acid (GABA) may be a transmitter of cochlear nerve fibers projecting from the cochlea to the cochlear nucleus, and of centrifugal fibers projecting to the cochlear nucleus via the trapezoid body and the acoustic striae of the medulla. The uptake and the electrically evoked release of exogenous [14C]GABA were measured, in vitro, in the three major subdivisions of the guinea pig cochlear nucleus: the anteroventral, posteroventral, and dorsal cochlear nuclei. These activities were compared using unlesioned animals, animals with bilateral cochlear ablations, and animals whose trapezoid body and acoustic striae were interrupted on the right side of the medulla. Subdivisions from unlesioned animals took up [14C]GABA, achieving concentrations in the tissues that were 11–19 times that in the medium. Electrical stimulation evoked a Ca2+‐dependent release of [14C]GABA from each subdivision. Bilateral cochlear ablation, which presumably destroyed the cochlear nerve fibers, had no effect on [14C]GABA uptake and release. Section of the trapezoid body and the acoustic striae on the right side of the medulla typically severed all known connections of the right posteroventral and dorsal cochlear nuclei with the rest of the brain, but left intact many connections involved with the right anteroventral cochlear nucleus. This lesion partially depressed [14C]GABA uptake and release in the right posteroventral and dorsal cochlear nuclei, but not in the right anteroventral cochlear nucleus. These findings suggest that one or more of the centrifugal tracts projecting to the cochlear nucleus may be GABAergic, 88% or more of the cochlear nerve fibers probably are not GABAergic, and some neurons of the cochlear nucleus are probably GABAergic.
Changes in Protein Content of Goldfish Optic Nerve During Degeneration and Regeneration Following Nerve Crush
Authors: Perry G. W. , Burmeister D. W. , Grafstein Bernice
Published: October 2006

Abstract: After the goldfish optic nerve was crushed, the total amount of protein in the nerve decreased by about 45% within 1 week as the axons degenerated, began to recover between 2 and 5 weeks as axonal regeneration occurred, and had returned to nearly normal by 12 weeks. Corresponding changes in the relative amounts of some individual proteins were investigated by separating the proteins by two‐dimensional gel electrophoresis and performing a quantitative analysis of the Coomassie Brilliant Blue staining patterns of the gels. In addition, labelling patterns showing incorporation of [3H]proline into individual proteins were examined to differentiate between locally synthesized proteins (presumably produced mainly by the glial cells) and axonal proteins carried by fast or slow axonal transport. Some prominent nerve proteins, ON1 and ON2 (50–55 kD, pI ∼6), decreased to almost undetectable levels and then reappeared with a time course corresponding to the changes in total protein content of the nerve. Similar changes were seen in a protein we have designated NF (∼130 kD, pI ∼5.2). These three proteins, which were labelled in association with slow axonal transport, may be neurofilament constituents. Large decreases following optic nerve crush were also seen in the relative amounts of α‐ and β‐tubulin, which suggests that they are localized mainly in the optic axons rather than the glial cells. Another group of proteins, W2, W3, and W4 (35–45 kD, pI 6.5–7.0), which showed a somewhat slower time course of disappearance and were intensely labelled in the local synthesis pattern, may be associated with myelin. A small number of proteins increased in relative amount following nerve crush. These included some, P1 and P2 (35–40 kD, pIs 6.1–6.2) and NT (∼50 kD, pI ∼5.5), that appeared to be synthesized by the glial cells. Increases were also seen in one axonal protein, B (∼45 kD, pI ∼4.5), that is carried by fast axonal transport, as well as in two axonal proteins, HA1 and HA2 (∼60 and 65 kD respectively, pIs 4.5–5.0), that are carried mainly by slow axonal transport. Other proteins, including actin, that showed no net changes in relative amount (but presumably changed in absolute amount in direct proportion to the changes in total protein content of the nerve), are apparently distributed in both the neuronal and nonneuronal compartments of the nerve.
Formation of Free Fatty Acid and Ceramide During Brain Handling: Lability of Sphingomyelin
Authors: Deshmukh Gayatri D. , Radin Norman S.
Published: October 2006

Abstract: Intact brain and brain homogenates readily form free fatty acids and ceramides, even in the cold during subcellular isolation procedures. The fatty acid formation is slightly stimulated by chelators and might be due to phospholipid hydrolysis by lysosomal phospholipases. The ceramide formation is accompanied by loss of sphingomyelin and is apparently due to the action of neutral, metal ion‐activated sphingomyelinase. The latter reaction is inhibited by EDTA whereas both degradative processes are inhibited by mercuriphenylsulfonate, the thiol‐reacting inhibitor. Cerebroside does not seem to be a source of accumulated ceramide.
Ammonia Intoxication: Effects on Cerebral Cortex and Spinal Cord
Authors: Lin Sping , Raabe Winfried
Published: October 2006

Abstract: The effect of an acute systemic ammonia intoxication on the metabolic states of the cerebral cortex and the spinal cord of the same animal was studied in the cat. The intravenous infusion of ammonium acetate (2 and 4 mmol/kg body weight/30 min) increased the gross levels of tissue NH4, glutamine, glutamine/glutamate ratio, lactate, and the lactate/pyruvate ratio in the cerebral cortex and the spinal cord. Pyruvate increased, but significantly only in the spinal cord; aspartate decreased, but significantly only in the cerebral cortex. The infusion of ammonium acetate did not significantly change the levels of phosphocreatine, ATP, ADP, AMP, total adenine nucleotides, adenylate energy charge, glucose, glutamate, α‐ketoglutarate, and malate in either tissue. The changes of NH4+, glutamine, and lactate levels as well as gutamine/glutamate and lactate/pyruvate ratios in the spinal cord correlated significantly with the corresponding changes of these metabolites in the cerebral cortex. Thus, cerebral cortex and spinal cord show certain specific and comparable metabolic changes in response to a systemic ammonia intoxication. The effect of ammonia intoxication on the increases of glutamine and lactate levels is discussed.
Substance P in Human Plasma Is Degraded by Dipeptidyl Peptidase IV, Not by Cholinesterase
Authors: Nausch Ingo , Heymann Eberhard
Published: October 2006

Abstract: Human serum cleaves two dipeptides from the N‐terminus of the neurohormone substance P. It has been suggested that this degrading activity is inherent to serum cholinesterase. We oppose this, because it turned out that highly purified serum cholinesterase contains traces of dipeptidyl peptidase IV, an enzyme known to attack the N‐terminus of substance P. The peptidase is incompletely separated from cholinesterase during the procainamidegel affinity chromatography as the last step of the usual purification procedure. Physostigmine completely inhibits the hydrolysis of butyrylthiocholine by such purified cholinesterase preparations, but not their substance P‐degrading activity. Vice versa, δ‐carbobenzoxy‐lysylproline, an inhibitor of dipeptidyl peptidase IV, inhibits the peptidase activity of these preparations more than their esterase activity. After rechromatography on procainamide gel the peptidase is completely separated and the remaining cholinesterase has lost its substance P‐degrading activity. We conclude that the N‐terminal region of substance P is not degraded by cholinesterase but by the contaminating dipeptidyl peptidase IV, a different serine enzyme.