bluuebunny/crossref_metadata_2025 huggingface dataset:
EVIDENCE FOR THE BIOSYNTHESIS OF MANNOSYLPHOSPHORYLDOLICHOL AND N‐ACETYLGLUCOSAMINYLPYROPHOSPHORYLDOLICHOL BY AN AXOLEMMA‐ENRICHED MEMBRANE PREPARATION FROM BOVINE WHITE MATTER
Authors: HarforD J. B. , Waechter C. J. , Saul R. , DeVries G. H.
Published: October 2006

An axolemma‐enriched membrane fraction prepared by an improved procedure from bovine white matter catalyzes the enzymatic transfer of [14C]mannose and N‐acetyl[14C]glucosamine from their nucleotide derivatives into a mannolipid and an N‐acetylglucosaminyl lipid in the presence of exogenous dolichyl monophosphate. The labeled glycolipid products have the chemical and chromatographic characteristics of mannosylphosphoryldolichol and N‐acetylglucosaminylpyrophosphoryldolichol. The initial rates of synthesis of the glycolipids by the axolemma‐enriched membrane fraction have been compared with the initial rates of glycolipid formation catalyzed by a microsomal preparation and myelin in the presence or absence of dolichyl monophosphate. Essentially no glycolipid synthesis was observed when either GDP‐[14C]mannose or UDP‐N‐acetyl[14C]glucosamine were incubated with myelin in the presence or absence of exogenous dolichyl monophosphate. A comparison of the initial rates of synthesis of the glycolipids using endogenous acceptor lipid revealed that the rate of formation of mannolipid was 7 times faster for the microsomal membranes than the axolemma‐enriched membranes. In the presence of an amount of dolichyl monophosphate approaching saturation the initial rate of glycolipid synthesis was markedly enhanced for both membrane preparations. However, due to a more dramatic enhancement in the axolemma‐enriched membranes the initial rate of mannolipid synthesis was only approx. 2.5 times greater in the microsomal membranes. A similar observation was made when the initial rates of N‐acetylglucosaminyl lipid synthesis were compared for axolemma‐enriched and microsomal preparations in the presence and absence of exogenous dolichyl monophosphate. These studies indicate that the axolemma‐enriched membranes have a relatively lower content of dolichyl monophosphate than the microsomal membranes although the difference in the amount of mannosyltransferase is only two to three‐fold lower.

The presence of a sugar nucleotide pyrophosphatase activity capable of degrading GDP‐mannose and UDP‐N‐acetylglucosamine has also been demonstrated in the axolemma‐enriched membrane fraction.
THE EFFECT OF TRIETHYLLEAD ON TOTAL AND MYELIN PROTEIN SYNTHESIS IN RAT FOREBRAIN SLICES
Authors: Konat G. , Offner H. , Clausen J.
Published: October 2006

Rats (20‐day‐old) were acutely intoxicated with triethyllead and their forebrains were studied during the following 14 days. All the lead in the tissue was found in the form of triethyllead, proving that the toxin per se was responsible for the pathological changes observed in the organ. The incorporation of [14C]leucine into the acid‐insoluble protein was suppressed in the forebrain slices prepared from the intoxicated animals as well as in the slices, to which PbEt3 was added in vitro. In both systems the synthesis of myelin protein was inhibited more than the total protein synthesis. The results suggest a specificity of triethyllead toward processes involved in the furnishing of the myelin membrane proteins.
RAPIDLY LABELED AND SECRETED PROTEINS OF THE CHICK BRAIN
Authors: Benowitz Larry I. , Shashoua Victor E.
Published: October 2006

Abstract— The extracellular and cerebrospinal fluids (ECF) of the chick brain were found to contain a distinctive group of rapidly labeled proteins. Gel staining patterns suggest that most ECF protein bands correspond with components also found in either the homogenized whole brain cytoplasmic fraction or the blood serum. The valine‐incorporation profiles of these three fractions, however, were entirely distinctive. Comparisons were carried out using a sensitive double‐labeling method, in which ECF proteins from chicks labeled for 1 h with [3H]valine were comigrated on SDS‐polyacrylamide gels with the cytoplasmic or serum proteins from a 14C‐labeled animal. Analyses of the 3H‐ and 14C‐labeling profiles from these gels showed that certain newly‐synthesized proteins are heavily enriched in the ECF relative to the other two fractions. Most prominently, material with an apparent molecular weight of # 17,000 was found to incorporate nearly one‐third of all the radioactivity appearing in the ECF proteins, but was not heavily labeled in either the cytoplasmic or serum fractions. The effects of a simple training experience on the pattern of chicks’ brain protein synthesis were also examined.
THE DISTRIBUTION OF MYELIN BASIC PROTEIN IN SUBCELLULAR FRACTIONS OF DEVELOPING JIMPY MOUSE BRAIN1
Authors: Zimmerman Thomas R. , Cohen Steven R.
Published: October 2006

Abstract— The amount of myelin basic protein in jimpy mutants and unaffected littermates was measured by radioimmunoassay during the period of most active myelination (11‐21 days). This protein was examined in whole brain homogenates and in four subcellular fractions (nuclear, 900 g pellet; heavy membrane, 11,500 g pellet; microsomal, 100,000 g pellet; and cytosol, 100,000g supernatant solution). At all ages examined, the mutants, which have very little myelin in the CNS, had only about 2% the amount of basic protein found in controls. As expected, the amount of myelin basic protein increased 4‐fold in the control animals during the developmental period studied. This was not the case in the jimpy mutants, where little increase in the whole brain basic protein was observed. In the jimpy mutants, all of the fractions had significantly less basic protein than control fractions, except the cytosol, where the amounts of basic protein were similar in controls and mutants. These results are discussed with respect to possible mechanisms of myelination and the site of the genetic lesion.
BIOCHEMICAL CHARACTERIZATION OF A MYELIN FRACTION ISOLATED FROM RAT BRAIN AGGREGATING CELL CULTURES
Authors: Matthieu J. M. , Honegger P. , Favrod P. , Gautier E. , Dolivo M.
Published: October 2006

Abstract— Subcellular fractions isolated from rat brain aggregating cell cultures were studied by electron microscopy and showed the presence of typical myelin membranes. The chemical composition of purified culture myelin was similar to the fraction isolated from rat brain in terms of CNP specific activity, protein and lipid composition. The ratio of small to large components of myelin basic protein was comparable in culture and in vivo. These two proteins incorporated radioactive phosphorus. The major myelin glycoprotein was present and during development in culture its apparent molecular weight decreased although it never reached the position observed in myelin isolated from adult rats. In culture, the yield of myelin did not increase substantially between 33 and 50 days and was comparable to that of 15‐day‐old rat brain. The ratio basic protein to proteolipid protein resembled immature myelin and the cerebroside content was very low. A ‘floating fraction’ was isolated from the cultures and contained some myelin but mostly single membranes. Although these results indicate that myelin maturation is delayed in vitro this culture system provides substantial amounts of purified myelin to allow a complete biochemical analysis and metabolic studies during development.
KINETICS OF ENTRY OF GALACTOLIPIDS AND PHOSPHOLIPIDS INTO MYELIN1
Authors: Benjamins Joyce A. , Iwata Roderick
Published: October 2006

Abstract— Brain slices from 17 day rats were incubated with [3H]galactose and [35S]sulphate to label cerebroside and sulphatide. Myelin was isolated by centrifugation on a sucrose density gradient. Following lipid extraction and alkaline methanolysis, cerebroside and sulphatide were isolated by tic, and radioactivity was measured. Appearance of [3H]cerebroside and [3H]sulphatide in myelin showed a lag of less than 15min, while appearance of [35S]sulphatide in myelin showed a longer lag of about 30min. In chase experiments, the rate of appearance of [3H]cerebroside and [3SS]sulphatide in the non‐myelin fraction and of [3H]cerebroside in the myelin fraction slowed markedly after the chase. In contrast, [35S]sulphatide continued to increase in myelin at a normal rate for 30min after the chase, then stopped, while 3H from galactose continued to accumulate in myelin sulphatides for 60 min. These data are interpreted to demonstrate an interval of 30 min between synthesis of cerebroside and its sulphation in the non‐myelin fraction, and another delay of 30 min between sulphation and appearance in myelin. The distribution of newly synthesized cerebroside and sulphatide between myelin and non‐myelin fractions also supported the concept that a complex metabolic pool of cerebroside in the non‐myelin fraction is precursor to sulphatide of myelin. For comparison, entry of phosphatidyl choline and phosphatidyl ethanolamine into myelin was followed with [2‐3H]glycerol as precursor. Like cerebroside, both phospholipids showed little delay in their initial appearance in myelin, and prompt cessation of their addition after a chase with unlabeled precursor. These results are consonant with either rapid entry of these three lipids into myelin after synthesis at an extra‐myelin site, or synthesis of the lipids within myelin itself.
K–STIMULATED ACETYLCHOLINE RELEASE: INHIBITION BY SEVERAL BARBITURATES AND CHLORAL HYDRATE BUT NOT BY ETHANOL, CHLORDIAZEPOXIDE OR 11‐OH‐Δ9‐TETRAHYDROCANNABINOL
Authors: Richter Judith A. , Werling Linda L.
Published: October 2006

Abstract— Thiamylal, methohexital, secobarbital, amobarbital, pentobarbital, phenobarbital and barbital, and also chloral hydrate inhibited K‐stimuiated acetylcholine (ACh) release from rat midbrain slices in a dose related manner. The concentrations required for 50% inhibition were related to the lipid partition coefficient. None of the barbiturates nor chloral hydrate inhibited unstimulated ACh release. Ethanol, chlordiazepoxide and 11‐OH‐Δ9‐tetrahydrocannabinol were without effect on both unstimulated and stimulated ACh release. The results are discussed in terms of other effects of these drugs on the cholinergic system.
EFFECT OF DIVALENT CATIONS ON THE AMPHETAMINE‐INDUCED STIMULATION OF [3H]CATECHOL SYNTHESIS IN THE STRIATUM1
Authors: Uretsky Norman J. , Kamal Linda , Snodgrass S. Robert
Published: October 2006

Abstract— The effects of divalent cations on the stimulation of [3H]catechol formation in striatal slices induced by d‐amphetamine was studied in order to determine the role of calcium in this action of amphetamine. In the absence of any divalent cations in the medium, amphetamine did not significantly stimulate [3H]catechol synthesis in striatal slices, but it produced a marked stimulation of synthesis when calcium (1.25 mm) was added to the medium. In the presence of calcium (1.25 mm), high concentrations of magnesium (15mm), other divalent cations (2.5 mm) such as barium, strontium, manganese and cobalt, as well as verapamil, inhibited the amphetamine‐induced stimulation. When the slices were incubated in medium containing no divalent cations, the addition to the medium of either strontium, cobalt, zinc, or magnesium (2.5 mm) could not support the amphetamine‐induced stimulation of [3H]catechol synthesis, while the addition of barium resulted in a significant stimulation of synthesis. In contrast, the stimulation produced by amphetamine in the presence of manganese was comparable to that observed when calcium had been added to the medium. Since amphetamine did not alter the specific activity of [3H]tyrosine in the tissue in the presence of any of the divalent cations tested, the amphetamine‐induced stimulation of [3H]catechol synthesis was probably due to an increase in tyrosine hydroxylase activity. Calcium and manganese were also able to support the stimulation of [3H]catechol synthesis in striatal slices induced by high potassium concentration. However, compared to the effects with amphetamine, manganese was much less effective than calcium in supporting the stimulation induced by high potassium concentration. These results show that specific divalent cations can support the stimulation of catechol synthesis induced by amphetamine in striatal slices, and suggest that the entry of these specific ions into cells, presumably dopamine neurons, is involved in this action.
COMPARATIVE STUDIES ON THE CHARACTERISTIC PROPERTIES OF TWO FORMS OF BRAIN ACTIN SEPARABLE BY ISOELECTRIC FOCUSSING
Authors: Flanagan Michael D. , Lin Shin
Published: October 2006

Abstract— Actin isolated from 10‐day embryonic chick brain produced a single band indistinguishable from that of muscle actin when electrophoresed in sodium dodecyl sulfate‐polyacrylamide gel. Isoelectric focussing in polyacrylamide gel showed that the isolated protein was composed of two components–the β and γ forms of actin previously detected in other mammalian nonmuscle cells and tissues–in a molar ratio of 1.1/1.0. The same ratio was observed in a sonicate of 10‐day embryonic chick brain and in various actin‐containing fractions at each step of an actin purification procedure which involved gel filtration chromatography and polymerization‐depolymerization of the protein. Additionally, the two forms of actin were found to co‐precipitate with muscle myosin and to bind to a DNase I‐agarose affinity column in this ratio. In contrast, a third isoelectrically distinct protein with the same electrophoretic mobility as actin was found in the whole brain sonicate, but not in any of the actin‐containing fractions examined. When cellular protein in neuronal and non‐neuronal cell populations isolated from 10‐day embryonic chick brain was analyzed, it was found that β and β actins were also present in each class of cells in the molar ratio of 1.1/1.0. However, this ratio decreased slightly during development of the chick brain. We conclude from this study that the β and γ forms of brain actin are very similar in several characteristic properties of actin, and that it is unlikely that brain cells utilize different forms of the protein for different types of cell motility.
LOCALIZATION OF GLUTAMINASE IN THE RAT NEOSTRIATUM
Authors: McGeer E. G. , McGeer P. L.
Published: October 2006

Abstract— Glutaminase activity in rat striatal tissue was not significantly decreased by lesions of the cortico‐striatal tract which depressed striatal glutamate uptake by 47% but was markedly decreased following intrastriatal injections of kainic acid. There also appeared to be a linear correlation between glutaminase and glutamic acid decarboxylase activities in the substantia nigra of rats injected intrastriatally with kainic acid. The results suggest that most of the glutaminase activity in these regions is localized in GABAergic structures and provide no evidence for the occurrence of this enzyme in nerve endings of the glutamergic cortico‐striatal tract.
CERAMIDE‐GALACTOSYLTRANSFERASE AND CEREBROSIDE‐SULPHOTRANSFERASE LOCALISATION IN GOLGI MEMBRANES ISOLATED BY A CONTINUOUS SUCROSE GRADIENT OF MOUSE BRAIN MICROSOMES
Authors: Siegrist H. P. , Burkart T. , Wiesmann U. N. , Herschkowitz N. N. , Spycher M. A.
Published: October 2006

Abstract— A 17,000 g supernatant of mouse brain microsomes was subfractionated on a continuous sucrose gradient in order to localise ceramide galactosyltransferase (CGalt, EC 2.4.1.47) and cerebroside sulphotransferase (CST, EC 2.8.2.11), both enzymes involved in the synthesis of myelin lipids. The submicrosomal fractions were analysed for marker enzymes of myelin, plasma membranes, Golgi membranes, endoplasmic reticulum and lysosomes, and their protein distribution was studied. The results and EM studies give evidence that CGalT and CST are located in the Golgi membranes of the brain.
EFFECT OF SENSORY STIMULATION ON AMINO ACID INCORPORATION INTO BRAIN PROTEINS IN VIVO
Authors: Abdul‐Ghani A.‐S. , Luqmani Y. A. , Bradford H. F.
Published: October 2006

Abstract— Stimulation (AES) of the brachial plexus of anaesthetised rats resulted in an increased incorporation of carbon from [U‐14C]glucose into TCA‐insoluble proteins in the contralateral cerebral hemisphere, as compared with the ipsi‐lateral hemisphere. The greatest change was observed in the sensori‐motor cortex grey matter.

Following intraventricular injections of [U‐14C]glucose, the changes caused by brachial plexus stimulation were variable, depending on which hemisphere received the label. The injection itself severely inhibited the incorporation into protein. Neither the injection, nor stimulation affected the conversion of [U‐14C]glucose into amino acids or its relative distribution between the two hemispheres.
THE EFFECT OF DELTA‐AMINOLAEVULINIC ACID ON THE UPTAKE AND EFFLUX OF AMINO ACID NEUROTRANSMITTERS IN RAT BRAIN SYNAPTOSOMES
Authors: Brennan M. J. W. , Cantrill R. C.
Published: October 2006

Abstract— δ‐Aminolaevulinic acid (δ‐ALA) is an omega amino acid structurally similar to γ‐aminobutyric acid (GABA) and l‐glutamate. We have examined the effect of δ‐ALA on the uptake and efflux of radiolabelled GABA and l‐glutamate in rat cortical synaptosomes and report: (1) low concentrations of δ‐ALA reduced the potassium‐stimulated release of [3H]GABA from the synaptosome preparation. This effect was reversed by the GABA receptor antagonist bicuculline. We postulate that GABA release is modulated by a feedback mechanism on presynaptic GABA receptors, and that δ‐ALA has agonist activity at these receptors. (2) δ‐ALA at high concentrations (0.75‐5.0 mm) stimulated the efflux of l‐[14C]glutamate from preloaded synaptosomes. (3) δ‐ALA had no effect on potassium‐stimulated release of l‐glutamate. (4) Uptake of labelled l‐glutamate was inhibited by δ‐ALA in a noncompetitive fashion. (5) Synaptosomes did not accumulate [14C]δ‐ALA in the range 0.5‐50 δm. These results are discussed in relation to the control of GABA release from nerve endings, and the role of δ‐ALA in the neuropsychiatric manifestations of the acute porphyric attack.
STEADY STATE AND REGENERATING LEVELS OF ACETYLCHOLINESTERASE IN THE SUPERIOR CERVICAL GANGLION OF THE RAT FOLLOWING SELECTIVE INACTIVATION OF PROPIONYLCHOLINESTERASE1
Authors: Koelle George B. , Rickard Kathleen Kitto , Smyrl Eloisegabel
Published: October 2006

Abstract— The effects of selective inactivation of propionylcholinesterase (PrChE) by tetramonoisopropylpyrophosphortetramide (iso‐OMPA) on the steady state and regenerating levels of acetylcholinesterase (AChE) were investigated on the superior cervical ganglion (SCG) of the rat. Over the dosage range of 1.5‐40.0 μmol iso‐OMPA/kg intraperitoneally, which produced nearly total inactivation of ganglionic PrChE and 0‐35% inactivation of AChE, there was no subsequent increase in AChE activity above the control level. Single or repeated injections of iso‐OMPA at total doses of 5.0‐40.0 μmol/kg intraperitoneally caused no reduction in the rate of regeneration of ganglionic AChE during the 24 h following its inactivation by sarin, 2.0 μmol/kg intravenously. Both sets of findings differ from those obtained previously in a similar study of ganglionic AChE and butyrylcholinesterase (BuChE) in the cat. Possible reasons for this distinct species difference are discussed.
CHARACTERIZATION AND SUBCELLULAR LOCALIZATION OF BRAIN MUSCARINIC RECEPTORS LABELLED IN VIVO BY [3H]DEXETIMIDE
Authors: Laduron P. M. , Janssen P. F. M.
Published: October 2006

Abstract— [3H]Dexetimide specifically labels brain muscarinic receptors in vivo. After i.v. injection of labelled drug into rats, radioactivity specifically accumulates in brain regions containing muscarinic receptors but not in cerebellum. This accumulation is stereospecific, saturable and displaceable by unhbelled dexetimide. In contrast, [3H]levetimide, the inactive enantiomer, does not show such preferential uptake or stereospecific displacement.

An analytical approach was used to study the subcellular distribution of [3H]dexetimide binding sites. After differential centrifugation the binding sites are mainly recovered in the microsomal fraction from different brain regions but not from the cerebellum. After displacement the radioactivity is found in the supernatant. After equilibration in a density gradient the distribution pattern of [3H]dexetimide is bimodal, like that of 5′‐nucleotidase, with a major peak in a region of low density.

When the microsomal fraction was treated with digitcnin, three groups of membrane were characterized by isopycnic centrifugation on the basis of their differential shift to higher densities. Evidence is provided that the postsynaptic membranes bearing muscarinic receptors belong to the class of plasma membranes. Finally, digitonin treatment may represent a useful tool to produce subfractions enriched in postsynaptic membranes which can now be identified biochemically in binding experiments.
SOME CHARACTERISTICS OF ATPase ACTIVITY IN A BRAIN MICROTUBULE PROTEIN PREPARATION
Authors: Larsson Harkan , Wallin Margareta , Edström Anders
Published: October 2006

Abstract— The Mg‐ and Ca‐ATPase activities in a brain tubulin preparation have been measured. The activity of the microtubule protein (MTP) preparation was optimal, 3‐4.5 nmol Pi/mg protein/min, at pH 8.0 in the presence of 1‐2 mm‐Mg2+ or Ca2+, with a half maximal stimulation at about 0.3 mm concentration of either of the divalent ions.

Phosphocellulose (PC) purified tubulin exhibited no or very low activity (0‐2 nmol Pi/mg protein/min).

The majority of ATPase activity was found in the microtubule associated proteins (MAP) fraction. It was stimulated by Mg2+ and Ca2+, inhibited by NaF or high ionic strength but unaffected by vanadate at 10−4m. In decreasing order of effectiveness ATP, GTP, UTP, CTP and ADP were hydrolyzed. p‐Npp was a poor substrate. Vmax values for Mg‐ and Ca‐ATPase activities were about 15 and 10 nmol Pi/mg protein/min, respectively with a Km value of about 25 μm. However, double reciprocal plots disclosed more complicated kinetics, which were not fully resolved.

The activity was largely confined to 30‐36S material (i.e.‘rings’and 'spirals'). The protein responsible for the ATPase activity is possibly the smaller one of the two (or three) high molecular weight (HMW) proteins of mol wt over 200,000.

There are similarities between this enzyme and both flagellar dynein and myosin. However, the present ATPase differs from myosin in several important aspects (i.e. ionic requirements). Furthermore, no peptides of the myosin type were found upon electrophoretic analysis of the MAP fraction.
MELATONIN: DEACETYLATION TO 5‐METHOXYTRYPTAMINE BY LIVER BUT NOT BRAIN ARYL ACYLAMIDASE
Authors: Rogawski Michael A. , Roth Robert H. , Aghajanian George K.
Published: October 2006

Abstract— Rat liver and brain slices were incubated in vitro with [3H]melatonin. Liver slices synthesized small amounts of [3H]5‐methoxyindoleacetic acid ([3H]5‐MIAA) along with other melatonin metabolites including 6‐hydroxymelatonin. Pretreatment of animals prior to killing with the irreversible monoamine oxidase inhibitor pargyline allowed [3H]5‐methoxytryptamine ([3H]5‐MT) to be recovered from the incubation. No [3H]5‐MIAA or [3H]5‐MT could be detected in incubations with hypothalamic slices or following intraventrieular injection of [3H]melatonin. The possibility that the deacetylase aryl acylamidase was in part responsible for the deacetylation occurring in liver slices was examined. Liver aryl acylamidase was able to utilize [3H]melatonin as substrate to produce [3H]5‐MT. Furthermore, the liver enzyme was inhibited by melatonin (Ki. 1 mm) when tested with the alternate substrate o‐nitroacetanalide. Brain aryl acylamidase did not generate any detectable [3H]5‐MT nor was it inhibited by melatonin. These results suggest that 5‐MT is not formed in brain from melatonin although trace amounts of 5‐MT in the periphery could be derived from this precursor.
SEMIAUTOMATED FLUORIMETRIC DETERMINATION OF TYROSINE IN RAT BRAIN AND HUMAN PLASMA
Authors: Evans Gwyneth , Hartmann Margot K. , Wood Liliana , Gaitonde M. K.
Published: October 2006

Abstract— The evidence is presented to show that the fiuorimetric method of Waalkes & Udenfriend (1957) cannot be used directly for the determination of free tyrosine in acid deproteinized extracts of rat brain, owing to the reaction of peptide bound tyrosine in some proteins present in the acid extracts. A method is given to obtain a fraction containing free tyrosine in the acid extracts of rat brain and to determine its tyrosine content fluorimetrically by autoanalysis. An autoanalysis method is also given for the fiuorimetric determination of free tyrosine directly in 50 trichloroacetic acid extracts of the plasma.
SUBCELLULAR DISTRIBUTION AND STRUCTURAL POLYMORPHISM OF MYELIN BASIC PROTEIN IN NORMAL AND JIMPY MOUSE BRAIN
Authors: Barbarese E. , Carson J. H. , Braun P. E.
Published: October 2006

Abstract— Brains from 20 day old normal and 20 day old Jimpy mice were fractionated by a modification of the procedure described by Eichberget al. (1964). Each of the fractions obtained was subjected to radioimmunoassay (RIA) for myelin basic protein (MBP). From both the normal brain and the Jimpy brain MBP was recovered in three separate membrane fractions designated P1A. P2A. and P3A. which differed in their sedimentation properties but which had similar densities (less than 1.08 g'ml). In the Jimpy brain compared to normal brain the amounts of P1A and P2A were greatly reduced but the amount of P3A was increased. During development in the normal brain the amount of MBP in the PIA fraction increased in parallel with the accumulation of myelin. The amount of MBP in P2A increased gradually during active myelination and decreased slightly in the adult. The amount of MBP in P3A increased sharply during the period of most active myelination and decreased approx 10‐fold as the rate of myelination in the brain declined. Electron microscopic examination revealed that the P1A and P2A fractions from normal brain contained myelin fragments while the P1A and P2A fractions from Jimpy brain contained numerous vesicular membranous structures with little if any identifiable myelin. The P3A fraction from both normal and Jimpy brain contained small vesicles of uniform size, some with polyribosomes attached. Each of the fractions was analyzed by a technique combining sodium dodecyl sulfate polyacrylamide gel electrophoresis with RIA for MBP in order to identify and quantitate the four different forms of MBP with molecular weights of 21.5 K. 18.5 K. 17 K and 14 K dalton. The proportions of the four MBPs were characteristic for each fraction. The relative proportions of the four proteins were 14 K > 18.5 K > 17 K > 21.5 K daltons in all the fractions except P1A Jimpy in which 21.5 K dalton protein was the predominant form of MBP present. The cellular origin of the MBP containing fractions from normal and Jimpy brain is discussed.
FORMATION OF A SUPPLEMENTAL LONG TIME‐CONSTANT RESERVOIR OF HIGH ENERGY PHOSPHATE BY BRAIN IN VIVO AND IN VITRO AND ITS REVERSIBLE DEPLETION BY POTASSIUM DEPOLARIZATION
Authors: Woznicki Dennis T. , Walker James B.
Published: October 2006

—Brains of mice fed a diet containing 1% cyclocreatine (1‐carboxymethyl‐2‐iminoimidazolidine) accumulated the high energy phosphate compound cyclocreatine‐P (1‐carboxymethyl‐2‐imino‐3‐phosphonoimidazolidine), an analogue of creatine‐P (phosphocreatine). During a 50‐day feeding period mouse brain cyclocreatine‐P increased linearly to 14 μmol/g fresh wt; during this time the total phosphagen level of brain, creatine‐P plus cyclocreatine‐P, increased from 3 to 15 μmol/g. When the blood‐brain barrier was circumvented, a more rapid accumulation of synthetic phosphagen was achieved. Minced brain preparations from 11 to 15‐day chick embryos incubated in vitro with 30 mm‐cyclocreatine accumulated 10 μmol/g of cyclocreatine‐P in 90 min, and this novel high energy phosphate pool could be depleted by incubation with 105 mm‐potassium ions or 3 μm‐valinomycin. Subsequent regeneration of the depleted pools could also be demonstrated. Brain tissue containing a supplemental reservoir of cyclocreatine‐P, which is utilized to regenerate ATP much more slowly than creatine‐P, might be better able to withstand anoxia and certain other metabolic stresses, but this has not been established. However, the marked delay of onset of rigor previously shown to occur in ischemic heart and skeletal muscle of cyclocreatine‐fed animals is compatible with this suggestion.
BIOSYNTHESIS OF HETEROGENEOUS FORMS OF MAMMALIAN BRAIN TUBULIN SUBUNITS BY MULTIPLE MESSENGER RNAs
Authors: Marotta Charles A. , Strocchi Paola , Gilbert Jeffrey M.
Published: October 2006

—Heterogeneity among the primary translation products of rat brain tubulin messenger RNA was examined. On two‐dimensional gels native cytoplasmic tubulin from randomly bred rats (PB21) consists of two groups of α tubulin subunits among which the most acidic forms, α1 and α2, are most abundant; and β tubulin consists of a minimum of two species, β1 and β2. In the same group of animals the primary translation products of rat brain tubulin mRNA consist of at least these four subunit forms (α1α2, β1 and β2); however, minor basic forms of α subunits were not synthesized. This same result was obtained from a homologous brain protein synthesizing system, a heterologous system prepared from brain polysomes and rabbit reticulocyte initiation factors, and a wheat germ lysate programmed with brain poly A mRNA. A variant form of brain tubulin was found in rats bred monogamously for over 30 generations (MB71 rats). MB71 brain polysomes synthesize overlapping a subunits which migrate in two‐dimensional gels to the α1 position, and the typical PB21 α2 is not present. The addition of PB21 brain mRNA to a protein synthesizing system composed of MB71 polysomes plus reticulocyte initiation factors allowed synthesis of the typical α2 tubulin in addition to the MB71 tubulin subunits. The structural relationship among subunits was examined by radioiodinated peptide mapping. The α subunits are structurally different from the β subunits; however, among the major tyrosine‐containing tryptic peptides no prominent differences were observed between α1 and α2, or between β1 and β2 by the radioiodination procedure. The results provide evidence for heterogeneity among the primary translation products of brain tubulin mRNA, and for the existence of multiple functional tubulin genes in rat brain.
NIACIN AND NIACINAMIDE ACCUMULATION BY RABBIT BRAIN SLICES AND CHOROID PLEXUS IN VITRO
Authors: SPECTQR REYNOLD , KELLEY PAUL
Published: October 2006

The transport into and release of14C‐labeled niacin and niacinamide from rabbit brain slices and isolated choroid plexuses were studied. In vitro, both brain slices and choroid plexus concentrated 14C by specific, energy‐dependent mechanisms when [14C]niacinamide was added to the incubation medium. The saturable accumulation velocities, which were linear for 30 min, depended, in part, on incorporation of the [14C]niacinamide into NAD. The XT and Ymax for 14C accumulation with [14C]niacinamide in the medium by brain slices and choroid plexus were 0.80 μM and 1.45 μmolkg−1 (30 min)−1, and 0.23 μM and 18.6 μmol kg−1 (30 min)−1 respectively. In vitro, the choroid plexus, unlike brain slices, vigorously concentrated 14C by a separate, specific energy‐dependent process when 14C niacin was added to the incubation medium. The saturable accumulation velocity, which was linear for 30 min, depended completely on the metabolism of [14C]niacin. The KT and Ymax for14C accumulation by choroid plexus with [14C]niacin in the medium were 18.1 μM and 439 μmol kg−1 (30 min)−1 respectively. Whether preincubated in [14C]niacin or [14C]niacinamide, choroid plexus released predominantly [14C]niacinamide.
Experimental Scrapie in the Mouse: Electrophoretic and Sedimentation Properties of the Partially Purified Agent
Authors: Prusiner Stanley B. , Garfin David E. , Cochran S. Patricia , McKinley Michael P. , Groth Darlene F. , Hadlow William J. , Race Richard E. , Eklund Carl M.
Published: October 2006

: Some biochemical and biophysical properties of the scrapie agent in a partially purified fraction P5 from murine spleen are described in this communication. The agent was stable in the nonionic detergents Triton X‐100 and Nonidet P40 and stable in the nondenaturing, anionic detergents sodium cholate and sodium N‐lauroyl sarcosinate. In contrast, sodium dodecyl sulfate (SDS) inactivated the agent at high concentrations (1% or >) when the detergent‐to‐protein ratio approached 1.5 g SDS/g protein. The agent was resistant to inactivation by nucleases and proteases, even in the presence of 0.1% SDS. A broad peak of infectivity was exhibited in modified colloidal silica (Percoll) density gradients. Maximal titers were found at a Percoll density of 1.10 g/cm3 in the presence and absence of 0.05% SDS. Gel electrophoresis of the agent in the presence of 0.1% SDS resulted in inactivation of >95% of the agent loaded onto the gel. Free‐flow electrophoresis showed that >99% of the agent in fraction P5 migrated toward the anode, but not as a discrete species. Sedimentation analysis of the agent in fraction P5 in the presence of 1% lysolecithin showed that the agent has a sedimentation coefficient of <300S but >30S. Heating P5 preparations caused the agent to associate with cellular elements and form aggregates with sedimentation coefficients >10,000S. Removal by differential centrifugation of the large forms of the agent produced upon heating permitted characterization of a discrete subpopulation of scrapie agent particles. Rate‐zonal sucrose gradient studies showed that >>95% of the infectivity in this subpopulation sedimented as uniform particles with a sedimentation coefficient of 240S.
Cerebral Blood Flow and Metabolism During Hypoglycemia in Newborn Dogs
Authors: Hernández Milton J. , Vannucci Robert C. , Salcedo Alberto , Brennan Robert W.
Published: October 2006

: Cerebral blood flow (CBF) and cerebral metabolic rates (CMR) were studied in newborn dogs during insulin‐induced hypoglycemia. Pups were anesthetized, paralyzed, and artificially ventilated with a mixture of 70% nitrous oxide and 30% oxygen to maintain normoxia and normocarbia. Experimental animals were given regular insulin (0.3 units/gm IV); controls received normal saline. CBF was determined using a modification of the Kety‐Schmidt technique employing 133Xe as indicator. Arteriovenous differences for oxygen, glucose, lactate, and β‐hydroxybutyrate (β‐OHB) were also measured, and CMRo2 and CMRsubstrates calculated. Two groups of hypoglycemic dogs were identified; those in which blood glucose levels were greater than 0.5 mm (group 1), and those in which they were less than 0.5 mm (group 2). CBF did not change significantly from control values of 23 ± 10 ml/min/100 g (mean ±s.d.) at both levels of hypoglycemia. Similarly, hypoglycemia did not alter CMRo2, significantly from its initial level of 1.05 ± 0.37 ml O2/min/100 g. Glucose consumption in brain during normoglycemia accounted for 95% of cerebral energy supply with minimal contributions from lactate (4%) and β‐OHB (0.5%). During hypoglycemia, CMRglucose. declined by 29 and 52% in groups 1 and 2, respectively, while CMR,lactate increased to the extent that this metabolite became the dominant fuel for oxidative metabolism in brain. The cerebral utilization of β‐OHB was unaltered by hypoglycemia. The findings indicate that insulin‐induced hypoglycemia in the newborn dog is associated with an increase in cerebral lactate utilization, supplementing glucose as the primary energy fuel and thereby preserving a normal CMRo2. These metabolic responses may contribute to the tolerance of the immature nervous system to the known deleterious effects of hypoglycemia.
Distribution of GABA Receptors in the Rat Cerebellum
Authors: Kingsbury A. E. , Wilkin G. P. , Patel A. J. , Balázs R.
Published: October 2006

: The distribution of GABA receptors in the cerebellum is not homogeneous. In comparison with detergent‐treated membranes from the whole tissue the number of [3H]muscimol binding sites per mg protein (Bmax) is about doubled in preparations enriched in large fragments of the cerebellar glomeruli, and it is about one‐third in the dissected deep nuclei. On the other hand, the apparent affinity (Kd) is similar in the different preparations. Comparison of the results with earlier studies suggests a heterogeneity in cerebellar GABA receptors and/or their control.
The Utilization of Choline and Acetyl Coenzyme A for the Synthesis of Acetylcholine
Authors: Jope Richard S. , Jenden Donald J.
Published: October 2006

Acetylcholine synthesis in rat brain synaptosomes was investigated with regard to the intracellular sources of its two precursors, acetyl coenzyme A and choline. Investigations with α‐cyano‐4‐hydroxycinnamate, an inhibitor of mitochondrial pyruvate transport, indicated that pyruvate must be utilized by pyruvate dehydrogenase located in the mitochondria, rather than in the cytoplasm, as recently proposed. Evidence for a small, intracellular pool of choline available for acetylcholine synthesis was obtained under three experimental conditions. (1) Bromopyruvate competitively inhibited high‐affinity choline transport, perhaps because of accumulation of intracellular choline which was not acetylated when acetyl coenzyme A production was blocked. (2) Choline that was accumulated under high‐affinity transport conditions while acetyl coenzyme A production was impaired was subsequently acetylated when acetyl coenzyme A production was resumed. (3) Newly synthesized acetylcholine had a lower specific activity than that of choline in the medium. These results indicate that the acetyl coenzyme A that is used for the synthesis of acetylcholine is derived from mitochondrial pyruvate dehydrogenase and that there is a small pool of choline within cholinergic nerve endings available for acetylcholine synthesis, supporting the proposal that the high‐affinity transport and acetylation of choline are kinetically coupled.
Suppressive Effect of Triethyllead on Entry of Proteins into the CNS Myelin Sheath In Vitro
Authors: Konat G. , Clausen J.
Published: October 2006

Incorporation of [14C]leucine into the myelin sheath was studied in brain stem slices prepared from 22‐day‐old rats. Individual major myelin proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. There was a time lag before incorporation of the label into proteolipid protein (PLP) and intermediate protein (IP) reached maximal rates. Labelling of basic proteins (BP) and Wolfgram proteins (WP) revealed a much shorter lag in entry. Appearance of radioactive proteins in the myelin sheath was significantly hampered by triethyllead (PbEt3) added to the incubation medium at micromolar concentrations. Inhibition values were highest in the case of PLP and were closely followed by the values for IP. BP and WP were less inhibited, although incorporation of these proteins into myelin was still suppressed more than was synthesis of total homogenate protein. Thus, myelin‐forming cells seem to be unduly vulnerable to the toxin relative to the rest of the tissue. Furthermore, the results indicate an interference of PbEt3 with certain posttranslational processes involved in furnishing of integral myelin proteins.
Bovine P2 Protein: Sequence at the NH2‐Terminal of the Protein
Authors: Weise Michael J. , Hsieh Diane L. , Levit Shimon , Brostoff Steven W.
Published: October 2006

Sequence data from key fragments of the P2 protein established the order of cyanogen bromide (CNBr) peptides in the structure of the protein and the primary structure for approximately one‐half of the molecule. Data were obtained from the three tryptic peptides of blocked NH2‐terminal CNBr peptide (CN3), the large CNBr peptide of P, protein (CN1), and a fragment obtained from P2 by cleavage at tryptophan with 2‐(2‐nitrophenylsulfenyl)‐3‐methyl‐3′‐bromoindolenine. This last fragment was found to contain an overlapping sequence that proved the juxtaposition of CN 1 and CN3 in P2 protein. Thus, based on this fact and the characteristics of the CNBr peptides, the P2 structure is composed of CNBr peptides in the order: CN3‐CN1‐CN2(Va1)‐CN2(Lys). A comparison was made between the partial sequence of P2 protein and the equivalent portion of the structure of bovine myelin basic protein. The structures of these two proteins were found to be distinctly different although certain similarities are found.
l‐Histidine Decarboxylase in the Human Brain: Properties and Localization
Authors: Barbin G. , Palacios J. M. , Garbarg M. , Schwartz J. C. , Gaspar P. , Javoy‐Agid F. , Agid Y.
Published: October 2006

The properties of the histamine‐forming enzyme in human brain samples were studied utilizing a radiochromatographic procedure. The influence of postmortem conditions was checked with rat brains, and the results indicated that the enzyme activity is not altered in situ for a delay not exceeding 4 h at ambient temperature. Moreover, tissue blocks or homogenates can be stored at low temperatures for up to 3 months with a good preservation of the enzyme activity. The data indicate that histamine synthesis in the human brain involves the „specific” histidine decarboxylase (HD, EC 4.1.1.22) and not the aromatic l‐amino acid decarboxylase; (1) the optimum pH is 7.4 at 10‐6m‐l‐histidine; (2) the apparent Km is about 3.10‐5m; (3) it is inhibited by α‐hydrazino histidine and brocresine but not affected by α‐methyl DOPA. Moreover, a major portion of the enzyme is localized in a subcellular fraction containing nerve terminals and it shows an uneven regional distribution which parallels that observed in the brain of other mammalian species. Taken together these data strongly suggest that histamine could play a neurotransmitter role in the human brain.
Brain Cyclic Nucleotide Responses to Anesthesia With Halothane Delivered in Air or Purified Oxygen
Authors: Divakaran P. , Rigor B. M. , Wiggins R. C.
Published: October 2006

Inhalation of either 0.5% or 1.0% halothane in air caused a slight decrease in the cAMP concentration in rat cerebral cortex and cerebellum. During recovery, concentrations returned to normal in 3 h, or less. In contrast, cGMP decreased sixfold in cerebellum, but increased twofold in cortex. Recovery time for cerebellum was several hours. When oxygen was used as the carrier gas for halothane delivery, cAMP in the cortex doubled, in striking contrast to the case with halothane in air. Oxygen alone had no apparent effect. The cGMP effect of halothane delivered in oxygen appeared the same as for halothane in air. Thus, the cAMP effects of brain halothane are related to the enrichment of oxygen.
Turnover of Molecular Species of Sphingomyelin in Microsomes and Myelin of Rat Brain
Authors: Freysz L. , Mandel P.
Published: October 2006

The metabolism of sphingomyelins containing short‐chain fatty acids (C18:sphingomyelin) and those containing long‐chain fatty acids (C24:sphingomyelin) in microsomes and myelin from rat brain were studied for periods from 1 to 60 days after intracisternal injection of [32P]Na2HPO4. In microsomes the half‐lives of both molecular species of sphingomyelin were 14 days. In myelin the half‐life of C18:sphingomyelins was 69 days, whereas C24: sphingomyelins exhibit a high degree of metabolic stability. The results suggest that in myelin the sphingomyelins containing short‐chain fatty acids may be located in the inner leaflet of the bilayer and those containing long‐chain fatty acids may be located in the outer leaflet.
3H‐Saxitoxin Binding to Nerve Membranes: Inhibition by Phospholipase A2 and by Unsaturated Fatty Acids
Authors: Baumgold Jesse
Published: October 2006

The mechanism by which phospholipase A2 (EC 3.1.1.4) inhibits the binding of 3H‐saxitoxin (STX) to membranes from lobster walking‐leg nerves was examined. It was concluded that this inhibition of the binding was due to the accumulation of unsaturated fatty acids and lysophosphatides, products of the lipase action, in the 3H‐STX binding site. The destruction of the lipids themselves did not appear to cause the loss of the binding. The following evidence was presented in support of these conclusions. (1) Among the phospholipases tested, phospholipase A2 was the most effective in inhibiting the binding. Phospholipase A2 is also the only lipase that produces fatty acids and lysophosphatides. (2) The addition of fatty acid‐free bovine serum albumin protected the 3H‐STX binding sites from the inhibitory action of phospholipase A2; fatty‐acid‐free bovine serum albumin is capable of binding free fatty acids and lysophosphatides, thus preventing these products from interacting with the STX binding sites. (3) The addition of exogenous unsaturated fatty acids or lysophosphatides to membrane material inhibited the binding. These findings show that the STX binding activity is sensitive to unsaturated fatty acids and to lysophosphatides.
Analysis of Messenger RNA Coding for S100 Protein in the Mammalian Brain
Authors: Mahony James B. , Brown Ian R.
Published: October 2006

S100 protein is a brain‐specific protein which is absent at birth and first appears in rabbit brain 2–3 days after birth. To determine how the synthesis of this brain‐specific protein is regulated, mRNA was isolated from brain polysomes and assayed for S100 protein mRNA activity by in vitro translation in a heterologous cell‐free system and immunoprecipitation of released polypeptides with rabbit anti‐S I00 protein antiserum. 5100 protein mRNA was detected primarily in small polysomes containing five to eight ribosomes, and virtually no S 100 protein mRNA was present in polysomes containing more than eight ribosomes. S100 protein mRNA was not detected in brain polysomes at stages prior to the induction of synthesis of S100 protein, i.e., in fetal brain or in 1‐day neonates. The amount of S100 protein mRNA in polysomes of the cerebral cortex and cerebellum was measured to see if it correlated with the level of S100 protein in the two regions of adult brain. The cerebellum, which contained three to four times the level of S100 protein in the cerebral cortex, contained four times more S100 protein mRNA.
Comparison of 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase and the Major Component of Wolfgram Protein W1
Authors: Drummond Robert J. , Dean Grace
Published: October 2006

Wolfgram protein preparations from myelin are heterogeneous mixtures having major components with molecular weights in the 43,000 to 60,000 range. Through the use of an SDS‐slab gel electrophoretic system of high resolving power, the Wolfgram 1 (W1) component described by Nussbaum et al. (1977) has been resolved into two components; bovine Wla and Wlb proteins are found to have mobilities identical to those of the two 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (2′,3′‐CN 3′‐ase) components, CNa and CNb (Drummond, 1979). Crossed immunoelectrophoresis with bovine 2′,3′‐CN 3′‐ase demonstrates that CNa and CNb are antigenically indistinguishable. Rat and bovine Wolfgram proteins Wla and Wlb, but not the W2 proteins, display cross reactivity with antiserum directed against bovine 2′,3′‐CN 3′‐ase. Chymotryptic and Staphylococcus aureus V8 protease peptide profiles indicate structural similarities between proteins CNa ‐Wla and CNb‐Wlb. It is concluded that an inactivated form of 2′,3′‐CN 3′‐ase is a major component of Wolfgram protein W1. These results are consistent with the studies on the histochemical localization of rat W1 protein by Roussel et al. (1977, 1978).