bluuebunny/crossref_metadata_2025 huggingface dataset:
POLYAMINE METABOLISM IN THE BRAIN AND LIVER OF THE DEVELOPING MONKEY
Authors: Sturman J. A. , Gaull G. E.
Published: October 2006

Abstract— The concentrations of putrescine and the polyamines, spermidine and spermine, along with the specific activities of the enzymes involved in their biosynthesis, ornithine decarboxylase, S‐adenosylmethionine decarboxylase and spermidine synthase have been measured in brain and liver of the developing Rhesus monkey from mid‐gestation, through birth and neonatal life to maturity. The results suggest that it is an increased concentration of putrescine and an increased specific activity of ornithine decarboxylase which are associated with the rapid growth of fetal brain during the middle of gestation. By the end of two‐thirds of gestation, both of these parameters have attained values similar to those found in mature brain. The concentration of spermidine in brain and the specific activities of S‐adenosylmethionine decarboxylase and spermidine synthase are lower in fetal brain than adult brain and increase slowly after birth to reach values similar to those of the adult only after several months. These results provide additional evidence that in the mature brain spermidine serves some function other than one associated with rapid growth.

Fetal liver at mid‐gestation was characterized by increased concentrations of both putrescine and spermidine and increased specific activities of the enzymes which synthesize them. By two‐thirds of gestation, values similar to those found in adult liver had been attained. Liver has thus reached maturity with regard to polyamine metabolism by this time.
FLOW OF GLUCOSE CARBON INTO BRAIN LIPIDS IN ADULT RATS FOLLOWING FOOD DEPRIVATION
Authors: Barkai A. , Rapport M. M.
Published: October 2006

Abstract— Rates of flow of glucose carbon in vivo into brain cholesterol, phospholipids, cerebrosides and gangliosides and concentrations of these lipids in the brain, were determined in adult rats after various periods of food deprivation. The rates were calculated from two measurements, the curve representing the decrease of plasma [14C]glucose specific activity with time and the specific activity of the brain lipid 180 min after intravenous injection of a tracer dose of d‐[U‐14C]glucose. Specific activities of brain lipids in rats deprived of food for 72h were significantly higher than in postabsorptive rats which were treated with the same dose of [14C]glucose. These higher specific activities were interpreted as a result of more labelled glucose available to lipid synthesis in the brain of fasted rats due to the substantial decrease in the rate of irreversible disposal of glucose by the whole body, commonly observed in fasted animals. The possibility that the higher specific activity values resulted from enhanced synthesis of brain lipids from glucose was ruled out since no changes were observed in the rate of flow of glucose carbon into brain lipids after food deprivation. The rate of flow of glucose carbon into gangliosides (15.4 ng C/min/mg C) was more than twice as fast as into either phospholipids or cerebrosides and about 4 times as fast as into cholesterol. The rates of carbon flow were used to calculate half lives of glucose carbon in the different classes of brain lipids. These half life values were 31 days for gangliosides, 72 days for phospholipids, 82 days for cerebrosides and 133 days for cholesterol. The results suggest that the synthesis of brain lipids from glucose is not affected by prolonged starvation in the adult rat.
TRANSPORT OF CHOLESTEROL IN THE CHICK OPTIC SYSTEM
Authors: Rostas J. A. P. , McGregor Angela , Jeffrey P. L. , Austin L.
Published: October 2006

Abstract—

After injection of radioactive cholesterol into the vitreous humour of chicks, cholesterol is axonally transported along the optic nerve to the contralateral optic tectum. In the optic nerve of young chicks cholesterol is only transported at the slow rate whereas in the sciatic nerve of adult chicks it is transported at both the fast and slow rates. This difference is not due to age or a peculiarity of avian species as the same difference exists in mice. Estimates of the cholesterol content of tissues and the amount of cholesterol transported suggests the existence of small rapidly turning over pools of cholesterol in nerves. Due to its great metabolic stability, cholesterol is a useful and specific marker for slow transport in the optic nerve and thus may prove suitable for the mapping of neuronal projections.
TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE IN HUMAN BRAIN
Authors: Viola M. V. , Cole Margery L. , Norton P.
Published: October 2006

Abstract—

A DNA polymerase lacking template direction for base selection has been partially purified from human brain. The molecular size, optimum reaction conditions, initiator preferences and chemical inhibitors of the brain enzyme were similar to calf thymus terminal deoxynucleotidyl transferase (TdT). TdT has been found only in the thymus, and now in brain. The possibility exists that its function is related to biological property unique to these two organs.
BIOCHEMICAL AND IMMUNOLOGICAL CHARACTERIZATION OF ALKALOID‐BINDING PROTEINS FROM IMMATURE RAT BRAIN1
Authors: Twomey S. L. , Raeburn S. , Baxter C. F.
Published: October 2006

Abstract— Vinblastine‐ and colchicine‐binding proteins in the soluble fraction of immature rat brains were characterized and compared. Based upon criteria of Sephadex G‐200 chromatography, electrofocusing and immunological reactivity, several separable species of vinblastine‐binding protein were isolated. By contrast, these same procedures yielded only one protein band or elution peak to which [14C]colchicine could be tightly bound. This colchicine‐binding protein peak coincided, in part, with one of the protein peaks to which [3H]vinblastine was tightly bound. Rabbit antiserum against soluble brain proteins precipitated by vinblastine sulfate contained antibodies which reacted with colchicine‐binding protein. Thus, despite apparent differences in physical properties between the bulk of the vinblastine‐binding proteins and the colchicine‐binding protein, the vinblastine sulfate‐precipitated protein antigens gave rise to antibodies capable of forming an immune complex with colchicine‐binding protein.
BIOSYNTHESIS OF RAT BRAIN PHOSPHATIDYLCHOLINES FROM INTRACEREBRALLY INJECTED CHOLINE1
Authors: Arienti G. , Corazzi L. , Woelk H. , Porcellati G.
Published: October 2006

Abstract— [Me‐3H] Choline was injected intracerebrally into male rats and the brains immediately removed by particular procedures at regular intervals over the first 1200 s. The incorporation of radioactivity into brain phosphorylcholine, CDP‐choline and phosphatidylcholines was examined and quantitated, in order to investigate the relative roles of net synthesis and base‐exchange reactions for choline incorporation into lipid. The molecular subspecies of phosphatidylcholines were also examined after isotope administration. Phosphorylcholine, CDP‐choline and phosphatidylcholines all became labelled as early as 5 s after the administration of labelled choline. The time course of incorporation of choline into brain lipid is biphasic with two flex points at about 20 and 120 s from the injection. The specific radioactivity of different phosphatidylcholines appears to be different at early and later intervals from injection. The suggestion is made that the base‐exchange pathway for choline incorporation into lipid might be operative in vivo in early periods after administration.
NEUROTRANSMITTER UPTAKE INTO SLICES OF RAT CEREBRAL CORTEX IN VITRO: EFFECT OF SLICE SIZE
Authors: Riddall D. R. , Leach M. J. , Davison A. N.
Published: October 2006

Abstract— The uptake of [14C]GABA, [14C]taurine, [3H]β‐alanine and [14C]dopamine was compared in slices of rat cerebral cortex of three different sizes (0.1 × 0.1 × 2 mm, 0.2 × 0.2 × 2 mm and 0.4 × 0.4 × 2 mm prepared with a mechanical tissue chopper). [14C]Taurine and [3H]β‐alanine uptake increased whereas [14C]GABA uptake decreased with increasing slice size. [14C]Dopamine uptake was optimal in 0.2 × 0.2 × 2 mm slices. Increasing slice size was shown to decrease inhibition of [3H]β‐alanine and [14C]GABA uptake by l‐2,4‐diaminobutyric acid. Lactate dehydrogenase activity increased with increasing slice size indicating decreased tissue damage or increased cellular integrity. The possibility that varying slice size can be used to distinguish between neuronal and glial uptake is discussed. It is suggested that taurine uptake in the cerebral cortex is predominantly glial.
5′‐ADENINE MONONUCLEOTIDES TN SYNAPTOSOMAL PREPARATIONS FROM GUINEA PIG NEOCORTEX: THEIR CHANGE ON INCUBATION, SUPERFUSION AND STIMULATION
Authors: Barberis C. , McIlwain H.
Published: October 2006

Abstract—

–(l) The contents of potassium and of ATP, ADP and AMP of homogenates of guinea pig neocortex in 0.32 M‐SUCROSE, and of synaptosomal preparations derived therefrom, were determined and effects of incubation, superfusion and stimulation of the preparation were examined. The synaptosome preparations in M‐SUCTOSe carried a smaller content of 5′‐nucleotide per unit protein than did the homogenate from which they were derived. However, the proportion of synaptosomal nucleotides present as ATP was markedly greater than in the homogenate as a whole, the adenylate energy‐charge being 83% greater in the synaptosomes than in the homogenate. Dilution of the synaptosomal preparation from the 1 M‐sucrose to isotonic sucrose, decreased the contcnt of ATP per unit protein, but did not change the sum ATP + ADP + AMP.

(2) Examined as deposited beds during 5 to 20 min incubation in oxygenated glucose‐bicarbonate salines, synaptosomal K content and adenylate energy charge increased. These changes were sustained during a subsequent 40 min of incubation and also during superfusion. During such continued superfusion, electrical stimulation caused diminution of the ATP and the adenylate energy charge of the beds, as also did superfusion with fluids of increased K‐content.

(3) Lactate formation by the superfused beds was relatively stable during 30 min incubation. By electrical stimulation, the rate of lactate formation was increased by up to 30%. Increase in the potassium content of superfusion fluids could however increase lactate production 2.4‐fold. The basis for these actions is discussed.
CYTOCHROMES IN BRAIN MITOCHONDRIA FROM LAMBS WITH ENZOOTIC ATAXIA
Authors: Smith R. M. , Osborne‐white W. S. , Odell B. L.
Published: October 2006

Mitochondria from the cerebral cortex of untreated lambs from areas in which enzootic ataxia occurred showed a lower content of haem a (from cytochromes a and a 3) than mitochondria from control lambs with adequate copper but the haems of cytochromcs b2 c1 and c were not depleted. Cytochrome oxidase activity was closely correlated with the content of haem a (P<0.001) but there was no correlation with total mitochondrial copper which was always present in molar exccss of haem a. It is concluded that the lowered cytochrome oxidase activity in mitochondria from untreated lambs is immediately attributable to depletion of haem a. In clinically ataxic lambs that showed degradation of myelin in thc spinal cord, brain cytochrome oxidase was depressed by no more t han 60%. Arguments are advanced that this depletion was not sufficiently severe to have led to respiratory constraint
OLIGOSACCHARIDE STORAGE IN BRAINS FROM PATIENTS WITH FUCOSIDOSIS, GM1‐GANGLIOSIDOSIS AND GM2‐GANGLIOSIDOSIS (SANDHOFF'S DISEASE)1
Authors: Tsay Grace Chen , Dawson Glyn
Published: October 2006

Abstract— Analysis of whole autopsy brain from a patient with fucosidosis (α‐fucosidase deficiency) revealed minor storage of H‐antigen glycolipid [Fuc (α, 1→2) Gal‐GlcNAc‐Gal‐Glc‐Ceramide] and a slightly abnormal ganglioside composition in the form of a two‐fold elevation of GM1 and the presence of a fucose‐containing glycolipid (a minor component) which co‐migrated with GD1a. The major storage materials in fucosidosis brain were an oligosaccharide (Fuc‐Gal‐GlcNAc‐Man[Fuc‐Gal‐GlcNAc‐Man]‐ManGlcNAc) and a disaccharide [Fuc(α, 1→6)‐GlcNAc] in the approximate ratio of 5:1. Lesser amounts of a related oligosaccharide (Gal‐GlcNAc‐Man[Gal‐GlcNAc‐Man]‐Man‐GlcNAc) were isolated from the brain of patients with GM1‐gangliosidosis (Types I and II) where the major storage material is known to be GM1‐ganglioside (Gal (β, 1→3)GalNAc(β, 1→4) [NeuNAcf(α, 2→3) Gal(β, 1→4)Glc‐Ceramide). Similarly, a related oligosaccharide (GlcNAc‐Man [GlcNAc‐Man]‐Man‐GlcNAc) was isolated from the brain of a patient with a total deficiency of N‐acetyl‐β‐d‐hexosaminidase (Sandhoff variant of GM2‐gangliosidosis) where the major storage products are known to be GM2‐ganglioside (GalNAc (β 1→4) [NeuNAc (α, 2→3)Gal(β, 1→4)Glc‐Ceramine) and its asialo derivative. These studies indicate that glycoproteins containing at least 2 mol of l‐fucose per oligosaccharide unit are normally catabolized in human brain. Further, it appears that such glycoproteins are initially catabolized by an endo‐N‐acetylglucosaminidase to release an oligosaccharide which is then degraded by the sequential action of exo‐glycosidases.
UPTAKE AND METABOLISM OF 5‐METHYL‐TETRAHYDROFOLIC ACID BY RAT BRAIN SLICES
Authors: Schnell Mercedes , Ordóñez Luis A.
Published: October 2006

Abstract— The uptake and metabolism of CH3‐[14C]tetrahydrofolic acid (MTHF) by rat brain was studied in vitro. At physiological concentrations, (10−10m), a small net uptake of the labeled compound was observed in whole brain, as well as in the cerebrum, cerebellum and brain stem slices. The passive diffusion observed in the presence of KCN proceeded slowly, equilibrium being achieved after 120 min. DEAE–Sephadex chromatography of the radioactive labeled material present in the tissue showed that a pseudo‐uptake mechanism was operative. Eighteen percent of the label was found in methionine, and after pre‐incubation with KCN this was reduced to approx 9%. Neither dietary folate deficiency, nor the availability of a methyl group acceptor (l‐DOPA), were able to increase the observed pseudo‐uptake. Nevertheless, l‐DOPA reduced the proportion of label found as unmetabolized MTHF in the slices at the end of the incubation (47% vs 59%) while increasing other (methylated ?) metabolites. The pseudo‐uptake process was not detected in newborn animals; it increased linearly with age, reaching adult levels at 10 days of age.

It is suggested that uptake mechanisms are not responsible for maintaining brain folate levels during folate deficiencies in the adult animal.
BINDING CHARACTERISTICS OF 125I‐LABELLED TETANUS TOXIN TO PRIMARY TISSUE CULTURES FROM MOUSE EMBRYONIC CNS
Authors: Dimpfel W. , Habermann E.
Published: October 2006

Abstract— The interaction of 125I‐labelled tetanus toxin with cells in tissue cultures derived from embryonic CNS has been studied.

The optimum toxin binding occurs about 2–3 weeks after transfer of the cells to culture conditions. The amount of label bound per culture was doubled at this time in comparison to the fourth day nfter inoculation.

The amount of toxin bound depends on the concentration applied. It reaches its maximum 8 h after application then decreases slowly. Low amounts of radioactivity were still detectable 97 h after washing off the unbound toxin. Up to 80% of the label can be replaced by simultaneous application of‘cold’toxin. Fixation of the toxin is higher at 4°C than at 37°C.

Preincubation of the cultures with neuraminidase prevents about 75% of the binding. The presence of cytochalasin B leads to a small but reproducible decrease of binding, whereas colchicine had no measurable effect.

The radioactive (1251) material was identified by a double‐isotope technique in disc gel electrophoresis before and after reductive cleavage of its disulphide bonds. In every test is was indistinguishable from 131I‐labelled toxin added as standard.

Our results largely parallel those obtained with synaptosomes and other systems. They suggest that gangliosides might be the acceptor molecules, and that the culture system will be suitable for studying the actions of this toxin in vitro.
ACTIONS OF l‐GLUTAMATE AND RELATED AMINO ACIDS ON OXYGEN UPTAKE, LACTATE PRODUCTION AND NADH LEVELS OF RAT BRAIN IN VITRO
Authors: Cox D. W. G. , Osborne R. H. , Watkins J. C.
Published: October 2006

Abstract— A range of acidic amino acids differing in (i) their potency as neuronal excitants, (ii) their transport properties and (iii) their ability to act as substrates for metabolism have been compared with respect to their effects on energy metabolism of rat cerebral cortex in vitro.l‐Glutamate, and d‐ and l‐homocysteate, increased tissue slice NADH levels, and the same three amino acids, together with d‐glutamate and kainate, increased oxygen uptake by the slices. It was concluded that these effects were predominantly due to neuronal depolarization and the ensuing activation of ion pump mechanisms. l‐Glutamate, d‐glutamate and l‐homocysteate increased lactate production by the slices, whereas d‐homocysteate and kainate did not. Since the two latter amino acids are the strongest neuroexcitants but probably the least rapidly transported, it is suggested that stimulation of lactate production in slices by amino acid excitants is a consequence of the energy requirements of active uptake of the amino acids, and probably occurs mainly in glial cells. Although the metabolism of l‐glutamate appeared not to be an essential requirement for the effects observed with this amino acid in the present work, such metabolism may make a proportionately greater contribution under sub‐optimal conditions of slice preparation and incubation, where electrical activity of the tissue may be impaired.
TAURINE IN DEVELOPING RAT BRAIN: SUBCELLULAR DISTRIBUTION AND ASSOCIATION WITH SYNAPTIC VESICLES OF [35S]TAURINE IN MATERNAL, FETAL AND NEONATAL RAT BRAIN
Authors: Rassin D. K. , Sturman J. A. , Gaull G. E.
Published: October 2006

Abstract—

The concentration of taurine in the brain of the fetus in several species is higher than that found in the mature animal. In order to explore the functional significance of this, we have studied the subcellular distribution of taurine and [35S]taurine in the brain of the mother, the fetus and the neonate after [35S]taurine was administered to pregnant rats.

In maternal brain, the distribution of taurine and of radioactivity (all of which was recovered from brain as taurine) in the subcellular fractions of maternal brain were essentially identical and were recovered primarily in two fractions (72% taurine, 71% [35S]taurine was soluble, S3; 16% and 17%, respectively, was in the crude mitochondrial and synaptosomal fraction, P2). After further fractionation of P2, most of the taurine and [35S]taurine were in the cytoplasmic, O, and the synaptosomal, B, fractions.

In the neonatal brain, shortly after birth there was a decrease in taurine and [35S]taurine recovered in the supernatant fraction, S3, accompanied by an increase in the percentage of taurine and [35S]taurine recovered in the crude mitochondrial fraction. A small percentage of taurine and [35S]taurine was consistently recovered in the synaptic vesicle fraction. Fractionation of the synaptic vesicles on a gel column separated the vesicle bound taurine completely from the free taurine: approx 1% of the taurine in the synaptic vesicle fraction was eluted with vesicles and could not be released by hypo‐osmotic shock.

The pattern of development in subcellular fractions of neonatal rat brain labelled with [35S]taurine via intraperitoneal injections of the pregnant mother may be an indication of maturation or protection of putative taurinergic nerve endings.
PRELIMINARY ESTIMATES OF THE DOPAMINE β‐HYDROXYLASE CONTENT AND ACTIVITY IN PURIFIED NORADRENERGIC VESICLES1
Authors: Klein R. L. , Kirksey D. F. , Rush R. A. , Goldstein M.
Published: October 2006

Abstract—

The dopamine β‐hydroxylase (DβH) content and activity of large dense‐core noradrenergic vesicles purified from bovine splenic nerve were determined using two assay procedures : enzymic activity expressed in Units per mg protein and homospecific activity based on radioimmunoassay expressed in Units per mg DβH antigen. Approximately two‐thirds of the total enzyme activity is latent in these vesicles, even after various treatments designed to compromise vesicle integrity. DβH can be completely unmasked by brief treatment with 0.01‐0.05% Triton X‐100 and activity increases from 0.20 to 0.64 Units per mg vesicle protein. Calculations based on both assay methods suggested that an average of 7% (range 3‐15%) of the total vesicle protein was DβH and that the average vesicle contained about 4 molecules of enzyme (range 2‐9 molecules). The estimated homospecific activities indicated an average of 25 and 50% (range 18‐72%) of the vesicle enzyme was inactive in the various samples using the two antibodies. The vesicle can synthesize up to 30 molecules of noradrenaline/s per molecule of DβH at near optimal substrate concentration, and 60‐270 molecules of norepinephrine/s per vesicle. The assumptions used in the various calculations were critically analyzed and, based on the methods employed, it is tentatively considered to be unlikely that there could be more than 5‐12 molecules of DβH per vesicle. The possibility that circulating DβH originates primarily, if not exclusively, from the large dense‐core vesicle type is considered and the functional implications of the data support the concept of vesicle reuse during several cycles of exocytosis involving a quantal size equal to a fraction of the vesicle transmitter content.
HIGH AFFINITY UPTAKE OF L‐GLUTAMATE AND L‐ASPARTATE BY GLIAL CELLS
Authors: Balcar V. J. , Borg J. , Mandel P.
Published: October 2006

Abstract—

The presence of an efficient, high affinity uptake system specific for L‐glutamate and L‐aspartate has been demonstrated in cultured glial cells originating from syrian hamster astroblasts (line NN). The system was found to be temperature dependent, to require sodium ions and its structural specificity is similar to systems studied previously in brain slices and synaptosomes. Co‐culturing of the glial cells with neuroblastoma cells brought about only minor changes in structural specificity while values of kinetic parameters remained virtually unchanged. The temperature dependence and the susceptibility to metabolic inhibitors suggest that the uptake is mediated by an active transport system.
DETECTION OF A SOLUBLE SEROTONIN‐BINDING PROTEIN IN THE MAMMALIAN MYENTERIC PLEXUS AND OTHER PERIPHERAL SITES OF SEROTONIN STORAGE
Authors: JONAKAIT G. M. , TAMIR H. , RAPPORT M. M. , GERSHON M. D.
Published: October 2006

Abstract—

Groups of neurons intrinsic to the mammalian myenteric plexus have been shown to have both tryptophan hydroxylase and a specific uptake mechanism for serotonin. They are probably serotonergic. A soluble protein with a high binding affinity for serotonin, similar to a protein previously found in rat brain by TAMIR & HUANG (1974), has now been found in the myenteric plexus of both rabbit and guinea pig. Partial purification of the protein from the rabbit's myenteric plexus by ammonium sulfate fractionation increased the ratio of specific to nonspecific serotonin binding almost 3‐fold. Two dissociation constants for serotonin binding were obtained by equilibrium dialysis: 6.7 × 10−10 M and 4.8 × 10−7 M. The protein was similar to the soluble serotonin‐binding protein of CNS: the indole derivatives 5, 6‐ and 5, 7‐dihydroxytryptamine, and 6‐hydroxytryptamine inhibited serotonin binding by 50% at 10−7 M; norepinephrine was a poor inhibitor of serotonin binding; most of the serotonin‐protein complex had a very high molecular weight and did not penetrate a 6.5% acrylamide gel. The appearance of the serotonin binding protein during development of the intestine in fetal rabbit correlates closely with the development of a serotonin uptake mechanism by nerves of this tissue and precedes the ingrowth of the adrenergic innervation. In‐vitro administration of 6‐hydroxydopamine to adult animals has no effect on the binding capacity for serotonin. Binding activity in denervated preparations is only 1/5 that of innervated tissue. It is concluded that the serotonin‐binding protein, which has been found associated with serotonergic pathways in the CNS, is found associated with serotonergic neurons in the periphery as well. Since a similar serotonin‐binding protein is also found in sheep thyroid, which stores but does not take up serotonin, the protein may be a component of the serotonin storage mechanism.
D‐ AND L‐STEREOISOMERS OF ALLYLGLYCINE: CONVULSIVE ACTION AND INHIBITION OF BRAIN L‐GLUTAMATE DECARBOXYLASE
Authors: ORLOWSKI MARIAN , REINGOLD DIANE F. , STANLEY M. E.
Published: October 2006

Abstract—

DL‐Allylglycine was resolved into the L‐ and D‐stereoisomers using hog kidney acylase. Both isomers were active as convulsants after administration to mice. The dose of D‐allylglycine required to induce convulsions was greater than that of the L‐isomer. Studies on the concentration of the two isomers in brain suggest that the lower effectiveness of D‐allylglycine is partially due to its slower penetration into the brain through the blood‐brain barrier.

Both isomers of allylglycine inhibited brain glutamate decarboxylase in vitro to approximately the same extent, however, in vivo L‐allylglycine inhibited the enzyme more strongly than the D isomer. Concentrations of allylglycine which caused a significant inhibition of L‐glutamate decarboxylase in vivo were ineffective in inhibiting the enzyme in vitro. Oxidation products derived from L‐ or D‐allylglycine by the action of either L‐ or D‐amino acid oxidase caused an almost complete inhibition of the enzyme in vitro. It is suggested that a common intermediate derived from the two isomers (possibly 2‐keto‐4‐pentenoic acid) is responsible for the in vivo inhibition of L‐glutamate decarboxylase and possibly also for the induction of convulsions.
PURIFICATION AND IMMUNOHISTOCHEMICAL LOCALIZATION OF RAT BRAIN MYELIN PROTEOLIPID PROTEIN1
Authors: Agrawal H. C. , Hartman B. K. , Shearer W. T. , Kalmbach S. , Margolis F. L.
Published: October 2006

Abstract— A homogeneous preparation of proteolipid protein (PLP) from rat brain myelin was isolated by preparative gel electrophoresis in sodium dodecyl sulfate and chemically characterized. The results of amino acid and N‐terminal amino acid analyses are reported. The same preparation of myelin PLP was used to produce specific precipitating antibodies. Rabbit and goat antisera to myelin PLP each gave a single precipitin line with purified PLP dissolved in Triton X‐100. Under identical conditions, no precipitation was observed with antiserum to myelin basic protein or with control serum. Immunofluorescence localization employing antiserum to PLP demonstrated bright specific fluorescence restricted to the myelin sheaths of axons in all anatomical areas of the rat brain examined. Neuronal cell bodies and their dendrites were completely negative with respect to the presence of proteolipid protein. PLP could not be localized in the cell bodies or fibrous processes in any of the glial elements in the adult rat brain. However, myelin PLP was clearly visible in the cytoplasm and processes of actively myelinating oligodendrocytes in the corpus callosum in the brains of 10‐day‐old rats.
OLIGODENDROGLIA FROM HUMAN AUTOPSIED BRAIN: BULK ISOLATION AND SOME CHEMICAL PROPERTIES1
Authors: Iqbal Khalid , Grundke‐Iqbal Inge , Wisniewski Henryk M.
Published: October 2006

Abstract— Oligodendroglia were isolated from fresh and frozen human autopsied brains by a modification of our technique for isolation of neuronal perikarya and astroglia (Iqbal & Tellez‐Nagel, 1972). Cerebral white matter was minced in a hypertonic hexose‐Ficoll buffered medium, passed through successive screens of decreasing pore size, and the cells were then separated on a discontinuous sucrose density gradient using low speed centrifugation. Cells could also be isolated by substituting sucrose for hexoses in the cell isolation medium. About 95% of the isolated cells had morphology characteristic of oligodendroglia. The usual contaminants in this fraction were capillary fragments, red blood cells, and a few astrocytes. Free myelin was only sparsely seen. About 5–10% of the isolated oligodendroglia had one or more loops of loose membranes extending from the cell plasma membrane. These membraneous loops resembled myelin. The ultrastructural preservation of the cells was poor. The average yield per gram of wet tissue was 52 million cells amounting to 2.4 mg protein, 283 μg DNA, and 94 μg RNA. The protein, DNA, and RNA contents per average cell were 47, 5.3, and 1.8 pg respectively. About 50% of the tissue DNA was recovered as isolated cells, suggesting a larger proportion of oligodendroglia to astroglia, the other principal cell type in human white matter.

Comparison of the total protein profiles of the isolated oligodendroglia with those of the purified human myelin on sodium dodecyl sulfate‐polyacrylamide gels revealed the presence of all human myelin constituent proteins in the isolated cells except the intermediate (or DM‐20) myelin proteins. Densitometry of the gels stained with Fast Green showed that the ratio of protein bands corresponding to myelin encephalitogenic basic protein (BP) to myelin proteolipid protein (PLP) in the isolated cells was three‐fold as high as in purified human myelin. The oligodendroglial BP which constituted about 14% of the total cell protein contents, also coelectrophoresed with the myelin BP on pH 4.3 polyacrylamide gels. Furthermore, the two dimensional peptide maps of the tryptic digest of the cell BP labelled with 125I were also identical to the similarly treated myelin BP. The data suggest that the BP can be used as a chemical marker for oligodendroglia.
CHOLINE KINASE ACTIVITY IN THE DEVELOPING RAT SPINAL CORD: DIFFERENTIAL DEVELOPMENT OF HEMICHOLINIUM‐3 SENSITIVE AND INSENSITIVE ACTIVITY1
Authors: Burt A. M.
Published: October 2006

Abstract— Choline kinase (ATP:cholinephosphotransferase; EC 2.7.1.32) activity was measured in preparations of lumbar spinal cord from rats ranging in development from 12 days of gestation to 46 days of age. The enzyme activity was measured with a radiochemical assay procedure suitable for whole tissue preparations which are rich in ATP‐metabolizing enzymes. Total choline kinase activity was further differentiated into hemicholinium‐3 (HC‐3) sensitive and HC‐3 insensitive components. The specific activity of choline kinase (unhibited) increased 3‐fold during the prenatal period and subsequently decreased to relatively low levels by birth. There was no significant change in choline kinase activity throughout the postnatal period. The ontogenetic patterns for the HC‐3 sensitive and HC‐3 insensitive components of choline kinase activity had transient peaks in activity during the prenatal period; however, these peaks in specific activity for the 2 components were 2–3 days out of phase temporally. HC‐3 insensitive activity reached a peak at 18 days of gestation while the HC‐3 sensitive activity peaked at 2CL21 days of gestation. In the 10‐day‐old rat, the apparent choline Km values were 0.56 and 0.16 MM for the total activity, 0.58 and 0.13 mM for the HC‐3 insensitive activity, and 0.47 for the HC‐3 sensitive activity.
CALCIUM FLUXES IN CULTURED AND BULK ISOLATED NEURONAL AND GLIAL CELLS
Authors: Lazarewicz J. W. , Kanje M. , Sellström A. , Hamberger A.
Published: October 2006

Abstract— The influx and efflux of 45Ca has been studied in cultured human glioma and mouse neuroblastoma cells and in isolated fractions enriched in synaptosomes, neuronal and astrocytic perikarya from rabbit brain.

The uptake of 45Ca was somewhat more efficient in glioma compared to neuroblastoma cells, whereas there was little difference in the rate of 45Ca uptake by isolated glial cells and neuronal perikarya. Isolated synaptosomes showed the highest rate of 45Ca accumulation. An increase of K concentration to 50 mm in the medium, with a corresponding lowering of Na, stimulated both glioma and glial as well as synaptosomal 45Ca uptake more markedly than the uptake by neuroblastoma cells and neuronal perikarya. Lowering the Na concentration and replacing it by choline had no effect on the cultured cells and astrocytes. Na‐free media caused massive stimulation of 45Ca influx in all fractions and cells tested.

The efflux of 45Ca was studied after preloading of cells. Three phases could be resolved from the desaturation curves. All cells had nearly similar half‐lives for 45Ca efflux under standard conditions. Pulses of media containing 50 mm‐K stimulated 45Ca efflux from glioma cells and astrocytes more efficiently than from neuroblastoma cells, neuronal perikarya and synaptosomes. The stimulated release was exclusively seen in Ca‐containing media in experiments with the cultured cells and in Ca‐free media in experiments with cell perikarya. The effect of transmitter pulses on the release of 45Ca was examined in a limited series. Acetylcholine and isoproterenol were found to stimulate 45Ca release more actively from glia than from neurons.
EFFECT OF DIAZEPAM AND PENTOBARBITAL ON AMINOOXYACETIC ACID‐INDUCED ACCUMULATION OF GABA
Authors: Peričić D. , Walters J. R. , Chase T. N.
Published: October 2006

Abstract— The effect of diazepam and pentobarbital on γ‐aminobutyric acid (GABA) levels, the aminooxyacetic acid (AOAA)‐induced accumulation of GABA, and the in vitro activity of l‐glutamate 1‐carboxyl‐lyase (EC 4.1.1.15) [GAD] were studied in various regions of rat brain. Diazepam increased GABA levels in the substantia nigra, diminished the AOAA‐induced accumulation of GABA in the caudate nucleus, cingulate, parietal and entorhinal cortex and had no effect on GABA accumulation in the pyriform and cerebellar cortex. After pentobarbital, GABA levels were elevated in the caudate nucleus but decreased in the parietal and pyriform cortex; the AOAA‐induced accumulation of GABA also diminished in all cortical regions studied. No correlation was found between the apparent changes in GABA synthesis, as estimated by accumulation after inhibition of 4‐aminobutyrate‐2‐oxoglu‐tarate (EC 2.6.1.19) [GABA‐T] with AOAA, and the changes in GABA levels induced by these drugs. The reduction in AOAA‐induced GABA accumulation after diazepam and pentobarbital treatment was most pronounced in regions which showed the greatest accumulation of GABA after AOAA administration. Neither diazepam nor pentobarbital administration affected the activity of GAD in homogenates of cingulate cortex. Chlorpromazine, at a dose which decreased spontaneous activity, enhanced the AOAA‐induced GABA accumulation in the cingulate cortex, suggesting that drug‐induced sedation is not necessarily associated with decreased GABA synthesis. While regional differences were observed in the effects of diazepam and pentobarbital on GABA synthesis, both agents appear to inhibit GABA synthesis in vivo and both do so, in at least some brain areas, at subsedative doses.
THYROID HORMONE INHIBITION OF SYNAPTOSOME AMINO ACID UPTAKE AND PROTEIN SYNTHESIS1
Authors: Verity M. A. , Brown W. J. , Cheung M. K. , Czer G. T.
Published: October 2006

Abstract— 3,3′,5‐Triiodothyronine (T3) inhibited L‐[14C]leucine uptake into synaptosomes. Inhibition was competitive with a Ki of 3.1 × 10−5m. Hofstee plot revealed an inverted hyperbolic curve suggestive of a two carrier or carrier plus diffusion mediated system for amino acid uptake. Both the carrier mediated and diffusional components were inhibited by thyroid analogues. l‐Thyroxine and analogues inhibited the incorporation of l‐[14C] leucine into cerebral synaptosome protein. At 50 μm, the triiodo‐compounds were more inhibitory than tetraiodo‐>3,5‐triiodo‐l‐thyronine >3,3′,5‐triiodothyropro‐pionic> l‐thyroxine >3,5‐diiodo‐l‐tyrosine. Thyroid analogue inhibition was not seen in liver or brain mitochondrial protein synthesis. 3,3′,5‐Triiodothyronine had no effect on respiratory control or 2,4‐DNP stimulated synaptosome respiration supported by malate plus pyruvate. Ouabain did not inhibit [14C]leucine uptake into adult synaptosomes. There was synergistic inhibition of synaptosome protein synthesis by thyroid analogues in the presence of 0.2 mm‐ouabain. 3,3′,5‐Triiodothyronine had no effect on synaptosome fraction ATPase or Na‐K ATPase. Addition of T3 induced further inhibition of synaptosome protein synthesis in the presence of either chloramphenicol (100μm) or cycloheximide (50μg/ml). [14C]Glycine uptake and incorporation into synaptosome protein was inhibited by 3,3′,5‐triiodothyronine. There was no inhibition of [14C]proline uptake or incorporation.

The above evidence and kinetic data strongly favor a selective competitive block in amino acid transport at the synaptosome membrane leading to a decreased rate of protein synthesis.
SUBCELLULAR ANALYSIS OF THE MOLECULAR FORMS OF ACETYLCHOLINESTERASE IN RAT SKELETAL MUSCLE
Authors: McLaughlin Jack , Engel W. King , Reddy N. Bojji
Published: October 2006

Acetylcholinesterase (AChE, EC 3.1.1.7) activity of rat gastrocnemius muscle homogenized in 1 M‐NaCl and 0.5% Triton X‐100 was separated by velocity sedimentation in sucrose gradients into three molecular forms with sedimentation coefficients of about 4S, 10S and 16S. The distribution of homogenate AChE activity among the three peaks was 53, 34 and 13% respectively. The different molecular forms were found to be heterogeneously distributed in subcellular fractions prepared from sucrose homogenates of muscle, as follows:

Subfractions of the crude sarcolemmal fraction were prepared by discontinuous sucrose gradient centrifugation. AChE was recovered in the greatest yield and with the highest specific activity in a light density subfraction (0.6/0.8 M‐sucrose interface). The AChE activity in this light density subfraction was mainly (81‐88%) the 10S form of the enzyme. The velocity sedimentation profiles of the AChE activity in the more dense subfractions were markedly different in that 16S AChE was a major component.
PURIFICATION AND COMPARISON OF 2′,3′‐CYCLIC NUCLEOTIDE 3′‐PHOSPHOHYDROLASES FROM BOVINE BRAIN AND SPINAL CORD
Authors: Drummond Robert J. , Hamill Elizabeth B. , Guha Arabinda
Published: October 2006

The membrane‐bound enzyme 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase has been purified from acetone powders of bovine white matter and spinal cord. Affinity chromatography on AMP‐Sepharose has been used as the final step in the chromatographic purifications. The yield was about 3 mg of purified enzyme per 100 g of tissue in each instance. The enzymes from the two sources were indistinguishable by chromatography, gel electrophoresis, and amino acid analysis; the enzyme from spinal cord, however, has shown a specific activity of 225 units/mg compared to 342 units/mg for the enzyme from white matter. Both proteins had a molecular weight of 100,000 by gel filtration and 50,000 by sodium dodecyl sulfate‐gel electrophoresis under reducing conditions. The properties of the enzyme, including amino acid composition determined on the purified soluble protein and on the protein purified by sodium dodecyl sulfate‐gel electrophoresis, were those of a basic hydrophobic protein.
RAPID AXOPLASMIC TRANSPORT OF PROTEINS IN REGENERATING SENSORY NERVE FIBERS
Authors: Theiler R. F. , McClure W. O.
Published: October 2006

Abstract— Utilizing an in vitro labeling procedure, the proteins carried by rapid axoplasmic transport in normal and regenerating sensory fibers of the rat sciatic nerve were compared. No statistically significant differences were found when the total amount of transported protein was compared in control and sectioned nerves at times from 2 to 76 days following axotomy. Fractionation of labeled proteins on polyacrylamide slab gels enabled the identification of some 25 individual transported proteins. By this criterion, no differences were detectable in the composition of proteins synthesized in the dorsal root ganglia from which sectioned vs control sciatic nerves project. When the electrophoretic distributions of transported proteins from control and sectioned nerves were compared, significant' differences were observed. The appearance and disappearance of two proteins were temporally related to chromatolytic changes in the nerve cell body. In addition, the composition of transported proteins in undamaged control nerves contralateral to the sectioned nerves exhibited changes which were not observed in either normal control nerves or sectioned nerves. Changes in the composition of transported proteins as a function of time following the onset of chromatolysis may be involved in controlling nerve regeneration in sensory nerve fibers.
SYNAPTOSOMAL PHOSPHORYLATION OF D‐GLUCOSAMINE
Authors: Tan C. H. , Raghupathy E. , Peterson N. A.
Published: October 2006

Abstract— Synaptosomal fractions from rat brains can phosphorylate d‐glucosamine once it is transported across the synaptosomal membrane. Intrasynaptosomal phosphorylation of d‐glucosamine was rapid; up to 80% of the d‐glucosamine was recovered as the phosphorylated amino sugar during 15 min of incubation with the substrate. Transport of d‐glucosamine was the rate‐limiting step in the uptake process since in the presence of cytochalasin B, a specific sugar transport inhibitor, the uptake of d‐glucosamine was inhibited to the same extent as its phosphorylation. The pH optimum for phosphorylation of d‐glucosamine by lysed synaptosomal preparations was between 7.5 and 9.0. The mean Km and Vmax values for this phosphorylation were 0.46 DIM and 676 nmol/mg protein per min, respectively. Phosphorylation of d‐glucosamine was inhibited competitively by d‐glucose, 2‐deoxy‐d‐glucose and N‐acetyl‐d‐glucosamine and uncompetitively by d‐glucose‐6‐phosphate. The phosphorylation was strongly inhibited by several sulfhydryl reagents, but was insensitive to product inhibition, cytochalasin B, phloretin and phloridzin.
ADENYLATE CYCLASE IN HELIX AND APLYSIA GANGLIA: CHARACTERISTICS OF ITS STIMULATION BY A PEPTIDE‐CONTAINING NERVOUS SYSTEM EXTRACT
Authors: Levitan I. B. , Bergstroem E. , Simonet Monique
Published: October 2006

Abstract— A crude particulate fraction, prepared from the central ganglia of Helix or Aplysia, contains levels of adenylate cyclase activity comparable to those in mammalian brain. This activity can be stimulated up to 50‐fold by NaF, and 4‐ to 10‐fold by guanyl nucleotides such as GTP and guanylylimidodiphosphate (Gpp(NH)p). A peptide‐containing extract from Helix or Aplysia nervous system also stimulates the adenylate cyclase, by 50‐400°. In contrast, a number of peptides known to occur in vertebrate and invertebrate nervous system are without effect. The adenylate cylase stimulation by the endogenous molluscan peptide‐containing extract may be receptor‐mediated, but the effect is not enhanced in the presence of guanyl nucleotides: in this respect it differs from many other hormone‐sensitive adenylate cyclases. The endogenous extracts prepared from Helix and Aplysia each stimulate both Helix and Aplysia adenylate cyclases, suggesting that the putative cyclase‐linked receptors may be similar in the two species. Furthermore, the active components in the extracts from Helis and Aplysia appear to be similar, since preliminary evidence suggests that they may interact with the same adenylate cyclase‐linked receptor in particulate fraction from Helix ganglia.
EFFECT OF BRAIN CORTEX PHOSPHOLIPIDS ON ADENYLATE‐CYCLASE ACTIVITY OF MOUSE BRAIN
Authors: Leon A. , Benvegnu D. , Toffano G. , Orlando P. , Massari P.
Published: October 2006

Abstract—

Intravenous injection of a sonicated dispersion of bovine brain phospholipids results in a significant change in both NaF‐dependent and dopamine dependent adenylate cyclase activity. High dosage of phospholipids inhibits the dopamine dependent, but not the NaF dependent, adenylate cyclase activity. The stimulation of cyclase activity is accompanied by an increased level of cAMP in mice brains. Treatment with haloperidol abolishes the increase in cAMP. Among individual phospholipids, phosphatidylserine is the most active component for inducing the activation of DA‐dependent adenylate cyclase activity.
SODIUM AND POTASSIUM IONS AND ACCUMULATION OF LABELLED d‐ASPARTATE AND GABA IN CRUDE SYNAPTOSOMAL FRACTION FROM RAT CEREBRAL CORTEX
Authors: Takagaki G.
Published: October 2006

Abstract—

The accumulation of labelled d‐aspartate into crude synaptosomal fraction (P2) prepared from the rat cerebral cortex proceeded by a ‘high affinity’ system (Km= 15.1 μm The maximal velocity of d‐aspartate uptake was higher than that of the ‘high affinity’ component of l‐aspartate uptake and almost equal to that of l‐glutamate under the same incubation conditions. Negligible metabolism of labelled d‐aspartate was observed in the P2 fraction. These findings are in accord with those which have been reported for rat cerebral cortical slices. The following observations were made on d‐aspartate uptake into rat cerebral P2 fraction. (1) The requirement of sodium is almost absolute and obligatory. (2) The affinity of the carrier for the substrate is increased by increasing sodium concentration in the medium, but the maximal velocity is not altered. (3) It is suggested that sodium ion is co‐transported mole for mole with the substrate molecule. (4) Omission of potassium from the medium inhibits the uptake competitively. (5) Ouabain is a competitive inhibitor on the uptake. (6) Whereas thallium, rubidium and ammonium are efficient substitutes for potassium in exhibiting Na–K ATPase activity of the P2 fraction, the uptake is activated only by rubidium in the absence of potassium. These observations were in common with the uptake of l‐aspartate as well as of l‐ and d‐glutamate, but not with GABA uptake. The requirement of sodium for the uptake of d‐glutamate was indicated to be higher than that in the uptake of the other amino acids. Mutual inhibitions of the uptake among l‐ and d‐isomers of glutamate and aspartate suggested that a common carrier is involved in the transport. Mechanisms of the transport of these amino acids in the crude synaptosomal fraction were discussed.
QUANTITATION AND CHARACTERIZATION OF BRAIN TUBULIN (COLCHICINE‐BINDING ACTIVITY) IN DEVELOPING HYPOTHYROID RATS
Authors: Gonzales Linda W. , Geel S. E.
Published: October 2006

Abstract—

The influence of early hypothyroidism on the concentration and biochemical properties of soluble and particulate tubulin from the cerebral cortex and cerebellum was investigated during development in the rat. Cellular soluble tubulin concentration (pmol colchicine bound/μg DNA) was approx 16% lower in both brain areas of hypothyroid animals compared to controls at 25 days of age. No effect of thyroid hormone deficiency was observed when tubulin concentration was expressed in terms of tissue protein or weight. The particulate tubulin concentration was approx 20% lower in the cerebral cortex of 25‐day‐old hypothyroid rats although the distribution of tubulin between soluble and particulate fractions was similar to controls. The incorporation of [14C]leucine into cerebral cortical tubulin in vitro (c.p.m. in tubulin/c.p.m. in total protein) was not significantly altered by the hormonal deficiency. Thus there was no apparent evidence of a selective defect in tubulin synthesis. Tubulin from hypothyroid rats behaved similarly to control samples with respect to the effects of pharmacological agents and temperature, lability of binding, chromatographic profile and electrophoretic mobility on sodium dodecyl sulfate polyacrylamide gels.