bluuebunny/crossref_metadata_2025 huggingface dataset:
N‐ACETYL‐ASPARTATE AMIDOHYDROLASE: PURIFICATION AND PROPERTIES1
Authors: D'Adamo A. F. , Peisach J. , Manner G. , Weiler C. T.
Published: October 2006

Abstract— The enzyme in rat brain responsible for the de‐acetylation of N‐acetyl‐aspartic acid has been partially purified. In contrast to the enzyme from hog kidney which is stable at 70°C, it rapidly denatures above 57°C. The rat brain enzyme has the same Km for its substrate and the same solubility in ammonium sulphate solution as the hog kidney enzyme. Results of migration on starch gel electro‐phoreses and isoelectric focusing indicate a pI for the amidohydrolase of 5.1. A variety of potential substrates, modulators, and inhibitors have been examined.
STUDIES ON MEVALONATE KINASE, PHOSPHOMEVALONATE KINASE AND PYROPHOSPHOMEVALONATE DECARBOXYLASE IN DEVELOPING RAT BRAIN
Authors: Ramachandran C. K. , Shah S. N.
Published: October 2006

Abstract— We have in the present study investigated the properties of mevalonate kinase, phosphomevalonate kinase and pyrophosphomevalonate decarboxylase in the 105,000 g supernatant fractions from rat brain, and determined whether the activities of these enzymes change during brain development. All three enzymes in brain showed a specific requirement for ATP for optimal activity. The presence of Mg2+ as divalent cation was also required for optimal activity of mevalonate kinase and phosphomevalonate kinase. Both Mg2+ and Mn2+ were equally effective divalent metal ions for pyrophosphomevalonate decarboxylase in brain. Mevalonate kinase as well as phosphomevalonate kinase were active in a broad pH range of 6.5–8 while the pH curve for pyrophosphomevalonate decarboxylase showed a peak activity at approx 6. No age‐dependent change occurred in the activities of mevalonate kinase and phosphomevalonate kinase in developing brain, whereas pyrophosphomevalonate decarboxylase activity in brain increased during the 1st week after birth, reached a peak value at about the 8th day of age and declined slowly thereafter. The Km for brain mevalonate kinase in 2, 13 and 52 day old rats were 312, 400 and 434 μM, respectively. The Vmax for the kinase in 2, 13 and 52 day old rats were in the range of 45–52 nmol/h/mg protein, respectively. This suggests that, like in liver (Ramachandran & Shah, 1976), pyrophosphomevalonate decarboxylase in brain may also be one regulatory step for cholesterol synthesis.
LEVELS OF CYCLIC NUCLEOTIDES IN MOUSE REGIONAL BRAIN FOLLOWING 300 ms MICROWAVE INACTIVATION1
Authors: Jones D. J. , Stavinoha W. B.
Published: October 2006

Abstract— The uniformity and speed of inactivation of mouse brain adenylate cyclase, guanylate cyclase and cyclic nucleotide phosphodiesterase were measured after 6 kW microwave irradiation (MWR). Inactivation of enzymes was uniform throughout the brain during heating and 100% loss of activity was evident after 300 ms. MWR. For comparison of effects of inactivation times on levels of cyclic nucleotides measured in regional brain areas, cyclic AMP and cyclic GMP were estimated after 1.5 kW MWR requiring 4 s of heating and 6 kW MWR requiring 300 ms. Except for corpus striatum, uniformly lower levels of cyclic AMP were measured following 300 ms vs. 4s MWR. There was no change in cyclic GMP levels in regional brain areas after 4s vs. 300 ms MWR. Cyclic AMP and cyclic GMP were measured from the same regional brain tissue samples after 300 ms and ratios calculated. The finding of much lower cyclic AMP:cyclic GMP ratios than had previously been reported suggests that slow inactivation times provide for the measurement of regional brain cyclic nucleotide values which are not consistent with the in‐vivo state.
GLYCOPROTEINS AND GLYCOLIPIDS OF SUBCELLULAR FRACTIONS FROM BOVINE RETINA. INCORPORATION OF MANNOSE AND SIALIC ACID1
Authors: Curtino J. A. , Maccioni H. J. , Caputto R.
Published: October 2006

Abstract— Endogenous lipids and proteins of bovine retina subcellular fractions were labelled from CMP‐[3H]NeuNAc and GDP‐[14C]mannose. The bulk of NeuNAc and mannose transfer activity was in membranes other than those from the rod outer segment (ROS). Lighter and heavier membranes, obtained from ROS free membranes by density gradient centrifugation, were the most active for the incorporation of NeuNAc and mannose, respectively. NeuNAc bound to a lipid indistinguishable from gangliosides, and a lipid that contains mannose (mannolipid‐I) were found in the fraction extractable with chloroform‐methanol (2:1, v/v). Mannose was also incorporated into a lipid fraction extractable with chloroform‐methanol‐water (1:1:0.3, by vol) (mannolipid‐II). Mannolipid‐I and mannolipid‐II were labile to mild acid hydrolysis. In the presence of ROS free membranes, radioactivity of mannoli‐pid‐I was transferred to mannolipid‐II and from this to proteins. Analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, the proteins labelled from GDP‐mannose migrated as a broad peak covering the range of molecular weights 20,000–30,000 and including the zone of rhodopsin migration. The proteins labelled from CMP‐NeuNAc showed four radioactive peaks that were coincident with three out of four periodic acid‐Schiff (PAS) positive bands.
NERVE GROWTH FACTOR AND THE ACTIVITY OF TYROSINE HYDROXYLASE IN ORGAN CULTURES OF RAT SUPERIOR CERVICAL GANGLIA
Authors: Yu M. W. , Nikodijevic B. , Lakshmanan J. , Rowe V. , MacDonnell P. , Guroff G.
Published: October 2006

Abstract— Superior cervical ganglia from young rats were cultured in the absence of serum. The effect of nerve growth factor on the level of tyrosine hydroxylase was studied. In the absence of nerve growth factor the specific activity of tyrosine hydroxylase fell by more than 50% within 48 h. In the presence of nerve growth factor the total and specific activities were maintained and even increased in the same period. Both the 2.5 S and the 7 S forms of nerve growth factor were effective. Oxidized nerve growth factor had no effect except when present in very high concentration. Purified antibody to nerve growth factor was inhibitory. Insulin had only a slight effect in this system, but dibutyryl CAMP elevated tyrosine hydroxylase activity substantially. Propranolol inhibited the action of nerve growth factor but its action appeared to be nonspecific and unrelated to its action on the β‐adrenergic receptor. Changes in the activity of dihydropteridine reductase paralleled those seen in tyrosine hydroxylase.
A LOW MOLECULAR W eight GLYCOPROTEIN ASSOCIATED WITH ISOLATED MYELIN: DISTINCTION FROM MYELIN PROTEOLIPID PROTEIN
Authors: Poduslo J. F. , Everly J. L. , Quarles R. H.
Published: October 2006

Abstract— A low molecular weight glycoprotein has been demonstrated in myelin isolated from immature rat brains. Both short term and long term fucose incorporation studies have identified this protein in the proteolipid protein region of a sodium dodecyl sulfate, polyacrylamide gel. A 1.7‐2.1 fold increase in radioactive fucose in this glycoprotein relative to the major myelin glycoprotein was seen after long term fucose incorporation (21 days) compared to short term incorporation (18–22 h). The demonstration that this fucose‐labelled protein is distinguishable from that of proteolipid protein was achieved by a variety of independent techniques. One technique involved a comparison of ether‐ethanol extracted, freshly isolated, myelin with myelin extracted with chloroform‐methanol. Treatment of isolated myelin with chloroform‐methanol results in the solubilization of the proteolopid protein and its subsequent absence on gel electrophoresis while, in contrast, an enhancement of fucose label was observed in the same region of the polyacrylamide gel. Another procedure involved the electrophoretic separation of the radioactive fucose peak from that of proteolipid protein by employing a continuous, phosphate buffered, gel system. Finally carbohydrate analysis by gas‐liquid chromatography of a partially purified proteolipid protein fraction did not reveal significant amounts of carbohydrates which are characteristic of glycoproteins. The identification of this minor glycoprotein comigrating with proteolipid protein indicates, therefore, a greater complexity associated with the purified myelin membrane than has been previously demonstrated.
ALTERATIONS IN ACETYLCHOLINE SYNTHESIS AND CYCLIC NUCLEOTIDES IN MILD CEREBRAL HYPOXIA
Authors: Gibson Gary E. , Shumada Masahisa , Blass John P.
Published: October 2006

In order to elucidate changes accompanying mild cerebral hypoxia, the synthesis of the neurotransmitter acetylcholine and the concentrations of cyclic AMP and cyclic GMP have been compared to changes in brain lactate in the forebrain of mice made mildly hypoxic. Both histotoxic hypoxia (injection of KCN) and anemic hypoxia (injection of NaNO2) were studied. Acetylcholine synthesis was followed by a double‐label technique using [U‐14C] glucose and [2H4] choline.

A 43%, decrease in the synthesis of acetylcholine from [U‐14C]glucose and an 80% increase of the level of cyclic GMP accompanied hypoxia so mild that there were no significant changes in cerebral lactate, or in cyclic AMP (or in AMP: Gibson & Blass, 1976b). Changes in glucose utilization do not account for the decrease in glucose incorporation into acetylcholine. Glucose utilization decreases and then increases with increasing hypoxia, whereas incorporation into acetylcholine decreased with increased hypoxia.
ACCUMULATION OF THE FOUR MYELIN BASIC PROTEINS IN MOUSE BRAIN DURING DEVELOPMENT1
Authors: Barbarese E. , Carson J. H. , Braun P. E.
Published: October 2006

Myelin was isolated from the brains of mice 15, 20, 30, and 60 days after birth. The total amount of basic protein present in the isolated myelin was determined by radioimmunoassay. The 4 myelin basic proteins, with molecular weights of 21,500, 18,500, 17,000 and 14,000, were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and their relative amounts were determined densitometrically. The absolute amount of each of the basic proteins was calculated from its relative amount on the gel and from the total amount of myelin basic protein in the sample as determined by radioimmunoassay. The results show that between 10 and 30 days after birth each protein accumulates at a characteristic rate so that the molar ratios among the 4 basic proteins are (in descending order according to their molecular weights) 1:5:2:10 during this period. Between 30 and 60 days after birth the 14 K and 18.5 K proteins continue to accumulate at reduced rates while the 21.5 K and 17 K proteins begin to disappear from the myelin membrane; 60 days after birth the molar ratios among the 4 basic proteins are 1:10:3.5:35. These developmental patterns of accumulation are discussed in relation to the possible role of each of the 4 myelin basic proteins in myelination.
A SIMPLE METHOD FOR THE PREPARATION OF THE IMMUNOGLOBULINS TO HYDROXYINDOLE O‐METHYLTRANSFERASE
Authors: Kuwano R. , Takahashi Y.
Published: October 2006

A simple procedure is described for the preparation of the monospecific immunoglobulins (Ig) to bovine pineal hydroxyindole O‐methyltransferase (HIOMT) (EC 2.1.1.4). We observed that anti‐serum to HIOMT was produced in rabbits which had received subcutaneous inoculations of the crude pineal extract. The monospecific Ig to HIOMT were isolated by an immunoadsorption technique in which the brain extract was coupled to the CNBr‐activated Sepharose. The purified Ig gave a single precipitin line against the pineal extract on an Ouchterlony double diffusion plate and also by immunoelectrophoresis. All other extracts tested, cerebral cortex, cerebellum, thalamus, caudate nuclei, superior coliculus, liver, adrenal gland, kidney, retina and serum did not express precipitin lines to the purified Ig. When immunochemical titration was used, the pineal HIOMT was quantitatively precipitated by the monospecific Ig.
THE EFFECT OF TRIALKYLTIN COMPOUNDS ON TUBULIN POLYMERISATION
Authors: Tan L. P. , Ng M. L. , Kumar Das V. G.
Published: October 2006

The effects of trialkyltin compounds on the colchicine‐binding activity and the in vitro polymerisation of rat brain tubulin have been investigated. Trialkyltins at concentrations between 10 and 100 μM inhibited the colchicine‐binding activity of purified tubulin preparations derived from rat brain. Viscometric studies have shown that the same concentrations of trialkyltins also inhibited the in vitro polymerisation of tubulin. Both the effects were found to be concentration dependent. However, trialkyltins did not interfere with the in vitro preassembled microtubules. The reasons which are believed to explain the inhibitory effects of these compounds are discussed.
THE UTILIZATION OF GLUTAMINE BY THE RETINA: AN AUTORADIOGRAPHIC AND METABOLIC STUDY
Authors: Voaden M. J. , Lake N. , Marshall J. , Morjaria B.
Published: October 2006

The cells able to accumulate exogenously applied [3H]glutamine in rat, cat, frog, pigeon and guinea pig retinas have been located by autoradiography, and the fate of the labelled glutamine, as regards its incorporation into aspartic, glutamic and γ‐amino‐butyric acids, followed for 60min. The results support the notion of glutamine as a precursor of transmitter amino acids in some neurones. In particular, it would appear to be a source of a relatively stable pool of GABA which may be located, with species variation, in amacrine or ganglion cells. In the pigeon retina a glutamate pool incorporates and retains a major percentage of the label, and perikarya in the middle of the inner nuclear layer of the tissue are predominantly labelled.
STUDIES ON AXOPLASMIC TRANSPORT OF INDIVIDUAL PROTEINS: 1–ACETYLCHOLINESTERASE (AChE) IN ACRYLAMIDE NEUROPATHY
Authors: Rasool C. G. , Bradley W. G.
Published: October 2006

Abstract— The axoplasmic transport rate and distribution of acetylcholinesterase (AChe, EC 3.1.1.7) was studied in the sciatic nerves of normal rats and those with a neuropathy due to acrylamide, by measuring the accumulation of the enzyme proximal to single and double ligatures. The single ligature experiments showed that the apparent transport rate of AChE was decreased in acrylamide neuropathy. The double ligature experiments indicated that only 8.1% of AChE was mobile in normal rat sciatic nerve. The mobility of the enzyme in acrylamide‐treated rat sciatic nerves was altered to 11.8%. The absolute transport rate of AChE in normal rat sciatic nerve was 567 mm/24 h, and in acrylamide neuropathy it was decreased to 287 mm/24 h.

The amount of AChE activity transported in normal rat sciatic nerve was 2.64 μmol/24 h. The rats with acrylamide neuropathy showed a decrease in the amount of AChE activity moving in the orthograde direction (2.03 μmol/24 h).

The colchicine‐binding properties of tubulin protein from sciatic nerves of normal and acrylamide‐treated rats were studied. In rats with acrylamide neuropathy, a marked decrease of 75% in tubulin‐colchicine binding was observed.
EFFECT OF OXOTREMORINE ON THE APPARENT REGIONAL TURNOVER OF ACETYLCHOLINE IN MOUSE BRAIN
Authors: Nordberg Agneta
Published: October 2006

Abstract—

The effect of oxotremorine (1 mg kg‐1i.p.) on the steady state concentration of acetylcholine (ACh) and choline (Ch) and the transformation of radioactive choline ([3H]Ch) was studied in different brain regions of the mouse following death by microwave irradiation of the head. Oxotremorine significantly increased the concentration of endogenous ACh in the cortex and hippocampus and of endogenous Ch in the cortex. Pretreatment with atropine (5 mg kg‐1i.p.) prevented the increase in ACh. The biosynthesis of radioactive ACh ([3H]ACh) was decreased in all brain regions. Atropine (5 mg kg‐1) pretreatment counteracted this effect of oxotremorine (1 mg kg‐1), while methylatropine (5 mg kg‐1) had no effect except in the striatum. A calculation of the apparent turnover rate of ACh showed that oxotremorine (1 mg kg‐1) decreased the turnover in the cortex, hippocampus, midbrain. and striatum.
A NEW INCUBATION TECHNIQUE FOR THE STUDY OF SYNAPTOSOMAL TRANSPORT
Authors: Hwang Shing‐Mei , Roth Karl S. , Segal Stanton
Published: October 2006

Abstract—

A simple, inexpensive method is described for the incubation and study of transport in synaptosomes. This method avoids the many potential sources of error inherent in the methods currently in use. The transport of two sugars and two amino acids has been studied, using this system, and the resulting data is compared to similar studies already published by others. A steady‐state was achieved and maintained for 60 min independent of tissue concentration, except in the case of γ‐aminobutyric acid. An analysis of the entry kinetics of 2‐deoxy‐d‐glucose has been performed, the results indicating that the present method offers the advantage of substantially increasing oxygenation and hence, efficiency of the uptake. Using α‐aminoisobutyric acid, analysis of substrate flux within the system was performed, illustrating the utility of the method. The specific advantages offered by the technique over other methods in use are discussed. It is concluded that adequate oxygenation of the synaptosome during study is essential to reliable results and can easily be achieved by means of the methodology described in this report.
RAT AND MOUSE BRAIN HISTAMINE N‐METHYLTRANSFERASE: MODULATION BY METHYLATED INDOLEAMINES1
Authors: Sellinger O. Z. , Schatz R. A. , Ohlsson W. G.
Published: October 2006

Abstract—

A purification procedure for rat and mouse brain histamine N‐methyltransferase (HMT, EC 2.1.1.8) is described which achieves the preparation of 87‐fold purified rat brain and 166‐fold purified mouse brain enzyme. The purified HMT (MW 29,000) is inhibited by a number of physiologically and pharmacologically active amines, among them several methylated indoleamines, at concentrations above 5 ± 10‐6M. At concentrations below 1 ± 10‐7M, most of the methylated indoleamines stimulate HMT, provided histamine is maintained at, or close to, its optimal concentration as an HMT substrate, namely 1 ± 10‐5M. A study of the nature of the inhibitory process revealed a non‐competitive inhibition of HMT by dopamine as against a competitive inhibition of the enzyme by most methylated indoleamines. Increasing the concentration of histamine beyond the optimal value, i.e. to inhibitory levels, resulted in less stimulation. The findings support the notion that MSO elicits the formation in selected brain cells of supranormal amounts of several methylated indoleamines which are able to stimulate HMT (and possibly other methyltransferases, see Salaset al., 1977), thereby causing the depletion of the cerebral levels of S‐adenosyl‐L‐methionine, reported previously (Schatz & Sellinger, 1975b).
ACTIVATION OF GUANYLATE CYCLASE IN SYNAPTIC PLASMA MEMBRANES OF CEREBRAL CORTEX BY FREE FATTY ACIDS
Authors: Asakawa T. , Takenoshta M. , Uchida S. , Tanaica S.
Published: October 2006

Abstract—

Guanylate cyclase (EC 4.6.1.2) of synaptic plasma membranes of rat cerebral cortex was stimulated about 6‐fold by several unsaturated fatty acids (arachidonic, linolenic, linoleic, oleic, palmitoleic and myristoleic acid). Ricinoleic acid (12‐hydroxyoleic acid) was much less effective. Saturated fatty acids (C10 and C14‐C20) and the methylester of linoleic acid were ineffective. Stimulation by linoleic acid was influenced by the concentration of enzyme protein. At 480 μg/ml of protein 0.6 mm‐linoleic acid produced maximal activation of 6‐fold_ Activity stimulated by linoleic acid examined with 1.0 mm‐GTP was maximal at pH 7.8‐7.9 and with 2 mm‐MnCl2, whereas basal activity showed broad optimal pH and Mn2+‐concentration dependence. Activation of the enzyme by linoleic acid was only partially reversed by washing. Particulate guanylate cyclase of heart, small intestine, adrenal medulla, liver and lung was also activated by linoleic acid. The extents of activation (1.5‐14.7‐fold) by linoleic acid and the concentrations (0.2‐1.0 mat) required for maximal activation depended on the tissues.
ACETYLCHOLINESTERASE IN THE SUBSTANTIA NIGRA AND CAUDATE‐PUTAMEN OF THE RAT: PROPERTIES AND LOCALIZATION IN DOPAMINERGIC NEURONS
Authors: Lehmann John , Fibiger H. C.
Published: October 2006

Abstract— In order to examine the hypothesis that acetylcholinesterase (AChE) is contained within dopaminergic neurons of the nigro‐striatal projection, the effects of selective destruction of these neurons by 6‐hydroxydopamine (6‐OHDA) on cholinesterase, tyrosine hydroxylase, and choline acetyltransferase in substantia nigra (SN) and caudate‐putamen (CP) were studied in the rat. Four to five weeks after intraventricular or intracerebral 6‐OHDA injections tyrosine hydroxylase in these structures was reduced by 90% or more. Choline acetyltransferase was not affected in the SN or CP, but cholinesterase was reduced by about 40% in the SN and by 12% in the CP. To determine that the observed decreases in cholinesterase activity reflected true AChE and not butyrylcholinesterase (BChE), further experiments were conducted on tissues from animals with intracerebral 6‐OHDA lesions. (1) Substrate specificity. Acetylcholine (ACh) was replaced by either acetyl‐β‐methyl‐choline (AcβMeCh) or butyrylcholine (BCh) in the cholinesterase assay. SN and CP from 6‐OHDA lesioned rats showed 54% and 92% of control tissue cholinesterase activity respectively with AcβMeCh as substrate, in good agreement with values found using ACh. No decrease in activity toward BCh was observed. (2) Kinetics. The decrease in cholinesterase activities at different concentrations of ACh was determined. Analysis of the data revealed that cholinesterase in dopaminergic neurons was inhibited by high ACh concentrations, a characteristic property of AChE but not BChE. (3) Selective inhibitors. In the SN, cholinesterase in dopaminergic neurons was inhibited by the selective AChE inhibitors BW284C51 and ambenonium with a dose‐response curve similar to erythrocyte AChE but different from serum BChE. The selective BChE inhibitor, tetraisopropylpyrophosphoramide, inhibited the enzyme in dopaminergic neurons only at concentrations which inhibited erythrocyte AChE, concentrations somewhat higher than those which inhibited serum BChE. These results support recent histochemical observations indicating that AChE is contained in dopaminergic neurons of the SN. Moreover, these experiments represent the first characterization of AChE from a homogeneous population of non‐cholinergic neurons in mammalian CNS.
EFFECTS OF N‐METHYLAMINOETHANOL, AND N,N‐DIMETHYL‐AMINOETHANOL IN THE DIET OF PREGNANT RATS ON NEONATAL RAT BRAIN CHOLINERGIC AND PHOSPHOLIPID PROFILE
Authors: Zahniser N. R. , Katyal S. L. , Shih T.‐M. , Hanin I. , Moossy J. , Martinez A. J. , Lombardi B.
Published: October 2006

Abstract— Pregnant rats were fed for 15 days predelivery until 15 days postpartum a choline (Ch)‐deficient diet (CD diet) or a CD diet supplemented with 0.8% Ch‐CI (CS), 1%N‐methylaminoethanol (MME) or 1%N,N‐dimethylaminoethanol (DME). Gestation and parturition of the pregnant rats proceeded normally. However, all the pups born of dams fed the MME diet, and most of those born of dams fed the DME diet, died within 36 h of birth. No histological or cytological alterations were detected in the brain of the pups. Levels of Ch and acetylcholine (ACh) were elevated in the brain of pups born of dams fed the MME and DME diets, but not the CS diet. The content of total phospholipids in the brain of the pups was not altered by the diet fed to the dams. However, the phosphatidyl‐Ch and phosphatidylaminoethanol (PAE) contents in the brain of the MME‐ and DME‐exposed pups were markedly reduced. At the same time, significant amounts of DME, phosphatidyl‐N‐monomethylaminoethanol (PMME) and of phosphatidyl‐N,N‐dimethylaminoethanol (PDME) were present in the same brain areas. These results are evaluated and discussed in terms of providing a cause for the death of the MME‐ and DME‐exposed neonatal rats.
HYPEROSMOLALITY‐INDUCED GABA RELEASE FROM RAT BRAIN SLICES: STUDIES OF CALCIUM DEPENDENCY AND SOURCES OF RELEASE
Authors: Chan P. H. , Wong Y. P. , Fishman R. A.
Published: October 2006

Abstract— The effects of hyperosmolal superfusion upon the release of preloaded, radio‐labeled GABA has been studied, using both first cortical and first pontine brain slices. GABA release was stimulated with either hyperosmolal Na+ or sucrose superfusion in cortical slices. This stimulated release of radio‐labeled GABA was partially Ca2+‐dependent in cortical slices. When barium ions replaced Ca2+ in hyperosmolal medium, a similar effect was seen. High concentration of magnesium in Ca2+ ‐free hyperosmolal medium did not induce stimulation. The increased release of α‐aminoisobutyric acid (AIBA), a non‐metabolized amino acid induced by hyperosmolality, was not Ca2+‐dependent.

GABA release was also stimulated with hyperosmolal sucrose superfusion in pontine slices. The effect of pre‐treatment of cortical and pontine slices with β‐alanine or L‐2,4‐diaminobutyric acid (DABA) was used to study the source of exogenous GABA release induced by hyperosmolality. In cortical slices, β‐alanine blocked the hyperosmolal release of GABA and also slightly inhibited GABA uptake. DABA did not change hyperosmolal GABA release, although it inhibited GABA uptake. In pontine slices, both DABA and β‐alanine inhibited GABA uptake, but were unable to inhibit the hyperosmolal release of GABA.

The data suggest that hyperosmolality causes increased release of GABA from neurons, analogous to that seen with K+‐depolarization. AIBA, unlike GABA, is released from brain cells as a non‐Ca2+ ‐dependent response to osmotic equilibration. The observation that pre‐treatment with β‐alanine inhibits the hyperosmolal release of GABA suggests that hyperosmolality alters glial cell function.
CHARACTERIZATION OF MULTIPLE FORMS OF BRAIN TUBULIN SUBUNITS
Authors: Marotta Charles A. , Harris Jame L. , Gilbert Jeffrey M.
Published: October 2006

Abstract— Microtubular protein was isolated from rat forebrain by biochemical purification (ammonium sulfate precipitation followed by DEAE cellulose chromatography) or by two cycles of aggregation‐disaggregation. The protein subunit structure was examined on two‐dimensional electrophoretograms: first dimension, urea isoelectric focusing gel; second dimension, sodium dodecyl sulfate exponential acrylamide slab gel. Two forms of α tubulin were separated in the second dimension on the basis of different rates of migration (α and α2). Each of these species was further separated into at least three forms with different isoelectric points. β Tubulin was separated into a minor species (BI) and a major species β2). Multiple subunits were observed using protein from either purification method and in a two‐dimensional electrophoretogram of total supernatant proteins from rat brain. Separation and visualization of multiple forms of α and β tubulin is consistent with reports that provide evidence for post‐translational modification of these proteins.
MYELIN BASIC PROTEIN MICROHETEROGENEITY IN SUBFRACTIONS OF RAT BRAIN MYELIN
Authors: Shults C. W. , Whitaker J. N. , Wood J. G.
Published: October 2006

Abstract— Myelin subfractions were prepared from adult rat brain by discontinuous sucrose gradient ultracentrifugation. Gel electrophoretic studies at pH 10.6 in the presence of urea revealed differences in basic protein microheterogeneity among subfractions. With increasing myelin density there was a decrease in the most positively charged components of both large BP and small BP. Since these components are the least modified by deamidation and phosphorylation, it seems likely that the heavier myelin subfractions are enriched in the more modified components of the microheterogeneous population of BP. These observed differences may be related to the regulatory processes controlling biosynthesis, organization, and catabolism of BP in CNS myelin.
THE IN VIVO INCORPORATION OF LINOLENIC ACID INTO NEURONAL and GLIAL CELLS and MYELIN1
Authors: Cohen Steven R. , Bernsohn Joseph
Published: October 2006

Abstract— Neurons, astrocytes, oligodendrocytes, and myelin were prepared from 21‐day‐old rat brain at various times after intracerebral injection of [1‐14C]linolenate. Comparisons of phospholipid specific radioactivity demonstrated that the oligodendrocytes were much more active than neuronal, astroglial, or myelin fractions. This is consistent with the concept that the oligodendrocyte is responsible for synthesis of the relatively large mass of myelin sheath. Initially the phosphatidylcholine fraction was more active than the phosphatidylethanolamine fraction, but during the 36 h after injection the former decreased in radioactivity while the latter fraction showed an increase. Fatty acid elongation occurred rapidly. Within 2h after injection, 2/3 of the label had been converted to elongated products (20:4. 20:5, 22:5 and 22:6). All three cell types apparently contained the enzymes necessary to incorporate, elongate, and desaturate linolenic acid and this occurred at similar rates in each cell type. No direct precursor‐product relationship was found between the lipids of oligodendrocytes and myelin. There was, however, a lag in the appearance of elongated fatty acids in the phosphoglycerides of myelin. indicating that the polyunsaturated fatty acids in myelin were synthesized elsewhere and transported into the myelin sheath.
COMPARATIVE STUDIES ON THE DEGRADATION OF GABA and TAURINE IN THE BRAIN
Authors: Lähdesmäki P. , Korhonen K.
Published: October 2006

Abstract— The degradation of taurine and GABA in mammalian brain was studied in vivo and in vitro. Small amounts of [35S]isethionate (10–20 pmol/g brain wet weight) and [35S]sulphate (about 2 pmol/g) were detected in mouse brain after intramuscular injection of [35S]taurine. Taurine also produced isethionate in rat brain homogenates (about 20 nmol/h/g protein) and subcellular fractions (about 40 nmol/h/g protein in synaptosomes and about 300 nmol/h/g in mitochondria), but the reaction was not stimulated either by external electrical pulses or by the addition of various cofactors (NAD and NADP in both oxidized and reduced forms, riboflavin, glutathione. pyridoxal‐5′‐phosphate, ATP) to the incubation medium. [14C]GABA was readily metabolized to [14C]succinate both in vivo and in vitro. Isethionate formation activity was concentrated in the mitochondrial fraction, as was also GABA‐T activity. Partially purified GABA‐T from calf brain also slightly catalysed the formation of [35S]isethionate (about 1.3 μmol/min/g protein) from [35S]taurine. It appears that the slight formation of isethionate from taurine is coupled to GABA‐T activity. The formation of isethionate from taurine is so small, that it apparently has no role in the control of the brain taurine pool.
EFFECTS OF AMINOOXYACETIC ACID AND l‐GLUTAMIC ACID‐γ‐HYDRAZIDE ON GABA METABOLISM IN SPECIFIC BRAIN REGIONS
Authors: Walters J. R. , Eng N. , Peričić D. , Miller L. P.
Published: October 2006

Abstract— Aminooxyacetic acid (AOAA) administration produced an increase in γ‐aminobutyric acid (GABA) levels in regions of cerebral cortex, subcortex and cerebellum. In some cortical areas studied, the maximal effect was observed with 25 mg/kg AOAA; in other regions GABA levels were increased further with 50 and 75 mg/kg AOAA. Pretreatment with 25 mg/kg AOAA effectively inhibited GABA:2‐oxoglutarate aminotransferase (GABA‐T) and partially inhibited glutamic acid decarboxylase (GAD) activity in regions of cerebral cortex. However, this dose did not affect GAD activity in substantia nigra while GABA‐T in the nigra and in the cerebellum was only partially inhibited. In both cortical and subcortical areas, the increase in GABA produced by 25 mg/kg of AOAA was linear. In contrast, l‐glutamic acid‐hydrazide (GAH) had no effect in the pyriform and cingulate cortex for the first 60 min after injection, and produced a biphasic GABA increase in caudate and substantia nigra over a 4 h period. Results suggest that GAH and AOAA affect regional GABA metabolism differentially and that there are several problems associated with estimating absolute GABA synthesis rates by measuring the rate or GABA accumulation after inhibition of GABA catabolism with these agents. This approach, however, may provide an easily obtainable indication of whether drugs or other manipulations are altering GABA synthesis in a given region.
PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM TORPEDO CALIFORNICA
Authors: Brandon Christopher , Wu Jang‐Yen
Published: October 2006

Abstract— Choline acetyltransferase (ChAT), the enzyme responsible for the biosynthesis of acetylcholine in nervous tissue, has been purified to apparent homogeneity from the electric organ of the electric fish Torpedo californica using ion‐exchange, gel filtration, and hydroxyapatite chromatography. The final preparation had been purified 8570‐fold to a specific activity of 30μmol ACh formed/min/mg protein. The purified protein has a pH optimum of 6.8 (phosphate buffer), is activated by low concentrations (ca. 10 mm) of ammonium or alkylammonium ions, and is strongly inhibited by a sulfhydryl blocking reagent (DTNB). ChAT has a mol. wt. of 63000 when measured by SDS‐polyacrylamide gel electrophoresis or gel filtration.

A new method for the rapid assay of ChAT activity is described in which unreacted substrate ([3H]acetyl‐CoA) is removed from reaction mixtures by adsorption to charcoal: some advantages of this technique are discussed.
α‐TOXIN BINDING PROTEINS IN THE ELECTRIC ORGAN OF TORPEDO MARMORATA STUDIED BY IMMUNOCHEMICAL METHODS
Authors: Mattsson C. , Heilbronn E. , Ramlau J. , Bock E.
Published: October 2006

Abstract—

—Crossed immunoelectrophoretic techniques were developed to study the efficiency of the various purification steps in the isolation of nicotinic acetylcholine receptor (nAChR) from Torpedo mormorata electric organ. A new α‐neurotoxin binding assay based on immunoelectrophoresis is also presented. In crude extracts of Torpedo electric organ membranes one type of receptor molecule (M ñ; 300, 000) was found; an earlier described higher molecular form was shown to be an artifact of affinity chromatography.

Polyvalent antibodies against Torpedo electroplaque membranes, antibodies against purified membrane proteins and against Naja naja siamensisα‐neurotoxin revealed four α‐neurotoxin binding antigens (including nAChR). Two of these, nAChR and T2, were specific for electroplaque membrane and showed partial immunoidentity but different biochemical and physical properties.
POSTMORTEM AUTOLYTIC RESPONSE IN RAT BRAIN LYSOSOMES
Authors: McKeown Stephanie R.
Published: October 2006

Abstract—

Rat cerebral lysosomes were investigated during postmortem autolysis to determine the effect of the length and temperature of storage. A lysosome enriched fraction was isolated by differential ultracentrifugation and there was found to be a progressive increase in fragility as the length of storage increased. However, those lysosomes which survived the homogenisation procedure exhibited similar properties for at least 6 h after death if storage was at 21°C. Changes apparent after further storage for 18 h at 4°C were also detected in a shorter period if the temperature of storage was increased. The results suggest that, coupled with an all‐or‐none rupture of the lysosomes, there is a small increase in the permeability of the lysosomes shortly after death. In many respects the characteristics of the intact lysosomes up to 6 h after death resemble those obtained immediately.
COMPARATIVE PEPTIDE MAPPING AND ISOELECTRIC FOCUSING OF ISOLATED SUBUNITS FROM CHICK EMBRYO BRAIN TUBULIN
Authors: Nelles Lynn P. , Bamburg James R.
Published: October 2006

Abstract—

The α‐ and β‐subunits of chick embryo brain tubulin have been isolated under denaturing conditions and compared with respect to their molecular weight, amino acid composition, tryptic peptide maps, amide content and isoelectric focusing properties. An 8 M‐Urea‐containing polyacrylamide gel system with varying acrylamide concentrations was used for calculation of the retardation coefficients (KR) of the tubulin subunits. A molecular weight of 53,000 was estimated for each subunit by comparison to KR values for standard proteins. Amide contents of approx 41% of the carboxyl groups of α‐tubulin and 48% of the carboxyl groups of β‐tubulin were calculated using the average PI value, the pKintrinsic for the ionizable side chains of the amino acids and the amino acid composition of each subunit. Comparative peptide maps of trypsin digested α‐ and β‐tubulin demonstrated 16 peptides unique to each subunit and 23 peptides which comigrate. Both subunits give rise to multiple species on electrofocusing gels. The average isoelectric points for the α‐ and β‐subunits are 5.4 and 5.2, respectively.
STEADY STATE MAINTENANCE OF ELECTROLYTES IN THE SPINAL CORD OF THE FROG
Authors: Bianchi C. Paul , Erulkar S. D.
Published: October 2006

Isolated frog spinal cords equilibrated from 3 to 24 h in Ringer's solution maintained steady state conditions with regard to electrolyte composition. Total sodium and chloride contents measured on the same spinal cords were found to be nearly equivalent (Na = 46.6 ± 1.4μmol/g wet wt vs Cl = 46.2 ± 1.2μmol/g wet wt). Calcium and magnesium contents were 3.0 ± 0.5 and 4.8 ± 0.2pμol/g wet wt respectively for fresh spinal cords and 2.1 ± 0.4 and 5.5 ± 0.9pmol/g wet wt respectively for spinal cords equilibrated for 24 h. Zinc content was 0.29 ± 0.01 μmol/g wet wt. Insulin space was found to be smaller than sucrose space (0.24 ml/g vs 0.37 ml/g). Sodium and chloride spaces were slightly higher than sucrose space. Sodium and chloride in the non‐sucrose space was 4.8 and 9.5 μmol/g wet wt. Residual radioactive sodium, or sodium content of spinal cords washed out for 180 min in non‐radioactive Ringer's solution or in choline or lithium Ringer's solution, was 4.2 ± 0.4μmol/g wet wt (n = 9). The agreement between residual sodium content and sodium in the non‐sucrose space suggests that the mean intracellular sodium content of the spinal cord neurons is low.
ACTION OF THE NEUROTOXIN KAINIC ACID ON HIGH AFFINITY UPTAKE OF l‐GLUTAMIC ACID IN RAT BRAIN SLICES
Authors: Johnston G. A. R. , Kennedy Sue M. E. , Twitchin B.
Published: October 2006

Kainic acid is a linear competitive inhibitor (Kis 250 μm) of the ‘high affinity’ uptake of l‐glutamic acid into rat brain slices. Kainic acid inhibits the ‘high affinity’ uptake of l‐glutamic, d‐aspartic and l‐aspartic acids to a similar extent. Kainic acid is not actively taken up into rat brain slices and is thus not a substrate for the ‘high affinity’ acidic amino acid transport system or any other transport system in rat brain slices. Kainic acid (300 μm) does not influence the steady‐state release or potassium‐stimulated release of preloaded d‐aspartic acid from rat brain slices.

Kainic acid binds to rat brain membranes in the absence of sodium ions in a manner indicating binding to a population of receptor sites for l‐glutamic acid. Only quisqualic and l‐glutamic acid inhibit kainic acid binding in a potent manner. The affinity of kainic acid for these receptor sites appears to be some 4 orders of magnitude higher than for the ‘high affinity’l‐glutamic acid transport carrier.

Dihydrokainic acid is approximately twice as potent as kainic acid as an inhibitor of ‘high affinity’l‐glutamic acid uptake but is some 500 times less potent as an inhibitor of kainic acid binding and at least 1000 times less potent as a convulsant of immature rats on intraperitoneal injection. Dihydrokainic acid might be useful as a ‘control uptake inhibitor’ for the effects of kainic acid on ‘high affinity’l‐glutamic acid uptake since it appears to have little action on excitatory receptors.

N‐Methyl‐d‐aspartic acid is a potent convulsant of immature rats, but does not inhibit kainic acid binding or ‘high affinity’l‐glutamic acid uptake. N‐Methyl‐d‐aspartic acid might be useful as a ‘control excitant’ that activates different excitatory receptors to kainic acid and does not influence ‘high affinity’l‐glutamic acid uptake.
MULTIPLE DOPAMINE BINDING SITES: SUBCELLULAR LOCALIZATION AND BIOCHEMICAL CHARACTERIZATION1
Authors: Clement–Cormier Yvonne C. , George Robert J.
Published: October 2006

Abstract— The biochemical and pharmacological characteristics of dopamine agonist and antagonist binding to rat striatal subcellular fractions were studied and compared to the localization of dopamine–sensitive adenylate cyclase activity. The highest specific activity of adenylate cyclase sensitive to dopamine was associated almost exclusively with the crude synaptic membrane fraction (P2). Using [3H]‐haloperidol, [3H]apomorphine and [3H]spiroperidol as markers for the dopamine receptor, high affinity and stereoselective specific binding was observed for the crude synaptic fraction and the microsomal fraction (P3). Analysis of the binding of [3H]haloperidol to the striatal microsomal preparation revealed a homogeneous receptor site with a Kd value of 3.0 nm. The data for [3H]haloperidol binding to the crude synaptosomal fraction showed two saturable binding sites with Kd values of 2.5 nm and 12.5 nm. A similar heterogeneous binding profile was observed in the P2 fraction using [3H]apomorphine. The Kd values for [3H]apomorphine in this fraction were determined to be 1.2 nm and 7.2 nm. The effects of various biochemical parameters including ionic strength, salt concentration and pH on the binding of [3H]haloperidol to the P2 fraction were also studied. Overall, these data show that the subcellular localization of multiple binding sites in the crude synaptosomal fraction and the identification of specific binding to purified synaptosomes correlate with the subcellular distribution of striatal dopamine‐sensitive adenylate cyclase activity.
ESTIMATION OF THE KINETIC PARAMETERS OF UNIDIRECTIONAL TRANSPORT ACROSS THE BLOOD‐BRAIN BARRIER
Authors: Cunningham V. J. , Sarna G. S.
Published: October 2006

Abstract— The unidirectional transport of metabolic substrates from blood to brain may be defined in terms of Michaelis‐Menten saturable (Km, Vmax) and non‐saturable (Kd) components of influx. Various computation procedures have been previously reported to estimate the kinetic parameters when an intracarotid injection technique is used. Transformations of the influx data which allow linear plots to obtain estimates were compared with estimates obtained directly from a best fit on a least means squares criterion for both experimental and simulated data. Large discrepancies were apparent between the various estimates of the kinetic parameters when an equal weight was given to transformed data. For pyruvate (21‐day‐old rats), Km, values varied between 1.02 and 6.25 mM and Vmax varied between 0.68 and 2.30 μmol g−1 min−1. The estimates were almost equivalent when pyruvate data was re‐analysed using a weighting scheme based on the finding that the absolute value of the S.D. of influx increased in proportion to influx. It is recommended that estimates of kinetic parameters be obtained by an iterative, non‐linear least squares method to fit appropriately weighted data directly.
DISAPPEARANCE OF NEWLY SYNTHESIZED AND TOTAL DOPAMINE FROM THE STRIATUM OF THE RAT AFTER INHIBITION OF SYNTHESIS: EVIDENCE FOR A HOMOGENEOUS KINETIC COMPARTMENT
Authors: Paden Charles M.
Published: October 2006

Abstract— The hypothesis that the biphasic disappearance of dopamine (DA) from the rat striatum following inhibition of synthesis with α‐methyl‐p‐tyrosine (AMPT) represents catabolism from separate‘functional’and 'storage’compartments (Javoy & Glowinski, 1971) was tested by administering 3H‐tyrosine i.v. 10 min before 400 mg per kg AMPT. The levels of newly synthesized 3H‐DA and total DA were then determined at 5‐min intervals. While both declined biphasically, the rate of decay of 3H‐DA was significantly less than that predicted by the two‐pool hypothesis. In fact, the patterns of change of 3H‐DA and total DA were identical and the specific activity of DA did not vary following AMPT. To determine if an increase in DA synthesis and release would result in the preferential catabolism of newly synthesized 3H‐DA, the experiment was repeated fol!owing treatment with 0.1 mg per kg haloperidol i.v. 3H‐DA and total DA still exhibited identical biphasic declines and there was no change in the specific activity of DA after AMPT even though 3H‐DA levels were increased three‐fold by haloperidol. Thus (a) no evidence for the preferential catabolism of newly synthesized 3H‐DA was obtained and (b) newly synthesized and total striatal DA behaved as if localized in a homogeneous kinetic compartment under all conditions employed.

Rates of striatal DA metabolism were estimated in both experiments from changes in total DA after AMPT and by the formation of 3H‐DA in the initial 10 min after 3H‐tyrosine injection. Results of both approaches were consistent and indicated that the rates of striatal DA synthesis and catabolism may vary rapidly between approx. 20 and 90 nmol per g per h without violating single compartment kinetics. It is proposed that any labile pool contains no more than a few per cent of the DA in the striatum and the implications of this hypothesis are discussed.
THE FORMATION OF GLUTAMATE, ASPARTATE AND GABA IN THE RAT RETINA; GLUCOSE AND GLUTAMINE AS PRECURSORS
Authors: Morjaria Bharti , Voaden Mary J.
Published: October 2006

Abstract— The distribution of the neuroactive amino acids taurine, GABA, glycine, glutamate and aspartate, together with glutamine, have been studied in the rat retina. Peak levels of taurine were found in photoreceptor cells and of GABA and glycine in a retinal fraction enriched in amacrine cells and, synaptic terminals. In vitro, GABA formation from [3H]glutamine and [14C]glucose was also most prominent in this fraction; at 500 μm [3H]glutamine was the better precursor.

Observations on metabolism in the photoreceptor cell layer of the tissue suggest an active turnover of glutamate, aspartate and GABA, and show that glutamine may serve as an alternative substrate to glucose here, perhaps via the GABA bypath.
GABA FLUXES IN PRESYNAPTIC NERVE ENDINGS FROM IMMATURE RATS
Authors: Levi Giulio , Gallo Vittorio , Ciotti Teresa , Raiteri Maurizio
Published: October 2006

Abstract— Several parameters of GABA Auxes across the synaptosomal membrane were studied using synaptosomes prepared from the brain of immature (8‐day‐old) rats. The following aspects of GABA carrier‐mediated transport were similar in immature and mature synaptosomes: (1) magnitude of [3H]GABA accumulation; (2) GABA homoexchange in normal ionic conditions; (3) GABA homoexchange in the presence of cationic fluxes (Na+ and Ca2+ influx, K+ efflux) characteristic of physiological depolarization. As in adult synaptosomes (Levi & Raiteri, 1978), in these conditions the stoichiometry of GABA homoexchange was in the direction of net outward transport (efflux > influx).

The essential differences between the behaviour of 8‐day‐old and adult synaptosomes were the following: (1) β‐alanine (a glial uptake inhibitor) inhibited GABA uptake in immature synaptosomes (the inhibition being greater in crude than in purified preparations) and was without a significant effect in adult synaptosomes. DABA and ACHC (two neuronal uptake inhibitors) depressed GABA uptake more efficiently in purified than in crude immature synaptosomes, but were as effective in crude and purified nerve endings from adult animals. The data suggest a greater uptake of GABA in the‘gliosomes’contaminating the synaptosomal preparations from immature animals. (2) In immature synaptosomes prelabelled with [3H]GABA the specific radioactivity of the GABA released spontaneously or by heteroexchange (with 300 μm‐OH‐GABA) was the same as that present in synaptosomes, while in adult synaptosomes OH‐GABA released GABA with increased specific radioactivity. The data suggest a homogeneous distribution of the [3H]GABA taken up within the endogenous GABA pool in immature, but not in mature synaptosomes. (3) In immature synaptosomes the release of GABA (radioactive and endogenous) induced by depolarization with high KC was not potentiated by Ca2+, unless the synaptosomes had been previously depleted of Na+ These data suggest that, although a Ca2+ sensitive pool of GABA may be present, this pool is not susceptible to being released in normal conditions, probably because the high intrasynaptosomal Na+ level prevents a sufficient depolarization. The possible significance of these findings in terms of functional activity of GABAergic neurotransmission in the immature brain is discussed.