bluuebunny/crossref_metadata_2025 huggingface dataset:
AXONAL TRANSPORT OF SELECTED PARTICLE‐SPECIFIC ENZYMES IN RAT SCIATIC NERVE IN VIVO AND ITS RESPONSE TO INJURY
Authors: Schmidt Robert E. , McDougal David B.
Published: October 2006

Abstract— Orthograde and retrograde axoplasmic transport of selected axonal organelles were examined by monitoring accumulation of enzyme activities residing in various types of particles proximal and distal to a ligature placed on rat sciatic nerve as a function of time after tying.

Proximal to the tie, activity of acetylcholinesterase (AChE, EC 3.1.1.7; probably in small endoplasmic reticulum‐like particles) accumulated for 2 days; then, during the next 5 days, the accumulation disappeared. Activities of glutamic dehydrogenase (GDH, EC 1.4.1.3) and monoamine oxidase (MAO, EC 1.4.3.4) (both located in mitochondria) accumulated steadily for 7 days. Accumulation of monoamine oxidase activity was more rapid than that of glutamic dehydrogenase during the first day or two. Acid phosphatase (acid P'tase, EC 3.1.3.2; in lysosomes) activity also accumulated throughout the week of observation. Accumulation of all four enzyme activities proximal to the ligature was blocked by nerve crush or subepineurial vinblastine injection 1 cm or more proximal to the site of the tie.

Distal to the ligature, AChE activity accumulated early (14 h), and then gradually disappeared in the course of the week. MAO activity also accumulated, with a maximum at 2 days, and no further change thereafter. GDH activity, on the other hand, showed little accumulation during the first 2 days, but did appear in modest amounts at the end of the week. Distal accumulation of acid P'tase kept pace with proximal accumulation for the first day, and continued more slowly for another day, after which there was no further change.

This system has been used to study the effects of axonal crush injury upon anterograde and retrograde axoplasmic transport. A tie applied at various times after injury, proximal to the site of injury, was used to show that orthograde transport of AChE was maintained for 1 day after tying, but at 2 days had fallen 50% or more, and within a week was down to 20–25% of control. At 3 days after injury retrograde transport of AChE activity was not different from the control. Orthograde transport of acid P'tase activity was depressed 35% by injury. Retrograde transport of acid P'tase was inhibited more than 50% both at 3 and at 7 days after injury. Transport of the mitochondrial enzymes was not measurably affected.
INCREASED BINDING OF [3H]COLCHICINE TO VISUAL CORTEX PROTEINS OF DARK‐REARED RATS ON FIRST EXPOSURE TO LIGHT
Authors: Stewart Michael G. , Rose Steven P. R.
Published: October 2006

Abstract— The binding of [3H]colchicine (or a functionally similar metabolite) to acid‐insoluble material in vivo was measured in the motor and visual cortices of littermates rats which were either dark‐reared (D), exposed to light for 3 h or 24 h (L), or raised normally (N) in 12 h light/12 h dark animal house conditions. No significant differences were found in the binding in the motor cortex of the 3 h or 24 h L, D or N animals, but in the visual cortex after 3 h of light exposure a 23% elevation in binding was measured in L compared with D animals and a small though non‐significant (10%) increase in binding was also observed in this region in L compared with N animals. After 24 h of light exposure, binding of the label in the L animals fell near to that of the N and D animals. The results of vinblastine precipitation experiments suggested that much of the radioactivity was bound to the protein tubulin, and this was confirmed when no increased binding of an analogue of colchicine, lumi‐colchicine, was observed after 3 h of light exposure in L compared with D animals. It is suggested that these experiments show that colchicine can be used as a marker for changes in the tubulin population in light exposed animals, and demonstrate the transient nature of the increase in tubulin quantity, as opposed to a lasting effect on its synthesis. Further, they argue strongly in support of the idea that a component of protein flow from neuronal cell body to axons and dendrites in light exposed animals, is subject to environmental modification.
BRAIN pH MEASUREMENTS USING A DIFFUSIBLE, LIPID SOLUBLE pH SENSITIVE FLUORESCENT INDICATOR
Authors: Sundt Thoralf M. , Anderson Robert E. , Van Dyke Russell A.
Published: October 2006

Abstract—

Brain tissue pH was measured in cats at normocapnia hypocapnia, hypercapnia, and death from anoxia using a pH sensitive fluorescent indicator (umbelliferone) with both molecular and ionic fluorophors. A ratio analysis of the indicator's calibrated 450 nm fluorescent tissue washout curves from 340 and 370 nm excitation permitted direct determinations using a nomogram. Possible errors in these measurements related to differential quenching, absorption, and changes in the redox state of the indicator were investigated in vitro and in vivo for brain tissue and blood. In animals with preserved autoregulation, brain pH varied linearly with arterial pH (art pH 7.0, brain pH 6.98: art pH 7.4, brain pH 7.24). Brain pH at death fell to 6.68. An analysis of the indicator clearance curves suggests these measurements reflect a component of the intracellular space and the lipid solubility of the indicator suggests this is a membranous component.
TAURINE UPTAKE BY RAT BRAIN SYNAPTOSOMES
Authors: Kontro P. , Oja S. S.
Published: October 2006

Abstract— Uptake of [3,5S]taurine by rat whole brain synaptosomes was studied at varying temperatures, under O2, and N2 atmospheres, during electrical stimulation and in the presence of dinitrophenol or variable taurine concentrations in the incubation medium. The morphology and purity of the synaptosomes was checked by electron microscopy. The respiration of the synaptosomes was linear for at least 90 min. The taurine uptake was energy‐ and temperature‐dependent and significantly enhanced by electrical stimulation. The total uptake of taurine could be divided into three components, non‐saturable influx and saturable high‐affinity (Km= 46 μmol/l) and low‐affinity (Km, = 6.3 mmol/l) transport systems. The efficacy of the high‐affinity transport appears small in view of the postulated neurotrans‐mitter role of taurine.
PRODUCT INHIBITION OF RAT BRAIN HISTAMINE‐N‐METHYLTRANSFERASE
Authors: Orr Edward L. , Quay W. B.
Published: October 2006

Abstract— The inhibition of S‐adenosylmenthionine: histamine‐N‐methyltransferase (EC 2.1.1.8; HMT) by its products, 3‐methylhistamine (3‐MetHm) and S‐adenosyl‐l‐homocysteine (SAH), was examined using a preparation of the enzyme which was partially purified from rat brains. SAH was found in in vitro experiments, to be a competative inhibitor of HMT in relation to S‐adenosyl‐l‐methionine (SAM), with a Ki= 5.6 μM. SAH was shown to be a non‐competitive inhibitor with respect to histamine (Hm) (Ki= 5.0 μM). The Km's for SAM and Hm were 10.2 and 3.0 μM respectively. On the other hand, 3‐MetHm was determined to be a non‐competitive inhibitor of HMT with respect to Hm (Ki= 8.7 μM) and an uncompetitive inhibitor with respect to SAM (Ki= 9.6 μM). These results suggest that the addition of the substrate to, and the release of products by, HMT occurs sequentially. In the nomenclature Of Cleland (1963) the reaction is seemingly of the ‘ordered Bi‐Bi’ type.
POST‐MORTEM AND AMINOOXYACETIC ACID‐INDUCED ACCUMULATION OF GABA: EFFECT OF GAMMA‐BUTYROLACTONE AND PICROTOXIN
Authors: Peričić D. , Eng N. , Walters J. R.
Published: October 2006

Abstract— The effects of γ‐butyrolactone (GBL) and picrotoxin on both the post‐mortem and amino‐oxyacetic acid (AOAA) induced accumulations of γ‐aminobutyric acid (GABA) were examined in rats. GBL produced a marked dose‐dependent decrease in AOAA‐induced GABA accumulation in caudate. globus pallidus, cerebellar and cerebral cortices. The cingulate cortex showed the greatest response to GBL treatment; subanesthetic doses completely blocked the effect of AOAA. Picrotoxin increased the AOAA‐induced accumulation of GABA in parietal, entorhinal and cerebellar cortices, and had no significant effect in pyriform or cingulate cortices. Neither drug significantly altered the post‐mortem accumulation of GABA. Results suggest that picrotoxin, a GABA antagonist and convulsant drug, causes an increase in GABA synthesis in vivo. The apparent decrease in GABA synthesis following GBL treatment was greater than that observed with anesthetic doses of chloral hydrate and was not blocked by picrotoxin. Alterations in the activity of GABA neurons, cerebral glucose metabolism and GAD activity may contribute to the apparent decrease in in vivo GABA synthesis caused by GBL.
A METHOD FOR DETERMINATION OF ACETYLCHOLINE BY SLOW PYROLYSIS COMBINED WITH MASS FRAGMENTOGRAPHY ON A PACKED CAPILLARY COLUMN
Authors: Polak R. L. , Molenaar P. C.
Published: October 2006

Abstract—

A new procedure is described for the estimation of ACh by pyrolysis‐gas chromatography/ mass spectrometry.

ACh iodide and the iodides of ACh‐d16 or propionylcholine are slowly demethylated at 250°C by pyrolysis on the tip of a glass probe after insertion into the heated entrance of a glass tube leading to a packed capillary column. The volatile tertiary amines are then carried by helium to the column and trapped in its initial part which is kept at about 60°C. After 2–3 min the chromatography is started when the amines are released by heating this part to the ambient temperature in the oven (165°C).

Peaks due to demethylated ACh and propionylcholine are well separated. The limit of detection is about 0.3pmol. After pyrolysis of mixtures of ACh and either ACh‐d16 or propionylcholine the peak amplitudes are linearly related to the doses.
DIHYDROMUSCIMOL, THIOMUSCIMOL AND RELATED HETEROCYCLIC COMPOUNDS AS GABA ANALOGUES
Authors: Krogsgaard‐Larsen P. , Hjeds H. , Curtis D. R. , Lodge D. , Johnston G. A. R.
Published: October 2006

A series of heterocyclic GABA analogues related to muscimol (5‐aminomethyl‐3‐isoxazolol) were tested as depressants of the firing of GABA sensitive neurones on the cat spinal cord, and as inhibitors of the sodium‐independent binding of GABA to rat brain membranes. Furthermore, the compounds were examined as inhibitors of GABA uptake into rat brain slices and as inhibitors of the activities of the GABA‐metabolizing enzymes L‐glutamate 1‐carboxylyase and GABA:2‐oxoglutarate aminotransferase.

Dihydromuscimol [(RS)‐4,5‐dihydromuscimol] and thiomuscimol (5‐aminomethyl‐3‐isothiazolol) were approximately equipotent to muscimol as bicuculline‐sensitive depressants of neuronal firing and as inhibitors of GABA binding. The structurally related compounds isomuscimol (3‐aminomethyl‐5‐isoxa‐zolol) and azamuscimol (5‐aminomethyl‐3‐pyrazolol) were much weaker than muscimol as GABA agonists. The affinity of the compounds for GABA receptor sites in vitro is in agreement with their relative potency as GABA receptor agonists in vivo. The rat brain synaptic membranes used for the GABA receptor binding studies were prepared by two procedures, which were shown to have a pronounced influence on the observed potency of the inhibitors of GABA binding.

The compounds were weak or inactive as inhibitors of the uptake of GABA into rat brain slices and of the activity of GABA: 2‐oxoglutarate aminotransferase in vitro. Azamuscimol and 2‐methylaza‐muscimol were moderately potent inhibitors.of the activity of L‐glutamate 1‐carboxylyase in vitro. This inhibition by azamuscimol was timedependent following pseudo‐first‐order kinetics, consistent with azamuscimol acting as a catalytic inhibitor.

The structure of the heterocyclic rings of these zwitterionic compounds is a factor of critical importance for interaction with GABA receptors. The present structure‐activity analysis demonstrates that heterocyclic GABA analogues having a high degree of delocalization of the negative charges have low affinity for the GABA receptors.
EFFECTS OF d‐ AND l‐AMPHETAMINE ON LOCAL CEREBRAL GLUCOSE UTILIZATION IN THE CONSCIOUS RAT1
Authors: Wechsler L. R. , Savaki H. E. , Sokoloff L.
Published: October 2006

Amphetamine, a potent sympathomimetic amine, has powerful stimulant actions in the central nervous system. These actions are believed to be related to the influence of amphetamine on release and uptake of catecholamine neurotransmitters. The [14C]deoxyglucose method makes it possible to study changes in cerebral metabolic rate in different areas of gray and white matter. Because of the close relationship between metabolic rate and functional activity, this method may be used to identify specific structures in the brain in which functional activity is altered. The [14C]deoxyglucose method was used to explore for changes in metabolic rate produced by d‐and l‐amphetamine (5 mg/kg) in forty gray and four white matter structures in normal conscious rats. d‐Amphetamine produced increases in local cerebral glucose utilization in a number of components of the extrapyramidal motor system, as well as in some other structures known to contain dopamine‐producing and/or dopaminoceptive cells. The largest increases after d‐amphetamine administration occurred in the subthalamic nucleus and the zona reticulata of the substantia nigra. l‐Amphetamine produced increases in some but not all of these same structures, and these were generally smaller than those observed with d‐amphetamine. Decreases in local cerebral glucose utilization after either d‐ or l‐amphetamine administration were found in the habenula and the suprachiasmatic nuclei of the hypothalamus. The effects in the suprachiasmatic nuclei may reflect their normal diurnal rhythm in metabolic rate. These results indicate that amphetamines may influence behavior through effects on specific regions of the brain. Only some of these regions have previously been studied as possible sites of action of amphetamine.
THE METABOLISM OF GABA AND OTHER AMINO ACIDS IN RAT SUBSTANTIA NIGRA SLICES FOLLOWING LESIONS OF THE STRIATO‐NIGRAL PATHWAY
Authors: Minchin M. C. W. , Fonnum F.
Published: October 2006

The metabolism of GABA and other amino acids from various radioactive precursors has been studied in the rat substantia nigra using a sensitive double isotope dansyl derivative assay. Labelled acetate gave greater labelling of glutamate than of glutamine in substantia nigra slices whereas the reverse was the case for cerebral cortex slices.

Unilateral transection of the striato‐nigral pathway caused a parallel decrease in the GABA and GAD content of the substantia nigra. It also reduced the total synthesis of GABA from all labelled precursors used, namely acetate, glutamate and glucose. After incubation with [1‐14C]acetate the specific activity of glutamate and aspartate, but not that of GABA, increased on the lesioned side compared with the normal side. The specific activity of glutamate, but not that of GABA or aspartate, decreased after incubation with [U‐14C]glucose on the lesioned side compared with the normal side. The results could be explained by the previously proposed hypothesis concerning differential labelling of metabolic pools by the two precursors. [U‐14C]Glutamate lead to increased labelling of GABA on the lesioned side relative to the normal side.

Incubation of slices from substantia nigra with β‐mercaptopropionic acid caused a decrease of labelling of GABA from glucose and acetate, probably as the result of GAD inhibition. The labelling pattern of the other amino acids, apart from that of glutamate which showed a decrease when synthesised from acetate, did not change appreciably.
INHIBITION PATTERN BY ANALOGS INDICATES THE PRESENCE OF TEN OR MORE TRANSPORT SYSTEMS FOR AMINO ACIDS IN BRAIN CELLS
Authors: Sershen Henry , Lajtha Abel
Published: October 2006

Abstract— We surveyed the transport systems present in the brain for amino acids. Cellular transport was measured in brain slices, and capillary transport was estimated by measuring in vivo the short‐term (15 s) extraction by brain from the blood. Specific analog inhibition of uptake was used to distinguish the classes. Amino acid levels (close to physiological) were such that primarily the ‘low‐affinity’ transport was measured.

In brain tissue we could distinguish 10 overlapping amino acid transport classes. Five of these, described in a number of tissues, were characterized by their substrates: alanine (A system), leucine (L system), alanine‐serine‐cysteine (ASC system), glutamic acid (Glu system), and arginine (Ly+ system), respectively. The others distinguished were each fairly specific for one of the following five amino acids: glycine, proline, γ‐aminobutyric acid (GABA), taurine, and lysine. Of these 10 systems only 4 could be clearly found in capillary transport: L, ASC, Ly +, and Glu.

The properties and the distribution of the transport systems are different. Examples are that at least one of the systems is present primarily only in neurons (GABA), and one primarily in glia (taurine). The specificity of some of the systems, e.g. A, is altered during development. In contrast to the properties of most other systems, there is little Na+, energy, or temperature dependence of the L system. This is reflected in the properties of capillary neutral amino acid transport when the L system is the predominant one.
ACETYLCHOLINE CONTENT OF THE SCHWANN CELL AND AXON IN THE GIANT NERVE FIBRE OF THE SQUID
Authors: Villegas Jorge , Jenden Donald J.
Published: October 2006

Abstract— Acetylcholine and choline were identified and their concentrations measured, by means of gas chromatography/mass spectrometry, in extracts obtained from nerve fibers of the hindmost stellar nerve of the squid Sepioteuthis sepioidea. These compounds were quantitated in samples of stellar nerve devoid of giant fiber, intact giant nerve fiber, extruded axoplasm, and axoplasm‐free giant nerve fiber sheaths. In 11 samples of stellar nerve devoid of giant fiber, weighing an average of 20.8 ± 2.3 mg (s.e.m.), 756 ± 91 pmol ACh and 8.65 ± 0.62 nmol of choline were found. The total ACh content of the largest fibre in this group (10 μm in diameter), for a 5 cm length of nerve, is in the order of 0.16 pmol. The average wet weights of a single giant nerve fiber (270‐420 μm in diameter) and its separate components (s.e.m.; in mg; number of fibers in parentheses) were: intact fiber, 4.58 ± 0.19 (25); extruded axoplasm, 3.38 ± 0.13 (20); sheaths, 1.21 ± 0.11 (16). The average ACh content per unit weight of sample was about 2‐3 times higher in the sheaths (5‐13 pmol‐mg−1) than in the axoplasm (2‐4 pmol mg−1), whereas the ACh concentrations estimated per unit volume of cellular water were about 40 times higher in the Schwann cell (107‐222 μm) than in the axon (2‐5μm). These experimental findings establish the presence of ACh in the giant nerve fiber of S. sepioidea. They also indicate the Schwann cells themselves as the main source for the release of ACh, responsible for their long‐lasting hyperpolarizations following the conduction of nerve impulse trains by the axon.
THE LACK OF SPECIFICITY TOWARDS SALTS IN THE ACTIVATION OF CHOLINE ACETYLTRANSFERASE FROM HUMAN PLACENTA
Authors: Hersh Louis B.
Published: October 2006

Abstract— The effects of monovalent and divalent anions on the choline acetyltransferase reaction have been determined at high (5.0 mM) and low (0.58 mM) choline. At 0.58 mM‐choline, both monovalent and divalent anions activate the enzyme ±9 fold; however, at 5.0mM‐choline, monovalent anions activate the enzyme ±25 fold, while divalent anions activate ±9 fold. Both monovalent and divalent anions show uncompetitive activation with respect to choline. When either dimethylaminoethanol, N‐(2‐hydroxyethyl)‐N‐methyl piperidinium iodide, or N‐(2‐hydroxyethyl)‐N‐propyl pyrrolidinium iodide was substituted for choline, activation by monovalent or divalent anions was only 2.5‐4 fold. With AcCoA as substrate the ChA reaction can be increased ±20 fold by increased salts; however, with acetyl dephosphoCoA as substrate, the reaction is insensitive to the salt concentration. Similar salt effects on the ChA reaction, as measured in the direction of acetylcholine synthesis, have been demonstrated in the reverse reaction. In addition, inhibition of the forward reaction by acetylcholine has been measured as a function of sodium chloride concentration. Although the K1 for acetylcholine increases with increasing salt, this change in K1, parallels the increase in the Km for choline. These results support the hypothesis that both monovalent and divalent anions activate choline acetyltransferase by the same singular mechanism; which is to increase the rate of dissociation of coenzyme A from the enzyme.
INFLUENCE OF TEMPERATURE ON THE VELOCITY AND ON THE ISOTOPE PROFILE OF SLOWLY TRANSPORTED LABELED PROTEINS
Authors: Cancalon Paul
Published: October 2006

Abstract— Slow intra‐axonal flow of [3H]leucine labeled proteins has been studied in the garfish olfactory nerve. Because of the homogeneity of the nerve a very well defined peak of slowly transported radioactivity is observed. The velocity of slow flow increases linearly with temperature. Between 14 and 28°C, the rate of the peak apex increases from 0.26 to 1.57 mm/day and the rate of the leading edge of the wavefront from 0.54 to 2.75 mm/day. Extrapolation of the rate‐temperature function indicates that slow flow should stop at 11°C. However, a velocity of 0.1 mm/day was determined for experiments conducted at 10°C. Between 15 and 25°C a Q10 of 3.7 was determined for the peak apex and of 3.3 for the leading edge of the wavefront. The Q10's are significantly larger than the value of 2.2 found for fast transport (Gross & Beidler, 1975) and support the possibility of at least partial differences between the mechanisms of fast and slow transport. A very small peak was found to migrate in front of the main peak. The positioning of this peak seems to be similar to one found by Lasek & Hoffman (1976) in rat ventral motor neurons.

A temperature dependent exponential decrease of the slow moving peak height was measured and it can be estimated that only 1% of the slowly transported radioactivity reaches the synapses. Most of the slow radioactivity appears to remain in the axon behind the peak. The plateau height was also found to decrease exponentially with time. The rate of disappearance greatly affects the profile determined by the slowly transported labeled proteins along the nerve.
PURIFICATION AND PROPERTIES OF A PROLYL ENDOPEPTIDASE FROM RABBIT BRAIN
Authors: Orlowski Marian , Wilk Elizabeth , Pearce Stevens , Wilk Sherwin
Published: October 2006

Abstract— An enzyme with the specificity of a prolyl endopeptidase was purified about 880‐fold from rabbit brain. The enzyme hydrolyzes peptidylprolyl‐peptide and peptidylprolyl‐amino acid bonds. Several biologically active peptides such as angiotensin, bradykinin, neurotensin. substance P and thyrotropin releasing hormone are degraded by hydrolysis of the bond between the carboxyl group of proline and the adjacent amino acid or ammonia respectively. The enzyme is activated by dithiothreitol and inhibited by heavy metals and thiol blocking agents. The serine protease inhibitor phenylmethanesulfonylfluoride has no effect on activity; however, inhibition was obtained with diisopropylfluorophosphate. Prolyl endopeptidase has a molecular weight of about 66,000 and a pH optimum of about 8.3. A new chromogenic substrate, N‐benzyloxycarbonylglycyl‐L‐prolylsulfamethoxazole, was used for determination of enzyme activity. The substrate is hydrolyzed to N‐benzyloxycarbonylglycyl‐L‐proline and free sulfamethoxazole which can be conveniently determined by a colorimetric procedure.
RIBONUCLEASE ACTIVITIES IN VARIOUS CLASSES OF NUCLEI FROM BOVINE CEREBRUM
Authors: Ittel M. E. , Mandel P.
Published: October 2006

Abstract— Nuclei of adult bovine cerebrum were separated by dissection of grey matter from white matter and discontinuous sucrose gradient centrifugation, into neuron, astrocyte‐enriched and oligodendrocyte fractions. The activities of alkaline and acidic RNAses were determined in the different nuclei types. The acidic and alkaline RNAse activities of the neuronal nuclei were much higher than those of the glial nuclei. In each type of nuclei the acidic RNAse activity was lower than the measured alkaline RNAse activity.
THE CONVULSANT ACTION OF HYDRAZIDES AND REGIONAL CHANGES IN CEREBRAL γ‐AMINOBUTYRIC ACID AND PYRIDOXAL PHOSPHATE CONCENTRATIONS
Authors: Horton R. W. , Chapman A. G. , Meldrum B. S.
Published: October 2006

Abstract— Regional changes in the concentration of GABA and pyridoxal phosphate were determined in rat brain after i.p. administration of convulsant doses of methyldithiocarbazinate (11 mg/kg), isonicotinic acid hydrazide (250 mg/kg) and thiosemicarbazide (25 mg/kg). At 15 and 30 min after methyldithiocarbazinate GABA concentrations were reduced in all brain regions (except ventral mid‐brain). After 30 min the largest decrease was in the cerebellum (41%) and the smallest decrease in the hypothalamus (20%). Pyridoxal phosphate concentrations were decreased by 39‐57%. After isonicotinic acid hydrazide. the regional decreases in GABA concentration were smaller and of slower onset than those seen after methyldithiocarbazinate. The pons‐medulla was the first region to show a decrease (at 15 min) whereas a decrease was not seen in the frontal cortex until 45 min. Regional decreases in pyridoxal phosphate were smaller than those seen after methyldithiocarbazinate. After thiosemicarbazide, small regional decreases in GABA concentration were observed only in the hypothalamus, cerebellum, pons‐medulla and posterior cortex (13‐18%) and there was no apparent correlation between regional decreases in pyridoxal phosphate and regional decreases in GABA.
IMMUNOCHEMICAL PROPERTIES OF S‐100 PROTEINS AND THEIR SUBUNITS
Authors: Mahadik S. P. , Graf L. , Korenovsky A. , Rapport M. M.
Published: October 2006

Abstract— The immunological activities of two populations of bovine S‐100 proteins with anti‐S‐100 serum were studied by complement fixation and rocket immunoelectrophoresis. The reactivities of subunits of these two populations were studied by crossed immunoelectrophoresis and rocket immunoelectrophoresis. Although the two populations conformed in all respects to the properties of S‐100 protein, the immunological reactivity of one, III‐IVa‐1, was significantly lower than that of the other, III‐IVb‐1. The difference was much larger when the S‐100 protein fractions were isolated in the absence of aids (mercaptoethanol, EDTA, EGTA, protease inhibitors). With bovine S‐100 fractions, the three subunits separated by differences in charge as well as the four subunits separated by differences in molecular weight all reacted with the same antibody molecules in the antiserum. The reactivities of the subunits showed large quantitative differences.

Two populations of S‐100 proteins from rat brain also showed differences in reactivity with anti‐S‐100 serum. The two subunits in each of these fractions reacted with anti‐S‐100 serum but with quantitative differences, the larger having almost double the activity of the smaller. These results provide firm evidence for the heterogeneity of S‐100 proteins based on immunological activity of their subunit components. Different molecular species of S‐100 proteins probably differ considerably in their reactivity with antibodies to S‐100 protein. Some of the more reactive molecular species also appear to be much more labile, since the reactivity of some S‐100 protein fractions was considerably reduced when they were isolated in the absence of aids.
EFFECTS OF SULFHYDRYL REAGENTS ON BRAIN MICROTUBULE‐ASSOCIATED ATPase ACTIVITY IN VITRO
Authors: Wallin Margareta , Larsson H. , Edström A.
Published: October 2006

Abstract— The effects of two sulfhydryl reagents, PCMBS (p‐chloromercuribenzene sulfonic acid) and NEM (N‐ethylmaleimide) on microtubule‐associated Mg2+ ‐and Ca2+ ‐ATPase activity were studied in a MTP (microtubule proteins) preparation and in a MAP (microtubule‐associated proteins) fraction. In the MTP preparation at pH 6.8, PCMBS stimulated the Mg2+ ‐ATPase activity at low concentrations and inhibited at higher, whereas the Ca2+ ‐ATPdse activity was only inhibited. NEM affected the activity in a similar way. At pH 8.0 PCMBS was only inhibitory. NEM showed stimulatory effects over a broader concentration range.

Preincubation in the presence of ATP counteracted the stimulatory effects of both PCMBS and NEM on Mg2+ ‐ATPase at pH 6.8.

In the MAP fraction at pH 6.8 PCMBS and NEM caused similar but less pronounced effects on the Mg2+ ‐and Ca2+ ‐ATPase.

The results show that brain microtubule‐associated ATPase activity is similar to dynein and myosin ATPases with respect to biphasic alteration by sulfhydryl reagents.
NEURONAL‐GLIAL CONTRIBUTIONS TO TRANSMITTER AMINO ACID METABOLISM: STUDIES WITH KAINIC ACID‐INDUCED LESIONS OF RAT STRIATUM
Authors: NICKLAS W. J. , Nunez R. , Berl S. , Duvoisin R.
Published: October 2006

Abstract– Various aspects of amino acid metabolism were studied in striatum of rats with unilateral, kainic acid‐induced lesions. Tissue slices were prepared from the lesioned and the contralateral, unlesioned, striatum. The preparations were incubated with a mixture of d‐[2‐14C]glucose and [3H]acetate in a Krebs‐Ringer bicarbonate medium to evaluate oxidative metabolism. Glutamate and aspartate levels were decreased in the slices prepared from the lesioned striata by 35‐40% and that of GABA by 75% compared to the levels found in the slices from the contralateral striata; glutamine levels were not different in the two preparations. Glucose utilization was decreased 60% in the slices from the lesioned striatum; this was caused not only by decreased levels of glutamate, aspartate and GABA but also by a decreased rate of labelling of glutamate and aspartate. On the other hand, the metabolism of [3H]acetate was greatly increased. The specific activities of glutamate and aspartate were 4‐5‐fold higher in the slices from kainic acid‐lesioned striata; those of glutamine and GABA were unchanged. Thus, there was a 6‐7‐fold increase in the ratio of 3H to 14C in the specific activities of glutamate, aspartate and GABA with no change in this ratio in glutamine. The labelling of glutamine relative to that of glutamate, especially from [3H]acetate, suggested that the compartmentation of the glutamate‐glutamine system was greatly altered in the kainate‐lesioned striatum which now more closely resembled a single compartment system. The activities of lactate dehydrogenase, glutamate dehydrogenase, GABA transaminase and ‘cytoplasmic’ aspartate aminotransferase were decreased in homogenates of lesioned striatum. Succinate dehydrogenase, glutaminase (phosphate‐activated) and ‘mitochondrial’ aspartate aminotransferase activities were unchanged whilst that of glutamine synthetase was increased. The results are consistent with hypotheses concerning the assignment of labelled acetate metabolism to glial cells as well as the distribution of the above enzymes between glia, neurones and nerve endings.
REDOX PROPERTIES OF CYTOCHROME OXIDASE AND VASCULAR REACTIVITY OF ASTROCYTOMAS AND NEUROBLASTOMAS IN VIVO
Authors: Sylvia Avis L. , Somjen George G. , Bigner Darell D. , Rosenthal Myron
Published: October 2006

Abstract— Dual wavelength reflection spectrophotometry was used to determine steady state changes in the reduction‐oxidation ratio of cytochrome c oxidase (a,a3) and vascular reactivity accompanying progressive growth of cortical and subcutaneous astrocytomas and neuroblastomas in vivo. Blood volume responses indicate that vessels invading the tumors retain regulatory reactivity typical of the body region of implantation and do not acquire those typical of the tissue of origin of the neoplastic cells. In comparison with non neoplastic tissue, early stage tumor growth was associated with highly oxidized ratios of cytochrome a,a3. A transition to highly reduced cytochrome a,a3 occurred during late stage tumor development. Such differences from normal cerebral tissues reflect alterations in micro‐circulation and respiratory chain function accompanying the dynamics of tumor growth, and could provide a basis for selective therapeutic measures.
γ‐AMINOBUTYRTC ACID RECEPTORS IN CEREBELLAR MEMBRANES OF RAT BRAIN AFTER A TREATMENT WITH TRITON X‐1001
Authors: Horng Jong S. , Wong David T.
Published: October 2006

Abstract— The treatment of cerebellar membranes of rat brain with a low concentration of Triton X‐100 followed by sufficient washing results in an increase of the Na+‐independent binding of [3H]GABA and a total loss of the Na +‐dependent binding of [3H]GABA. The Na+‐independent binding of [3H]GABA was more abundant in membranes of cerebellum than in membranes of other rat brain regions and mainly localized in the synaptic membrane fraction of a cerebellar homogenate. In the Triton‐treated membranes, the Na+‐independent binding of [3H]GABA was a saturable process, which could be resolved into two components, a high and a low affinity component with dissociation constants of 4.5 and 30 nm, respectively. The neurophysiological agonists, muscimol, GABA, and imidazole acetic acid, and the antagonist, bicuculline, inhibited the high affinity Na+‐independent binding of [3H]GABA by 50% at 0.003, 0.012, 0.3 and 10 μm respectively. These data suggest that the Na+‐independent binding of [3H]GABA in the Triton‐treated cerebellar membranes represents the synaptic receptors of GABA. It is emphasized that extensive washing of the membranes after a Triton treatment is necessary in order to detect the high affinity Na+‐independent binding of [3H]GABA.
ISOLATION OF AXONAL VARICOSITIES (AUTONOMIC SYNAPTOSOMES) FROM THE ENTERIC NERVOUS SYSTEM
Authors: Jonakait G. Miller , Gintzler Alan R. , Gershon Michael D.
Published: October 2006

Abstract— A subcellular fraction enriched with varicosities of autonomic axons was obtained from homogenates of strips of guinea‐pig longitudinal muscle and adherent myenteric plexus using differential and sucrose‐or Ficoll‐density gradient ultracentrifugation. This fraction contained the marker, [G‐3H]5‐hydroxytryptamine (5‐HT), taken up by serotonergic axon terminals present in the preparation during a period of incubation prior to homogenization. Electron microscopic examination showed that the isolated varicosities resembled CNS synaptosomes in containing vesicles and mitochondria but, as is characteristic of autonomic postganglionic terminals, they lacked synaptic membrane specializations. Quantitative electron microscopic radioautography revealed that [G‐3H]5‐HT was confined to isolated varicosities. Isolated varicosities were capable of taking up and sequestering 5‐HT from the surrounding medium; this uptake was temperature‐sensitive and blocked by fluoxetine. The subcellular distribution of [G‐3H]l‐tryptophan was also studied by subfractionation of strips in an attempt to determine which structures were responsible for the high‐affinity uptake of that amino acid. Although most of the [G‐3H]l‐tryptophan was found in the high‐speed supernatant, particulate [G‐3H]l‐tryptophan was most concentrated in the subcellular fraction containing isolated axonal varicosities. These studies indicate that a fraction containing functional serotonergic varicosities can be isolated from the gut. These varicosities are probably one of the elements responsible for the high‐affinity uptake of l‐tryptophan by the myenteric layer of the gut.
THE INCORPORATION OF GLUCOSE INTO α‐1, 4‐GLUCAN PROTEINS OF BOVINE RETINA MEMBRANES 1
Authors: Curtino Juan A. , Maccioni Hugo J. , Caputto Ranwel
Published: October 2006

—Incubation of bovine retina membranes with UDP‐[14C]glucose resulted in the incorporation of [14C]glucose into endogenous α‐1, 4‐glucan proteins. The transferring system was concentrated in membranes that floated at 0.94 and 1.10m‐sucrose when centrifuged in a discontinuous sucrose density gradient and was almost absent in the rod outer segment (ROS) and the 100, 000 g supernatant fractions. The glucan proteins labelled by incubation with the radioactive sugar nucleotide at micromolar concentrations were distinguished in two fractions by their solubilities in trichloroacetic acid (TCA): glucan protein‐I (GP‐I), insoluble in TCA, and glucan protein‐II (GP‐II), soluble in TCA and precipitable by ethanol from the TCA soluble fraction. GP‐I and GP‐II were precipitated by trichloroacetic acid‐phosphotungstic acid (TCA‐PTA). A third fraction, glucan protein‐III (GP‐III) was found when incubations were carried out with UDP‐[14C]glucose at millimolar instead of micromolar concentrations. GP‐III was soluble in TCA and in TCA‐PTA and precipitable by ethanol from the TCA soluble fraction. GP‐II was excluded from a Sephadex G‐200 column and showed a greater size than GP‐I in a Sepharose 2B column. The radioactive residues obtained from the glucan proteins after digestion with pronase were totally included in a Sephadex G‐25 column and were of a greater size than the labelled residues released with salivary α‐amylase. Only radioactive maltose was found after a‐amylase treatment. When membranes containing labelled GP‐I and GP‐II were incubated with unlabelled UDP‐glucose at millimolar concentrations, GP‐I was converted into GP‐II and GP‐III was formed.
FRACTIONATION OF SOLUBLE BRAIN GLYCOPROTEINS BY AFFINITY AND HYDROXYLAPATITE CHROMATOGRAPHY
Authors: Langley O. K.
Published: October 2006

—A separation of soluble brain proteins and Con A‐binding glycoproteins by chromatography on calcium hydroxylapatite in the presence of SDS is described. Seventeen Coomassie Blue‐stained bands were detected by polyacrylamide gel electrophoresis in SDS of Con A‐binding glycoproteins of the soluble fraction of rat brain: 16 of these were found by in vivo uptake of [3H]fucose to be fucosylglycoproteins. Hydroxylapatite chromatography yielded several glycoprotein pools, each of which was shown by gel electrophoresis to contain between 4 and 8 individual glycoproteins. Such pools were enriched in [36H]fucose relative to the brain soluble fraction by factors of between 6 and 21. Preliminary experiments demonstrate that this method is also applicable to the fractionation of membrane‐bound glycoproteins.
The Effects of Scorpion Venom Toxin on the Release of Amino Acid Neurotransmitters from Cerebral Cortex In Vivo and In Vitro
Authors: Coutinho‐Netto J. , Abdul‐Ghani A.‐S. , Norris P. J. , Thomas A. J. , Bradford H. F.
Published: October 2006

: Tityustoxin, the active component of the venom of the Brazilian yellow scorpion Tityus serrulatus, caused specific release of the neurotransmitter amino acids glutamate, aspartate and GABA in vivo from the superfused sensori‐motor cortex of conscious unanesthetised rats and in vitro from rat cortical synaptosomes. The effects on synaptosomes appear to be due to a depolarising action. Synaptosomal potassium levels were depleted by the toxin. The action was also blocked both in vivo and in vitro by tetrodotoxin and was Ca2+ ‐dependent. The uptake of [U‐14C]GABA was inhibited by tityustoxin but this action was prevented by tetrodotoxin (1 μm). Since the release of [U‐14C]GABA from synaptosomes due to the tityustoxin was also prevented by ietrodotoxin under identical circumstances, it is concluded that the tityustoxin has a primary action on release of neurotransmitters rather than on uptake.
Accumulation of Cyclooxygenase Products of Arachidonic Acid Metabolism in Gerbil Brain During Reperfusion After Bilateral Common Carotid Artery Occlusion
Authors: Gaudet Robert J. , Alam Iftekhar , Levine Lawrence
Published: October 2006

: Several of the cyclooxygenase products of arachidonic acid were measured in the cerebral hemispheres of gerbils subjected to transient interruption of the cerebral circulation. The levels of PGD2, PGF2α, PGE2, TXB2, 13,14‐H2‐15‐keto‐PGE2, and the stable nonenzymic product of prostacyclin, 6‐keto‐PGF1α, were not altered at the end of a 5‐min period of ischemia. However, the onset of reperfusion was accompanied by a rapid accumulation of these products. Levels were highest during the initial period of reperfusion, then decreased to approach control levels after 120 min. PGD2, PGF2α, and PGE2 were the predominant metabolites detected. This postischemic accumulation of arachidonic acid metabolites could be blocked by prior administration of inhibitors of cyclooxygenase activity.
Localization of Hexokinase in Neural Tissue: Electron Microscopic Studies of Rat Cerebellar Cortex
Authors: Kao‐Jen Judy , Wilson John E.
Published: October 2006

: The distribution of hexokinase (ATP:d‐hexose 6‐phosphotransferase, EC 2.7.1.1) in the rat cerebellar cortex has been studied at the electron microscopic level using the peroxidase‐antiperoxidase procedure. Extensive staining of cytoplasmic regions, with some increased staining at mitochondrial profiles, was seen in the cell bodies of both neurons (basket, stellate, Lugaro, Golgi, and granule cells) and astrocytes. Oligodendrocytes showed little or no detectable staining. Purkinje cell perikarya were much less intensely stained than were the perikarya of other neurons. The initial portion of the Purkinje dendrite was, like the perikaryon from which it emerged, lightly stained. More intense staining was seen in the secondary and tertiary branches of the Purkinje dendrite, but the terminal branches were devoid of stain. Granule cell dendrites were well stained in their initial portions but devoid of stain in their terminal dendritic digits which form part of the cerebellar glomeruli. In contrast to the unstained granule cell dendritic digits, the central mossy fiber nerve terminal of the glomerulus exhibited intense staining of the mitochondrial profiles and of synaptic vesicles adjacent to the mitochondria. Axons of basket cells showed intense staining in the segments adjacent to the Purkinje cell soma, while terminal twigs of the basket axons in the pinceau surrounding the (unstained) initial segment of the Purkinje axon showed markedly decreased staining intensity. These results indicate that there may be substantial variation in hexokinase levels between the various regions of neuronal processes. Hexokinase was seen at both cytoplasmic and mitochondrial locations in a variety of cells. It does not appear likely that location of hexokinase can be directly correlated with cell type, i.e., with neurons versus glia.
Regional and Subcellular Distribution of S‐Adenosylhomocysteine Hydrolase in the Adult Rat Brain
Authors: Broch Ole Jacob , Ueland Per Magne
Published: October 2006

The regional distribution of S‐adenosylhomocysteine hydrolase was determined in the rat brain. Small variations in enzyme activity between different regions were observed. Highest activity was found in hypothalamus and bulbus olfactorius, the least in pons and medulla. About 70% of the enzyme activity was recovered in the soluble fraction of the tissue homogenate and 25% was localized to the crude mitochondrial fraction. The corresponding values for lactate dehydrogenase were 40% and 50%, respectively. The small amount of enzyme (5%) sedimenting with the nuclear fraction could be explained by contamination of this fraction with soluble proteins and synaptosomes. Further separation of the crude mitochondrial fraction by discontinuous sucrose gradient centrifugation showed that most of the enzyme activity was localized to the synaptosomes, but a substantial amount was found in the top layer of the gradient. The relative specific activity of lactate dehydrogenase in the top layer was less than that of S‐adenosylhomocysteine hydrolase. No time‐dependent leakage of S‐adenosylhomocysteine hydrolase from the synaptosomes could be demonstrated. After hypoosmotic treatment of the crude mitochondrial fraction and separation of this fraction on a discontinuous sucrose gradient, S‐adenosylhomocysteine hydrolase and DOPA decarboxylase showed the same distribution in the gradient and were recovered in the cytoplasmic fraction.
A Sensitive and Reliable Assay for Dopamine (β‐Hydroxylase in Tissue
Authors: Sperk Günther , Galhaup Ingrid , Schlögl Elisabeth , Hörtnagl Heide , Hornykiewicz Oleh
Published: October 2006

A new assay procedure for dopamine β‐hydroxylase (DBH) in tissue extracts is described. Solubilized DBH was adsorbed from crude extracts on Concanavalin A‐Sepharose (Con A‐Sepharose), resulting in enrichment of the enzyme as well as removal of endogenous catecholamines and inhibitory substances. The enzymatic assay was carried out with DBH still adsorbed to Con A‐Sepharose. The adsorption of the DBH to Con A‐Sepharose offers three advantages over previous assay procedures. (1) Because of removal of the endogenous inhibitory substances, a single Cu2+ concentration can be used for the determination of DBH activity, regardless of the tissue dilution or inhibitor content of the analysed sample. Using this procedure, the optimal Cu2+ concentration for DBH of bovine adrenal gland extracts was 3 μM and for rat brain 10 μM. (2) Because of removal of endogenous catecholamines, dopamine, the main physiological substrate of DBH in noradrenergic neurons, can be used for the assay. The enzymatic reaction product, noradrenaline, was determined by high performance liquid chromatography and electrochemical detection (hplc‐ec). This procedure resulted in an approx. 10‐fold increase in sensitivity of the assay compared with other procedures, e.g., the radioenzymatic assay. (3) Direct determination of the immediate product of the enzymatic reaction (noradrenaline) permits kinetic analysis. It was found that the Michaelis constants for the substrate (dopamine) and co‐factor (ascorbic acid) (2 mM and 0.65 mM, respectively) determined in bovine adrenal tissue extracts by the described procedure were identical with the values for the purified DBH preparation.
Accessibility of Galactosyl Ceramides to Probe Reagents in Central Nervous System Myelin
Authors: Linington C. , Rumsby M. G.
Published: October 2006

The suitability of isolated central nerve myelin preparations for probe labelling studies was assessed and the accessibility of galactosyl ceramides in myelin to galactose oxidase and sodium periodate was determined. Isolated myelin preparations present a uniform external membrane surface to added probes because lamellae in the myelin sheath separate at their external apposition surfaces exclusively during isolation. The cytoplasmic apposition remains intact in isolated myelin. Cationised ferritin can gain access along external apposition regions of inner lamellae in multilamellar fragments of isolated myelin, indicating that proteins and lipids on the external membrane surface will be accessible to probes. Over 50% of the total galactosyl ceramides of myelin are accessible to galactose oxidase attack; hydroxy fatty acid‐ and nonhydroxy fatty acid‐containing cerebrosides are equally attacked. Sodium periodate attacks over 90% of the galactosyl ceramides in isolated myelin at 20°C and electron micrographs of the periodate‐treated myelin reveal changes at the external apposition only. Galactosyl ceramides in vesicles of myelin lipid vesicles are not so readily attacked by periodate. The disposition of galactosyl ceramides in the myelin lamellae is discussed.