bluuebunny/crossref_metadata_2025 huggingface dataset:
Vasoactive intestinal peptide 1‐12: A ligand for the CD4 (T4)/human immunodeficiency virus receptor
Authors: Sacerdote P. , Ruff M. R. , Pert C. B.
Published: January 2005

A five‐amino‐acid (TDNYT) sequence of vasoactive intestinal polypeptide (VIP) shares homology with the proposed attachment sequences of the human immunodeficiency virus (HIV). Synthetic peptides with these wequences have previously been shown to block viral envelop (gp120) binding and HIV infectivity and to serve as agonists of the CD4 (or T4) receptor. Utilizing an in vitro human monocyte chemotaxis bioassy we examined novel synthetic VIP and gp120 sequences and characterized their pharmacological activities on minocyte chemotaxis. CD4 receptor activity is primarily specified by N‐terminal (VIP [1–12]) amino acids. The profound immunosuppression of AIDS is not easily explained solely by virus infection. Recently described immunological functions of VIP are similar to some of the immunological abnormalities shown by patients with AIDS. An overproduction of a VIP‐like molecule from viral sources (e.g. gp120) could explain some of the immune system impairments which have been described in AIDS.
Purification of the 33,000‐dalton ligand binding‐protein constituent of the lymphoblast substance P receptor
Authors: McGillis J. P. , Organist M. L. , Scriven K. H. , Payan D. G.
Published: January 2005

Substance P (SP) acts as an immunoregulator by binding to specific funcitonal cell surface receptors on a subpopulation of human T‐helper lymphocytes. Receptors with similar properties have also been characterized on the human IM‐9 B‐lymphoblast cell line. Four distinct proteins of molecular weight (MW) 33,000, 58,000, 78,000, and 116,000 can be specifically affinity labeled using [125I]‐SP Bolton‐Hunter reagent and disuccinymidyl suberate (DSS). Peptide‐mapping studies of these individually purified affinity labeled proteins have shown that the 33,000 MW membrane protein is present in the higher molecular weight cross‐linked proteins. In the present studies, the 33,000 MW affinity‐labeled protein was purified using semi‐preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) followed by high‐performance hydroxylapatite chromatography. Starting with 800 mg of affinity‐labeled protein, the final yield was 39.0 μg of 33,000 MW affinity‐labeled protein. Based on an estimate of 53 mg receptor per 800 mg membrane protein, this represents an overall yield of greater than 70%.

Peptide mapping was done by digesting 20 μg of the receptor protein with bovine trypsin. The proteolytic fragments were separated by reverse‐phase high‐performance liquid chromatography, and the amino acid content of 13 distinct peptides was determined. With this procedure, sufficient receptor can now be purified so that partial amino acid sequences can be obtained for further structural studies.
Rat and mouse identifier sequences are preferentially but not exclusively located in cortical neuronal genes expressed postnatally
Authors: Brown I. R. , Danielson P. , Rush S. , Godbout M. , Sutcliffe J. G.
Published: January 2005

A genetic element called the identifier (ID) sequence, highly repeated in the rat genome, has previously been reported to be located in the introns of some genes transcribed in the adult rat brain by RNA polymerase II (Pol II). We show that nuclear RNA isolated from neurons of cerebral hemispheres (cortex) of 14‐day old rats is enrlched more than 10‐fold in ID sequences compared to nuclear RNA from liver, kidney, cerebellum, or cortical glia. The developmental onset of the difference is during the first 2 weeks after birth. Mouse cortical neuronal nuclear RNA is similarly enriched in an element related but not identical to the rat ID element, and the enrichment also has postnatal onset. The enriched appearnce of ID sequences in transcripts whose expression is increased postnatally in cortical neurons correlates developmentally and spatially with the transcription of ID elements by RNA polymerase III (Pol III) and with a change in chromatin structure.
Microsequencing of dopamine beta‐monoozygenase
Authors: McCafferty B. , Angeletti R. H.
Published: January 2005

Tryptic peptides and cyanogen bromide fragments of dopamine beta‐monooxygenase (DBH) were prepared and separated on C‐8 reverse phase columns by high pressure liquid chromatography. Absorbance profiles at both 220 nm and 280 nm were monitored so that peptides with aromatic residues could be isolated. These peptides were subjected to automated Edman degradation with a gas phase microsequencer.
Morphine dependence in the rat: The appearance in the spinal cord of a dorsal root ganglion cell neurotrophic factor
Authors: Hendry I. A. , Duggan A. W. , Hall J. G.
Published: January 2005

An increase in neurotrophic activity for the survival of dissociated sensory neurones has been found in the spinal cord of morphine dependent rats. This activity is not due to morphine itself and is not due to an increase in the nerve growth factor as antibodies to nerve growth factor fail to block the response. A new hypothesis is presented that some of the spinal effects of morphine dependence may be due to and increase in the level of neurotrophic factors as a response to an apparent denervation of the target cells in the spinal cord.
Arachidonic acid, stearic acid, and diacylglycerol accumulation correlates with the loss of phosphatidylinositol 4,5‐bisphosphate in cerebrum 2 seconds after electroconvulsive shock: Complete reversion of changes 5 minutes after stimulation
Authors: Reddy T. S. , Bazan N. G.
Published: January 2005

The effects of electroconvulsive shock (750 msec, 130 V, 150 pps) on the endogenous content of rat cerebral lipids were studied 2, 5, 10, 20, 30, 60, and 300 sec after stimulation. Rapid enzyme inactivation in situ was attained by high‐power head‐focused microwave irradiation (6.5 kW, 2450 MHz). At 10 sec, phosphatidylinositol 4,5‐bisphosphate (PIP2) mass had decreased by 249 nmol per g wet wt, mainly due to loss of arachidonate and stearate. At the same time, the stearoyl‐arachidonoyl glycerol accumulated, although to a lesser extent than the loss exhibited in PIP2. Changes in phosphatidylinositol and in phosphatidylinositol 4‐phosphate mass were not statistically significant. Free fatty acids and diacylglycerold accumulated to 395 nmol per g wet wt; arachidonic and stearic acids composed 322 nmol of these lipids. Hence, the reduction in content PIP2 is sufficient to account 80% of the increases in free fatty acid and diacylglycerol mass. Thirty‐three and 12 nmol of accumulated free palmitic and scosahexaenoic acids, respectively, are not accounted for by the loss of PIP2. Sixty seconds after stimulation, PIP2 content returned to 90% of control levels, while diacylglycerol tended to remain below control levels. Free fatty acids had not returned to control levels by 60 sec, with the exception of docosahexaenoic acid. At 300 sec, PIP2, diacylglycerol, and free fatty acids had all returned to control levels. It is proposed that the elctroshock‐induced release of neurotransmitters is associated with stimulation of phospholipases A1 and A2 at the presynaptic membrane followed by receptor‐linked phosphodiesteratic cleavage of PIP2 at the postsynaptic membrane, and that Ca2+ influx resulting from membrane depolarization activates the deacylation of stearoyl‐arachiodonoyl glycerol.
Ultrastructure of ependymal cells in primary cultures of cerebral cortex
Authors: Rieke G. K. , Wynder H. J. , Jordan F. L. , Thomas W. E.
Published: January 2005

Ultrastructural features of ependymal cells growing in primary cultures of dissociated cerebral cortical tissue were investigated using electron microscopy. The ependyma exhibited a specific orientation in tissue culture such that the cell surface corresponding to the apical surface in situ was directed toward the culture medium. The membrane on the apical surface of cultured ependyma was characterized by the presence of cilia and microvilli. The lateral margins were marked by a variety of junctions: zonula occludens, zonula adherens, and membrane interdigitations. The cell cytoplasm contained pleomorphic mitochondria, Golgi profiles, clusters of ribosomes, rough endoplasmic reticulum, and particularly profuse scattered plasmic reticulum, and particularly profuse scattered microfilaments and microtubules. These features support the identity of the cells as ependymal in the cultures and establish them as a relatively accurate reflection of ependyma in situ.
Effect of collagenase treatment and subsequent culture on rat muscle fiber acetylcholinesterase activity
Authors: Glavinović M. I. , Lee S. , Miledi R.
Published: January 2005

After collagenase treatment and mechanical disruption, acetycholinestrase (AChE) activity on the surface of individual flexor brevis muscle fibers fell by 88%. During the next 48 hr in culture, surface AChE activity continued to decline, while intracellular activity changed little. After 1 week in culture total muscle fiber AChE activity fell to very low levels and intracellular AChe activity could no longer be detected, probably as a result of reduced synthesis and rapid externalization of intracellular AChE. Apart from the removal of most of the surface activity, cultured muscle fibers had similar AChE activity to muscle fibers that had been denervated in vivo, suggesting that the change observed in culture reflect the loss of neuro muscular interaction and not to any contributory effects of the dissociation process.

It is to be hoped that these results, along with the published results of Bekoff and Betz [J. Physiol, 271:25–40, 537‐547], will serve as useful background data for those continuing to use adult dissociated muscle fibers in their studies.
Changes in the pattern of expression of pp60c‐src in cerebellar mutants of mice
Authors: Brugge J. S. , Lustig A. , Messer A.
Published: January 2005

Cultures of neurons from rat embroyos have been show previously to express high levels of a unique from of pp60c‐src [Brugge et al (1985): Nature 316:524‐526], the cellular homologue of the transforming protien of Rous sarcoma virus. This altered form of pp60c‐src(+), designated pp60c‐src(+), displays a retarded electroretic mobility due to a structural alteration within the aminoterminal region of the molecule [Brugge et al, 1985]. In order to investigate the distribution and possible role of pp60c‐src(+) in intact brain, we have examined the expression of pp60c‐src in extracts from developing cerebella from wild‐type mice and mutant mice that display progressive degeneration of specific classes of cerebellar neurons. The loss of pp60c‐src(+) generally correlated with the loss of granule cells and Purkinje cells from the cerebella of mice carrying the staggerer (sg/sg) and Lurcher (Lc/+) mutation, with the most pronounced changes observed in cerebella from the more severely affected sg/sg mice. The experssion of pp60c‐src(+) in weaver wv/wv mice is qualitatively and quantitatively quite different. From the earliest time points, there was a significant reduction in the levels of pp60c‐src(+), with no further loss of this form during the period of maximal neuronal differentiation. This suggests an maximal neuronal differentiation. This suggests an early, predegenerative absence of pp60c‐src(+) in this mutant strian, which is defective in granule‐cell migration.
Neuron‐Enriched cultures of adult rat dorsal root ganglia: Establishment, characterization, survival, and neuropeptide expression in response to trophic factors
Authors: Grothe C. , Unsicker K.
Published: January 2005

It is unknown whether adult dorsal root ganglion (DRG) neurons require trophic factors for their survival and maintenance of neuropeptide phenotypes. We have established and characterized neuron‐enriched cultures of adult rat DRGs and investigated their responses to nerve growth factor (NGF), ciliary neuronotrophic factor (CNTF), pig brain extract (PBE, crude fraction of brain‐derived neuronotrophic factor, BDNF), and laminin (LN).

DRGs were dissected from levels C1 through L6 and dissociated and freed from myelin fragments and most satellite (S‐100‐immunoreactive) cells by Centrifugation on Percoll and preplating. The enriched neurons, characterized by their morphology and immunoreactivity for neuron‐specific enolase, constityted a population representative of the in vivo situation with regard to expression of substance P (SP), somatostatin (SOM), and cholecystokinin‐8 (CCK) immunoreactivities. In the absence of trophic factors and using polyornithine (PORN) as a substratum, 60‐70% of the neurons present initially (0.5 days) had died after 7 days.

LN as a substratum did not prevent a 30% loss of neurons up to day 4.5, but it subsequently maintanied DRG neurons at a plateau. This behavior might reflect a cotrophic effect of LN and factors provided by non‐neuronal cells, whose proliferation between 4.5 and 7 days could not be prevented by addition of mitotic inhibitors of γ‐irradiation.

CNTF, but not NGF, slightly enhanced survival at 7 days on either PORN or LN. No neuronal losses were found in non‐enriched cultures or when enriched neurons were supplemented with PBE, indicating that non‐neuronal cells and PBE provide factor (S) essential for adult DRG neuron survival. Proportions of SP‐, SOM‐, and CCK‐immunoreactive cells were unaltered under any experimental condition, with the exception of a numerical decline in SP cells in 7‐days cultures with LN, but not PORN, as the substratum.

Our data, considered in the context of recent in vivo and vitro studies suggest that a combination of tropic factors or an unidentified factor, rather than the established NGF, CNTFl, and BDNF, which address embryonic and neonatal DRG neurons, are required for the in vitro maintenance of adult DRG neurons.
Capacity for substrate utilization in oxidative metabolism by neurons, astrocytes, and oligodendrocytes from developing brain in primary culture
Authors: Edmond J. , Robbins R. A. , Bergstrom J. D. , Cole R. A. , de Vellis J.
Published: January 2005

Neuron, astrocyte, and oligodendrocyte cultures which were established from developing rat brain were examined for their utlization of glucose, ketone bodies, and free fatty acids by oxidative processes. 14CO2 production was measured in these cells from [1‐14C] or [6‐14C]glucose; [1‐14C] and [1‐14C], [6‐14C], or [6‐14C]palmitate; and [3‐14C]acetoacetate and D(‐)‐3‐hydroxy[3‐14C]butyrate. Pyruvate dehydrogenase (EC 1.2.4.1.) abd 3‐oxoacid‐CoA transferase (EC 2.8.3.5.) activites were found at high levels in each of the cell population. Astrocytes and oligodendrocytes produced much more 14CO2 from [1‐14C]glucose than from [6‐14C]glucose, indcating substantial hexose monophosphate shunt activity. This process was not as active in neurons. All three cell populations readily utilized the ketone bodies for oxidative metabolism at rates 7–9 times greater than they utilize glucose. Only astrocytes were able to utilize fatty acids for 14CO2 production, and the rate of utilization was greater than of the ketone bodies. We found that the metabolic patterns of thesse brain cells which were derived from the developing brain complement the nature of the diet of the duckling animal which is rich fat and low in carbohydrate. They readily utilized the ketone bodies or fatty acids and spared glucose for processes that metabolites of fat cannot fulfill.
Selective enhancement of tubulingene expression and increase in oligo (dT)‐bound RNA in the rat brain after nigrostriatal pathway unilateral lesion and treatment with ganglioside
Authors: Yavin E. , Gil S. , Consolazione A. , dal Toso R. , Leon A.
Published: January 2005

Partial hemitransection of the rat nigrostriatal pathway has been applied to study changes in the expression of tulbulin and actin cytoskeletal genes. RNA was isolated from ipsilateral and contralateral structures of substantia nigra and striatum tissues of lesioned or sham‐operated animals by a combined proteinase K and oligo(dT)‐celluloseaffinity purification procedure. A significant increase in the oligo(dT)‐cellulose‐eluted RNA was observed in the lesioned substantia nigra and striatal tissues compared to naive controls. By 5 days, the RNA content of the lesioned nigra reached a value of 96–114.5 μg/mg DNA compared to 45.7–52.9 μg/mg DNA.

Administration of GMI gangliosides at daily intervals resulted by day 5 in a further increase (131.8–141.5 μg/mg DNA) in the RNA content of the lesioned but also of the sham‐operated (81.9–97.8 μg/mg DNA) animals. By 18 hr, the lesioned nigra exhibited a fourfold increase in tubulin messenger RNA (mRNAtub) gene expression in comparison to the sham‐operated animals. The substantia nigra tissue of GM1‐treated animals exhibited a hybridization value for mRNAtub twice as high compared to GM1‐untreated, lesioned animals. The effectof GM1 appearedselective for α‐tubulin and β‐tubulin compared to actin transcript. The latter was also elevated in the lesioned nigra tissue above the naive or sham‐operated animals.

The data suggest that, after injury, cytoskeletal RNA transcripts are elevated and that GM1 may play a role in the regulation of their steady‐state levels.
Ultrastructural reversible changes in fish neuromuscular junctions after chronic exercise
Authors: Palacios‐Prü E. , Colasante C.
Published: January 2005

Neuromuscular junctions (NJs) of fin muscles of teleostean fishes, Lebistes reticulatus, were ultrastructurally analyzed during 60 min of chronic exercise and a subsequent period of 90 min of induced recovery. NJs from 30‐min‐exercised fishes showed an almost complete depletion of synaptic vesicles (SVs), corresponding to 83% of SV consumption; 76% of axon terminals were branched at the end of this period. During the recovery period, it was possible to observe the reversibility of the changes induced by the exercise and the transitory events that lead to the reacquirement of the normal NJ morphology. After 15 min of rest, SV population increased to a value of 54.6 SVs/μm2 and the percentage of branched axons was 66.5%. At 60 min of recovery the number of SVs reached a value of 84.6 SVs/νm2. The SV population was fully reestablished at 80 min of rest, while the percentage of branched axons was found within normal ranges after 90 min of recovery. These results demonstrate that chronic exercise induced physiological depletion of NJ SVs and other axon terminal morphological changes, as well as that postexercise rest induces the reestablishment of the normal NJ morphology.
Region‐specifi neuro‐astroglial interactions: Ultrastructural study of the in vitro expression of neuronal polarity
Authors: Autillo‐Touati A. , Chamak B. , Araud D. , Vuillet J. , Seite R. , Prochiantz A.
Published: January 2005

Mesencephal;ic neurons were cultured for 2 days on mesencephalic or striatal astrocyte monolayers. The morphology of these neurons was studied in electron microscopy. The number of dendritic profiles was higher on mesencephalic astrocytes (homotopic neuroastroglial co‐cultures) than on striatal astrocytes (heterotopic co‐cultures). This increase in the number of dendrites correlated with a more mature aspect of the meurons. Striatal neurons were also cultured on the astrocytic monolayers. The state of maturation of these neurons was more advanced, and the number of their dendrites was higher on striatal than on mesencephalic astrocytes. These results confirm and extend the fact that neuronal maturation and dendritic growth can be regulated through region‐specific neuro‐astroglial interactions (Denis‐Donini et al., 1984; Chamak et al., 1987).
Distribution of glutamic acid decarbocylase and gamma‐aminobutyric acid in thehypoglossal nucleus in the rat
Authors: Aldes L. D. , Chronister R. B. , Marco L. A.
Published: January 2005

Immunocytochemistry was used to investigate the distribution of glutamic acid decarboxylase (GAD) and gamma‐aminobutyric acid (GABA) in the hupoglossal nucleus (XII) of the adult rat. The distribution of GAD and GABA was found to be co‐extensive throughout XII. Although immunoreactivity was moderately dense in all regions, the intensity of staining was greatest in the ventral district of XII particularly ventromedially in the caudal half of the nucleus. Immunoreactive terminal‐like profiles were observed around motoneuron somata and dendrites. There also was evidence of sparse mediolaterally‐oriented densities of immunoreactivety at the junction of XII and the dorsal vagal nucleus and between dorsal and ventral districts of XII. In addition, neurons staining positive for GABA were found scattered within XII laterally and immediately outside of XII in and around the nucleus of Roller. These observations suggest a complex, differential distribution of GABA in XII and are discussed in relation to tongue motor behavior.
Maternal dietary restriction causes myelin and lipid deficits in the brain of offspring
Authors: Yeh Y.‐Y.
Published: January 2005

The relationship between brain myelination and nutritional insufficiencey in suckling rats whose dams had a restricted dietary intake was studied. The undernourished pups were characterized by body weight and brain weight that were 40‐70% and 82‐88% that6 of controls througthout the suckling period. Myelin concentrations, whether expressred as mg protein or as mg myelin dry weight per g wet brain, were 30‐40% of normally fed controls. Myelin of undernourished rats contained total lipid, cholesterol, phospholipids, and sphingolipids, that were 42, 54, 39, and 48%, respectively of the control counterparts. There was no change in the mole ratio of cholesterol:phospholipids:sphingolipids (i.e., cerebroside + sulfatide) in myelin from undernoufished rats. Concentrations of individual phospholipids and sphingolipids were lowered by approximately the same percentage. Despite long‐lasting, irreversible stunting of whole‐body and brain growth, concentrations of myelin and myelin lipids returned to control levels after nutritional rehabilitation. Since the observed effects are different from those of more commonly used models, the present form of undernutrition may offer a useful system for studying the relationship between myelin lipids and brain development.
Development expression of glial‐specific mRNAs in primary cultures of rat brain visualized by in situ hybridization
Authors: Holmes E. , Hermanson G. , Cole R. , de Vellis J.
Published: January 2005

The localization of mRNAs which encode the gilialspecific maker proteins, glial fibrillary acidic protein (GFAP), glycerol phosphate dehydrogenase (GPDH, EC 1.1.1.8), and myelin basic protein (MBP), was mapped by in situ hybridization in primary cultures of 1–2‐day‐old rat brain in serum‐supplemented medium. Developmental changes of these expressed mRNAs were examined after various times in culture ranging from 8 to 5 days and were correlated with the histological morphological and positional characteristics of the cell. By day 8, the culture stratified into a population of flat polygonal astrocytes covered by another populatioan of phase‐dark process‐bearing cells. When counterstained with May‐Grunwald histological stain, astrocytes appeared pale blue, whereas two subpopulations of phase‐dark cells stained differentially; one was dark blue while the other was red and smaller GFAP‐specific sequences were abundant at day 8, increased in the astrocyte bedlayer as the culture became confluent, and plateaued at approximately day 16. A minor proportion of blue phase‐dark cell contained GFAP mRNA although at a lower abundance. In contrast, GPDH mRNA positive blue phase‐dark cells were seem scattered throughout the upper layer of the culture and also around the perimeter of large clumps of red phase‐dark cells. These cells were infrequent at day 8 but increased in number at later time points. The expression of MBP mRNA differed from GPDH in that it was more abundant at early time points, plateaued between day 20 and day 24, and was predominantly localized in red phase‐dark cells. Initially small and round, the MBP mRNA‐positive cells extended long processes, which then appeared to make connections with other process‐bearing cells and reorganize into clumps that dominated the culture. By day 40, MBP mRNA was scattered over membranous expanses. Thus, in situ hybridization has revealed differential gene expression and abundance of glial‐specific mRNAs in morphologically and histochemically distinct cell populations in uninduced mixed glial cultures. In future studies, the hormonal regulation of glial‐specific mRNAs will be compared to the basal genes activation presented here.
In vitro nerve‐growth‐promoting activity of human plasma α1‐acid glycoprotein
Authors: Liu H. M. , Takagaki K. , Schmid K.
Published: January 2005

Human plasma α1‐acid glycoprotein or orosomucoid (OR), and its derivatives, prepared by sequential enzymatic cleavage of the carbohydrate units, were tested for their nerve‐growth‐promoting activities with explants of whole dorsal root ganglia from chick embryos. The results showed that the OR derivatives with terminal galactose, N‐acetylglucosamine, or mannose have marked neurite‐promoting activities. These preparations at a concentration of 100 μg/ml are equivalent to 5% fetal bovine serum (protein concentration 3,000 μg/ml) in their ability to elicit extensive neurite outgrowth and collateral branching. The asialo‐OR, or ASOR, is the most potent form: its activity is estimated to be 20 times higher than that of transferrin and 100 times over that of fibronectin; it is approximately 1/1,500 that of NGF. The neuritepromoting activity of OR is independent of the nonneuronal cells and their products and can be blocked by a specific antiserum against OR. The mode of action of OR on the in vitro nerve growth is discussed and the pathophysiological significance of this plasma glycoprotein is considered in light of data from recent clinical and pathological studies.
Traumatic spinal cord injury in rats causes increases in tissue thromboxane but not peptidoleukotrienes
Authors: Demediuk Paul , Faden A. I.
Published: January 2005

Spinal cord samples from rats subjected to three different levels of impact trauma (25, 50, 100 g‐cm) were examined for immunoreactive thromboxane B2 and 6‐sulfidopeptide‐containing leukotrienes, using specific radioimmunoassays. Trauma resulted in pronounced increases in thromboxane levels as early as 5 min after injury, with maximum values at 1 hr. Although thromboxane values then slowly declined, they remained significantly above control values for up to 7 days. Significantly smaller thromboxane values were found in rats subjected to mild injury (25 g‐cm) than in those that received more severe, irreversible impact injury (50 and 100 g‐cm). No statistically significant changes were observed in leukotriene levels in any of the experimental groups. These findings are consistent with the hypothesis that cyclooxygenase products of arachidonic acid metabolism may contribute to secondary injudy after spinal cord trauma and provides the rationale for the use of cyclooxygenase inhibitors in the treatment of such injury.
Effects of acetylcholine and other agents on 32P‐prelabeled phosphoinositides and phosphatidate in crude synaptosomal preparations
Authors: White H. L.
Published: January 2005

Experimental conditions are described which permit effects of various agents on polyphosphoinositides and phosphatidic acid (PA) to be evaluated simultaneously in crude nerve‐ending preparations from rat brain. Acetylcholine (3‐100 μM) or carbachol (30‐1,000 μM) induced the hydrolysis of prelabeled polyphosphoinositides and, at the same time, stimulated the net label incorporated in phosphatidic acid. All muscarinic effects were blocked by atropine or pirenzepine. Nonmuscarinic agonists (glutamate, adenosine, norepinephrine) stimulated polyphosphoinositide hydrolysis in this preparation, but of these only norepinephrine affected phosphatidic acid turnover. A Potentiation of acetylcholine‐induced phosphoinositide turnover by KCI was observed, as well as an apparent selective inhibition of PIP2 hydrolysis by LiCl. Acetylcholinestimulated turnover of PA was not necessarily coupled to phosphoinositide hydrolysis.
cDNA coding for the tail region of the high molecular weight rabbit neurofilament protein NF‐H
Authors: Mack K. , Currie J. R. , Soifer David
Published: January 2005

A cDNA library was prepared from rabbit brain mRNA, in the expression vector, lambda gt11. The library was screened with polyclonal antibodies raised against the neurofilament protein NF‐H, and a cloned cDNA (KMRH‐1) was selected and characterized. The fusion protein coded for by KMRH‐1 includes epitopes for two monoclonal antibodies which react with nonphosphorylated sites in the tail region of NF‐H. The selected cDNA includes 891 base pairs. It hybridizes to human genomic DNA, as well as to rabbit genomic DNA, and to a rabbit brain mRNA with a size of 4.7 kilobases (kb). The sequence of KMRH‐1 includes extensive repeating regions, including one duplicated 60‐base segment. Within the first 196 bases, one 13‐base segment is repeated 9 times. The cDNA codes for the carboxy‐terminal 184 amino acid residues of NF‐H, including a series of 9 serines, each surrounded by a similar group of amino acids: Ala.Lys.Ser.Pro.{Glu./Val.}.Lys. Comparison of the derived amino acid sequence for KMRH‐1 indicates considerable divergence from the sequence information available for rodent NF‐H (Robinson et al.: FEBS Lett 209:203‐205, 1986). This diversity in amino acid sequence may account for the failure to induce tangles of neurofilaments in animals, such as rats, following treatment with doses of aluminum which are sufficient to induce such tangles in rabbits and to bring on seizures and behavioral pathology in both species.
Identification of a human glial fibrillary acidic protein cDNA: A tool for the molecular analysis of reactive gliosis in the mammalian central nervous system
Authors: Rataboul P. , Biguet N. Faucon , Vernier P. , De Vitry F. , Boularand S. , Privat A. , Mallet J.
Published: January 2005

Two clones encoding human glial fibrillary acidic protein (GFAP) were isolated from a human astrocytoma cDNA library. The clones pHGFAP1 and pHGFAP2 were selected by the combined use of differential colony hybridization and hybridization‐selection technique with polyclonal anti GFAP antiserum. The longer one, pHGFAP1, encompasses 3.0 kb and includes the 1.8 kb long 3′ untranslated region specific to the human mRNA. Sequence data disclosed an extensive homology within the coding region of human and mouse GFAP cDNAs even in the end domains. Blot hybridization analysis of RNAs from human, rat and mouse brain revealed a single GFAP mRNA species of 3.1, 2.8 and 2.7 kb respectively and Southern blot experiments indicated that this mRNA is most probably transcribed from a unique gene. In situ hybridization performed with biotinylated probes on cultured mouse brain cells suggests both the sorting and the transport of GFAP mRNA throughout the cytoplasm and processes of the astrocytes. As a model of reactive gliosis secondary to degenerative disorders, 6‐hydroxydopamine (6‐OHDA) lesion of the substantia nigra in the rat was performed. GFAP mRNA increased 1.4 fold in the ipsilateral striatum on day 10 after the lesion. It then declined to the control level 4 months later contrasting with the lower and more sustained increase in preproenkephalin (PPE) mRNA. The interspecies cross‐reactivity of the HGFAP probes make them useful as a tool for the molecular analysis of reactive gliosis in various experimental models.
Identification of horizontal cells generating different spectral responses in the retina of a teleost fish (Eugerres plumieri)
Authors: Negishi K. , Salas R. , Parthe V. , Drujan B. D.
Published: January 2005

Six different types of spectral responses were recorded from horizontal cells under mesopic conditions in perfused retina, isolated from the dark‐adapted mojarra (Eugerres plumieri). They were tentatively termed photopic Lr‐, Lg1‐, Lg2‐, Lb‐, and C‐type, and scotopic L‐type. The Lr‐, Lg‐, and Lb‐type responses showed a maximum peak at 605, 550, and 516 nm respectively, while the C‐type was composed of hyperpolarizing potentials in response to shorter wavelengths and depolarizing potentials in response to longer wavelengths (so‐called R/G‐type). The scotopic L‐type has a peak at 516 nm in the spectral response and a slow decay phase in the waveform response. Following a brief period of diffuse illumination, it was found that the Lgl‐type response is altered to the Lr‐type, while both Lg2‐ and Lb‐type responses change to the C‐type. Intracellular marking with Lucifer or Procion yellow identified the cellular origins of different response types: external (He) and medial horizontal (Hm) cells for the Lr‐type, internal horizontal (Hi) cells for the C‐type, and rod‐horizontal (Hr) cells for the scotopic L‐type. Only He cells were found to possess an axon, while dye coupling was seen between axonless Hm, Hi, or Hr cells but not between He cells. The morphology of these fluorescent dye‐marked cells was the same as that of the respective cells observed in Golgi‐stained materials.
Effects of radioligand oxidation and ascorbate‐induced lipid peroxidation on serotonin‐1 receptor assay: Use of ascorbate and ethylenediamine tetraacetic acid buffers to prevent (3H)‐5‐HT binding artifacts
Authors: May Patrick C. , Morgan D. G. , Salo D. , Goss J. R. , Finch C. E.
Published: January 2005

The effects of ascorbic acid and ascorbate‐induced lipd peroxidation upon (3H)‐5‐HT binding were examined in total membrane fractions prepared from mouse cortex and hippocampus. Low concentrations of ascorbic acid promoted lipid peroxidation of membranes as assessed by malondialdehyde production relative to intermediate concentrations of ascorbate. The actual concentration of ascorbate required for expression of pro‐oxidative and antioxidative properties was dependent upon assay conditions such as temperature and ionic constituents. Ascorbate‐induced lipid peroxidation was completely prevented by addition of 1 mM ethylenediamine tetraacetic acid (EDTA) and enhanced by GTP at concentrations typically used in binding assays. Marked lipid peroxidation was associated with loss of (3H)‐5‐HT binding sites with little effects upon affinity of the receptor for the ligand. In contrast, mild lipid peroxidation occurring during the binding assay with the ligand present was associated with a decreased affinity for (3H)‐5‐HT and the appearance of a curvilinear Scatchard plot. Under assay conditions that prevented ascorbateinduced lipid peroxidation, Scatchard analysis indicated only a single high‐affinity binding site for (3H)‐5‐HT even when assayed with an expanded range of ligand concentrations. Preparation of ligand and assay of (3H)‐5‐HT binding in the absence of ascorbate resulted in shallow, markedly curvilinear Scatchard plots. These data support the continued use of ascorbate in (3H)‐5‐HT binding assays to prevent ligand degradation and the addition of 1 mM EDTA to prevent lipid peroxidation from occurring during the binding assay.
Localization of choline acetyltransferase in perikarya and dendrites within the nuclei of the solitary tracts
Authors: Armstrong David M. , Rotler A. , Hersh L. B. , Pickel V. M.
Published: January 2005

Immunocytochemistry was used to establish the cellular localization of choline acetyltransferase [ChAT] throughout the rostrocaudal portions of the nuclei of the solitary tracts [NTS] in rat brain. By light microscopy, two distinct populations of ChAT‐positive cells were identified. The first consisted of relatively few, medium‐sized neurons located in the caudal one‐half of the medial NTS just dorsal to the dorsal motor nucleus of the vagus. The second population of ChAT‐labeled neurons was located more anteriorly and surrounded the medial and dorsal borders of the tractus solitarius. These cells were more abundant and smaller diameter than those located more caudally. Thick, non‐varicose processes with the light microscopic characteristics of dendrites also were selectively labeled for ChAT. A few of these processes were located near or were continuous with the labeled perikarya of the NTS. However, the vast majority of the immunoreactive processes could be traced from ChAT‐labeled perikarya in the ventrally adjacent dorsal motor nucleus of the vagus. These dorsally directed dendrites aborized extensively throughout the NTS, but they were densest in the rostral two‐thirds of the nucleus. Caudally, the labeled dendrites coursed horizontally, forming a commissure‐like structure between the two vagal motor nuclei.

Electron microscopy confirmed the perikaryal and dendritic localization of ChAT in the NTS. The perikarya were characterized by dense peroxidase immunoreactivity throughout the cytoplasm, infolded nuclear membranes, and somatic synapses. The labeled dendritic profiles also were intensely immunoreactive and received synaptic input from unlabeled terminals. The unlabeled afferents to somata and dendrites contained large populations of small clear vesicles. The abundance of cholinergic dendrites and relative sparsity or absence of identified cholinergic axons suggests that the release of acetylcholine from dendrites of intrinsic neurons or from dendrites of neurons located within the dorsal motor nucleus of the vagus may be important for cholinergic modulation of visceral functions within the NTS.
Fibroblast growth factor effects on peripheral nerve regeneration in a silicone chamber model
Authors: Danielsen N. , Pettmann B. , Vahlsing H. L. , Manthorpe M. , Varon Silvio
Published: January 2005

We have developed a silicone nerve regeneration chamber that is partitioned into two compartments by a strip of nitrocellulose paper. The modified two‐compartment chamber allows the investigation of the effects on rat sciatic nerve regeneration of trophic or growth factors that are initially bound to the nitrocellulose partition. In this study we compared the effects of untreated nitrocellulose, a siliconized nitrocellulose strip, and a strip that had been soaked in a basic fibroblast growth factor (FGF) solution. FGF is a known angiogenic factor and a mitogen for endothelial cells, fibroblasts, and Schwann cells. All of these cell types are present in the peripheral nerve. In vitro analyses, using 3T3 cells as test cells, showed that some of the bound FGF remained active on the nitrocellulose paper for at least 8–10 days. In vivo experiments, examined at 16 days post‐implantation, revealed that spatial migration of all cellular elements (perineurial‐like cells, vasculature, and Schwann cells) across the chamber gap was slower with untreated nitrocellulose strips than with siliconized strips but was most advanced with FGF‐treated ones. Most striking was the well‐developed vascular arborization of the regenerate within the FGF chambers. Histologic sections from the proximal one‐half of the chamber revealed that the regenerate in untreated strip chambers consisted of fibrin matrix and erythrocytes, whereas a welldeveloped structure with all the cellular elements of a regenerating nerve was seen in several of the FGF strip chambers. We conclude that FGF stimulates peripheral nerve regeneration in this model.
Glutamate decarboxylase inhibition and vitamin B6 metabolism in brain of cirrhotic rats chronically treated with carbon tetrachloride
Authors: Díaaz‐Muñtoz M. , Tapia Ricardo
Published: January 2005

In a previous work we found that the activity of glutamate decarboxylase (GAD), the enzyme responsible for the synthesis of the inhibitory neurotransmitter γ‐aminobutyric acid (GABA), is decreased in comatose cirrhotic rats after chronic treatment with CCl4. In the present report we studied the participation of pyridoxal phosphate in the inhibition of GAD, as well as the concentration of this coenzyme and the activity of its synthesizing enzyme, pyridoxal kinase, in the brain of the cirrhotic rats. Furthermore, cirrhotic animals were treated with three inhibitors of GAD, and the effects of such treatment were compared to those of ammonium. Liver failure resulted in a 25% inhibition of GAD activity when measured in the absence of added pyridoxal phosphate. Treatment with the GAD inhibitors thiosemicarbazide or 3‐mercaptopropionic acid enhanced this inhibition and produced convulsions at a dose that had no behavioral effects in control rats. Treatment with ammonia resulted in a comatose state and in a 25–40% inhibition of GAD. Both pyridoxal kinase activity and pyridoxal phosphate levels were found to be decreased by 15–20% in the brain of the cirrhotic rats. We concluded that chronic liver failure results in a decreased pyridoxal phosphate and GABA synthesis in brain, with a consequent diminished efficiency of GA‐BAergic neurotransmission; these effects are probably related to the manifestations of neuronal hyperexcitability that are frequently seen in human hepatic encephalopathy.
Embryonic exposure to high taurine: A possible nutritional contribution to Friedreich's ataxia
Authors: van Gelder N. M. , Bélanger F.
Published: January 2005

When fertilized eggs (50 g) are injected on day 1 with 100 μmol taurine (0.2 ml), 15‐day‐old embryos exhibit increased taurine content in heart and brain. Lethal toxicity was no higher than with equimolar injections of NaCl (50 μmol) or valine (100 μmol) of the same volume. That same dose of taurine injected either on embryonic day 1 or 7 produced in hatchlings a typical syndrome of ataxia, reduced muscle strength and motor incoordination. When injected on embryo day 15 (E15) most chicks appeared incapable of pecking out of the egg, and the resulting delay in hatching, if if did occur, precluded presuming that the poor condition of such chicks was exclusively due to the late taurine injections.

In view of the tendency of Friedreich's ataxia patients to exhibit increased alimentary absorption of taurine, and to demonstrate an excessive accumulation of taurine in the cerebellum and heart tissue on autopsy, fetal exposure to high taurine levels or neonatal high taurine milk ingestion may, by analogy, contribute to the slowly progressing disease process.
Protein synthesis by rat hippocampal slices maintained in vitro
Authors: Phillips L. L. , Steward O.
Published: January 2005

The present study evaluates protein synthesis in rat hippocampal slices maintained in vitro. Transverse slices of hippocampus were prepared from both adult rats and rat pups during postnatal development and incubated in a gassed (95% O2/5% CO2) balanced salt medium containing 5 nM 3H‐leucine. The time course of 3H‐leucine incorporation into TCA‐precipitable protein was determined using slices removed from the media after 5, 10, 20, 30, 40, 60, and 120 min of incubation. The pattern of 3H‐amino acid incorporation was evaluated by fixing slices with paraformaldehyde, embedding the slices in plastic, and sectioning the slices end on and en face for autoradiographic analysis.

Biochemical analysis of 300 and 400 μm slices revealed that incorporation of leucine into protein proceeds at a constant rate. The autoradiographic analysis revealed that in adult hippocampal slices of 300–600 μm thickness there was complete penetration of 3H‐leucine with no indication of a gradient in the extent of incorporation throughout the slice. The pattern of grain density within 300–600 μm slices matches that previously reported after in vivo injections of radio‐labeled amino acid, where grain density is highest over neuronal cell bodies and lower over the laminae that contain dendritic processes and axons (Phillips et al: Mol Brain Res 2:251–261, 1987). Hippocampal slices of 200, 800, and 1,000 μm thickness showed irregular labeling. Slices of 200 μm were filled with pyknotic nuclei and vacuoles and exhibited patchy labeling. In 800 μm slices there were isolated areas of good preservation within the slice core, but these areas exhibited little incorporation. Relative to the 300–600 μm slices, there was a higher number of pyknotic nuclei and a much deeper layer of necrosis along the cut edges. Slices of 1,000 μm thickness showed poor preservation throughout and low levels of incorporation. Biochemical studies revealed a much higher rate of incorporation in the slices prepared from postnatal animals. Autoradiography of the slices from developing rats revealed that penetration was excellent and incorporation appeared to be greater as judged by an overall higher grain density.

We believe that rat hippocampal slices provide a good in vitro model of protein metabolism that will be useful for studies of protein synthesis in isolated cell body and dendritic laminae and for the evaluation of whether protein synthesis in particular laminae is regulated by synaptic activity.
Migration‐related changes in the cytoskeleton of cultured neural crest cells visualized by the monoclonal antibody I‐5G9
Authors: Wright J. , Cooley B. , Duwell J. , Sieber‐Blum M.
Published: January 2005

An epitope recognized by the monoclonal antibody I‐5G9 was expressed by all neural crest cells shortly after explantation into culture. At this time all neural crest cells actively migrated away from the neural tube. Immunoreactivity was localized intracellularly and organized into stress fiber‐like filaments. Often, immunofluorescence was particularly high in short fibers in the lamellipodia of the reading edge of migrating cells. Two‐week‐old cultures had a diameter of 8–10 mm. At that stage a ring of immunoreactive cells was present at the periphery of each culture, an area where cells were still migratory. An inner concentric circle had reduced and more granular staining. In this area cells had ceased to migrate. In the center of the culture cells were multilayered, nonmigratory, and did not bind I‐5G9. After creating a lesion in the nonreactive central region, some cells resumed migration into the lesioned area and reexpressed the epitope. I‐5G9 staining and phalloidin fluorescence colocalized partially in some cells and completely in others. It is concluded that the epitope recognized by I‐5G9 is expressed in a migration‐dependent manner. The partial colocalization of I‐5G9 and phalloidin fluorescence supports the notion that the epitope recognized by I‐5G9 is specifically expressed in stress fibers of migratory cells, possibly in one of the actin‐associated proteins or an F actin‐associated protein complex.
Insulin‐like growth factor I promotes cell proliferation and oligodendroglial commitment in rat glial progenitor cells developing in vitro
Authors: McMorris F. A. , Dubois‐Dalcq M.
Published: January 2005

We investigated the mechanisms by which insulin‐like growth factor I (IGF‐I) acts to increase the number of oligodendrocytes that develop in cultures of cells explanted from perinatal rat cerebrum. Fluorescence‐activated cell sorting was used to isolate bipotential A2B5‐positive oligodendrocyte‐type 2 astrocyte (O‐2A) progenitor cells, which were then inoculated as single cells into microculture wells containing feeder layers of X‐irradiated type 1 astrocytes. Addition of 100 ng/ml IGF‐I to the culture medium increased the growth rate and the ultimate size reached by the resulting clones during the 18‐day experimental period. Moreover, 75–80% of the cells in the IGF‐I‐treated clones differentiated into galactocerebroside (GC)‐positive oligodendrocytes, whereas only 25–30% became oligodendrocytes in the absence of IGF‐I. IGF‐I did not increase the number of type 2 astrocytes that developed in the clones. IGF‐I appeared to have the greatest effect on growth and differentiation at a stage when the majority of the cells in the clones were at an intermediate stage of development, characterized by the expression of A2B5 and O4 glycolipid antigens but not GC. Analysis of the effects of IGF‐I on O4‐positive, GC‐negative intermediate precursor cells revealed a two to fivefold increase in the number of cells that incorporated 3H‐thymidine into their DNA during a 5‐h pulse. Moreover, IGF‐I increased the number of cell sorter‐purified O4‐positive cells that developed into oligodendrocytes 4–8 days later. Therefore, IGF‐I acts in two different ways to promote oligodendrocyte development: It promotes proliferation of precursor cells in the O‐2A lineage, and it induces precursors to become committed to develop into oligodendrocytes.