bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential effects of kainic‐acid‐induced hippocampal lesions on the acquisition of the jump‐box avoidance task in mice
Authors: Ozaki H. S. , Murakami T. H. , Toyoshima T. , Shimada M.
Published: January 2005

This research examined the effects of kainic‐acid (KA)‐induced hippocampal lesions on the acquisition of a jump‐box avoidance task in mice. When the hippocampal lesion was unilateral, mice with lesions in the lateral portion of the CAI region showed deficits in acquisition while those with lesions in the CA3 region and/or the medial portion of the CA1 region did not. When the hippocampal lesion was bilateral, on the other hand, mice with lesions in the CA3 region and/or the lateral portion of the CA1 region were deficient in acquisition while those with lesions in the medial portion of th CA1 region were not. Among the mice with various KA‐induced hippocampal lesions avoidance responses were not correlated with the level of open field activity, indicating that acquisition differences observed are not dependent upon, nor secondary to, levels of spontaneous activity. These results strongly suggest that there exist regional differences in the involvement of hippocampal pyramidal cell layers in acquisition of the jump‐box avoidance task: both the CA3 region and the lateral portion of the CA1 region are involved in acquisition, while the medial portion of the CA1 region is not.
Developmental features of rat cerebellar neural cells cultured in a chemically defined medium
Authors: Gallo V. , Ciotti M. T. , Aloisi F. , Levi G.
Published: January 2005

We studied some aspects of the differentiation of rat cerebellar neural cells obtained from 8‐day postnatal animals and cultured in a serum‐free, chemically defined medium (CDM). The ability of the cells to take up radioactive transmitter amino acids was analyzed autoradiographically. The L‐glutamate analogue 3H‐D‐aspartate was taken up by astroglial cells, but not by granule neurons, even in late cultures (20 days in vitro). This is in agreement with the lack of depolarization‐induced release of 3H‐D‐aspartate previously observed in this type of culture. In contrast, 3H‐(GABA) was scarcely accumulated by glial‐fibrillary‐acidic‐protein (GFAP)‐positive astrocytes, but taken up by glutamate‐decarboxylase‐positive inhibitory interneurons and was released in a Ca2+‐dependent way upon depolarization: 3H‐GABA evoked release progressively increased with time in culture. Interestingly, the expression of the vesicle‐associated protein synapsin I was much reduced in granule cells cultured in CDM as compared to those maintained in the presence of serum. These data would indicate that in CDM the differentiation of granule neurons is not complete, while that of GABAergic neurons is not greatly affected. Whether the diminished differentiation of granule cells must be attributed only to serum deprivation or also to other differences in the composition of the culture medium remains to be established. 3H‐GABA was avidly taken up also by a population of cells which were not recognized by antibodies raised against GFAP, glutamate decarboxylase, and microtubule‐associated protein 2. These cells exhibited a stellate morphology, were stained by the monoclonal antibody A2B5, and have been characterized elsewhere [Levi et al, 1986] as bipotential precursors of oligodendrocytes and of a subpopulation of astrocytes bearing a stellate shape and capable of high‐affinity 3H‐GABA uptake.
GM1 ganglioside counteracts selective neurotoxin‐induced lesion of developing serotonin neurons in rat spinal cord
Authors: Fusco M. , Donà M. , Tessari F. , Hallman H. , Jonsson G. , Gorio A.
Published: January 2005

The effect of exogenous monosialo ganglioside GM1 on neurotoxin‐induced lesioning of bulbo‐spinal serotonergic neurons of newborn rats was studied by means of biochemical and immunocytochemical techniques. 5,7‐dihydroxytrypatamine (5,7‐HT, a selective serotonin neurotoxin) treatment of newborn rats caused a pronounced reduction of 5‐hydroxytryptamine (5‐HT) and 5‐hydroxyindoleacetic acid (5‐HIAA) levels in the thoracic and lumbar spinal cord, while an increase of 5‐HT and 5‐HIAA was found in the pons medulla. These biochemical alterations were regionally correlated with similar changes in 5‐HT nerve terminal density analyzed by image analysis. GM1 administration (30 mg/kg for 4 consecutive days) antagonized the reduction of 5‐HT and 5‐HIAA levels induced by 5, 7‐HT treatment in the lumbar spinal cord of 2‐month‐old rats, as well as the decrease of 5‐HT nerve terminal density in both thoracic and lumbar spinal cord of 1‐ and 2‐month‐old rats. A minor counteracting effect of GM1 was found in the pons medulla where the neurotoxin induced an increase of 5‐HT and 5‐HIAA levels. These data support the hypothesis that GM1 may have a preventing action on retrograde degenerative processes following chemical lesion and/or a growth‐stimulating effect on injured 5‐HT neurons.
Theiler's virus in brain cell cultures: Lysis of neurons and oligodendrocytes and persistence in astrocytes and macrophages
Authors: Graves M. C. , Bologa L. , Siegel L. , Londe H.
Published: January 2005

The mechanisms of presistence and of demyelination in Theiler's virus (TV)‐induced chronic neurologic disease (a murine model for multiple scelerosis) are, as yet, disputed. We investigated the tropism and persistence of TV in brain cell culture to better understand the pathogenesis of this disease. Using anti‐genic markers to identify specific cells in culture, we have demonstrated that TV infects, lytically, neurons and oligodendrocytes and persistently astrocytes and macrophages. These results suggest that host cell factors play a key role in the mechanism of demyelination and the persistence of TV in the nervous system.
Regulation of myelin basic protein in oligodendrocytes by a soluble neuronal factor
Authors: Bologa L. , Aizenman Y. , Chiappelli F. , de Vellis J.
Published: January 2005

Myelin basic protein (MBP) is one of the most important myelin components. Based on our previous studies, we hypothesized that neurons might have regulatory effects on the production of MBP by oligodendrocytes, and we conducted studies designed to verify this hypothesis. Oligodendroglia‐rich cultures from total brain of neonatal rats or mice and pure cultures of embryonic rats or chicks were prepared. Cultures of mouse fibroblasts and astrocytes were prepared as well. We show here that MBP production by oligodendrocytes was greatly enhanced by treatment with either pure neurons, rat neuronal conditioned medium, or chick neuronal conditioned medium, while chemically defined, hormonally supplemented medium or medium conditioned by astroctyes and fibroblasts had no effect on MBP expression. We conclude that the production of MBP by oligodendrocytes is regulated by a nonspecies specific soluble neuronal factor. The conservation of this phenomenon from avian to rodent species implies its critical role in myelination and suggests its potential application as a treatment in demyelination.
Rapid, high‐resolution in situ hybridization histochemistry with radioiodinated synthetic oligonucleotides
Authors: Lewis M.E. , Arentzen R. , Baldino F.
Published: January 2005

In situ hybridization histochemistry is a valuable technique for localizing specific messenger RNA (mRNA) and detecting changes in gene expression. Generally, the mRNA of interest has been detected by probes obtained from cloned DNA and labelled to high specific activity by nick translation. Such probes have a number of disadvantages which can be circumvented by the use of short synthetic oligonucleotides designed to be complementary to a known mRNA sequence. We report here that synthetic oligonucleotides complementary to part of the mRNA coding for rat arginine vasopressin (AVP) can be labelled to high specific activity with [125I], using either the primer extension method with the Klenow fragment of DNA polymerase I or the 3′‐tailing method with terminal deoxynucleotidyl transferase. Both AVP probes hybridized well to the magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei. A strong autoradiographic signal was present by 2 days, with grains largely confined to the perikaryon. These results compare favorably to those obtained with [32P]‐ or [3H]‐labelled probes. Given the ease of the 3′‐tailing method, [125I]‐labelled oligonucleotides appear to be especially useful probes for in situ hybridization histochemistry.
In situ hybridization histochemistry for the analysis of gene expression in the endocrine and central nervous system tissues: A 3‐year experience
Authors: Bloch B. , Popovici T. , Le Guellec D. , Normand E. , Chouham S. , Guitteny A.F. , Bohlen P.
Published: January 2005

We report our experience in development of the in situ hybridization (ISH) procedure to detect messenger RNAs (mRNAs) coding for various molecules involved in endocrine glands and central nervous system activity, including mRNAs coding for endorphin precursors [preproenkephalin A (PPA), pro‐opiocortin (POMC)], vasopressin, and transferrin. Various conditions of fixation and handling of the tissues were tested to establish optimal parameters for mRNA detection. Double‐stranded DNA probes labeled by nick translation, synthetic oligonucleotides labeled at their 5′ end, as well as single‐stranded RNA probes were used, after incorporation of 32P‐ or 35S‐labeled nucleotides. Specific requirements for efficient and reproducible ISH investigations are discussed. Cells expressing the PPA gene in the adrenal medulla and in the brain were detected by ISH. The results show that ISH is as sensitive as immunohistochemistry in detecting peptide‐producing cells in the adrenal and that it allows detection of PPA cell bodies in brain in conditions in which they are inconstantly detected by immunohistochemistry. Unilateral destruction of substantia nigra provokes a dramatic decrease in the number of neurons expressing the PPA gene in the controlateral striatum. Cells expressing the POMC gene were detected in the pituitary of various species including man and in the rat arcuate nucleus. Neurons containing vasopressin mRNA were visualized in the supraoptic paraventricular and suprachiasmatic nucleus of the adult rat by using a synthetic oligonucleotide probe. Transferrin gene expression was shown in the central nervous system of the rat brain in two cell populations, the oligodendrocytes and the epithelial cells of the choroid plexus, by demonstration of simultaneous presence in them of transferrin immunoreactivity together with transferrin mRNA. These results show that the ISH procedure is a technique that can be routinely used to investigate gene transcription anatomically in complex heterocellular tissues such as the endocrine glands and the nervous system.
Chromaffin cell heterogeneity of process formation and neuropeptide content under control and nerve growth factor‐altered conditions in cultures of chick embryonic adrenal gland
Authors: Shaw T.J. , Letourneau P.C.
Published: January 2005

Adrenal glands from embryonic day 11 (E‐11) chicks were cryostat‐sectiond, and it was determined that tyrosine hydroxylase‐like immunoreactive (TLI) cells, somatostain‐like immunoreactive (SLI) cells, and methionnie‐enkephalin‐like immunoreactive (ELI) cells occupied chromaffin regions of the gland. Similar age adrenals were dissociated, and the cells were cultured under serum‐free conditons. Cultured TLI cells, ELI cells, and SLI cells were characterized according to cell size, cell number, and neurite formation. ELI and SLI cells composed two largely separate populations, with SLI cells tending to have larger cell areas, to be more numerous, and to be less likely to from neurits than ELI cells. The population of TLI cells, although unique in itself, was diverse and numerous enough to include all or portions of the neuropeptide‐immunoreactive populations. Neurites of some cells from each of the above populations were strongly immunoreactive for alpha neurofilament protein, and for NAPA73 neurofilament‐associated protein. However, neurities could also be observed in all populations that showed poor immunoreactivity for these cytoskeletal proteins. Exogenously added NGF significantly increased nurite‐like process formation among TLI and ELI cells, but not among SLI cells. Reductions in the number of neurite‐like processes following treatment with anti‐nerve growth factor (NGF) were not singnificant for any of the populations. However, if shorter and broader process were included, ELI cells. Anti‐NGF inhibition of process formation among ELI cells could be reversed with exogenous NGF. Neither NGF or anti‐NGF treatments showed a significant effect on cell numbers among TLI and ELI populations. The implications are that a compound of antigenic and physiologic similarity to mouse salivary NGF is made by embryonic chick adrenal cells in culture, but the effects of NGF do not appear to be the same for all neural‐crest‐derived cells from the adrenal, and greater heterogeneity of phenotypes may exist among chromaffin cells than has previously been accepted. Some quesitons are also raised concerning the neurite‐like nature of processes formed by some chromaffin cells in vitro.
β‐Adrenergic stimulation of protein (arginine) methyltransferase acitivity in cultured cererbral cells from embryonic mice
Authors: Amur S.G. , Shanker G. , Pieringer R.A.
Published: January 2005

Several adrenergic effectors and neurotransmitters were tested as potential regulators of myelin basic protein (MBP) and histone methyltransferase activities. Both enzymes were specifically activated by β‐adrenergic agonists in a stereospecific manner. Cyclic AMP (but not AMP) stimulated the enzymes to the same extent as did the β‐adrenergic agonist, (−) isoproterenol. The studies suggest that β‐adrenergic agonists stimulate adenylate cyclase thereby causing an increased production of cyclic AMP which stimulates the methyltransferases. Cycloheximide addition to the reaction mixture did not affect the stimulation due to cyclic AMP, indicating that new protein synthesis is not involved in the cyclic AMP stimulation of the methyltransferases. Thyroid hormone (T3) has been shown to stimulate MBP methyltransferase [Amur et al, 1984] and could exert its stimulatory effect through β‐adrenergic‐dependent systems. Thus, the methylation of MBP seems to be regulated both by T3 and by neurotransmitters and/or hormones mediating their effects through cyclic AMP production, whereas the methylation of histones seems to be regulated only by the latter.
Decreased acetylcholine receptor content in denervated skeletal muscles infused with nerve extract
Authors: Sayers S.T. , Yeoh H.C. , McLane J.A. , Held I.R.
Published: January 2005

The influence of a concentrated extract of soluble substances from the sciatic nerve upon the acetylcholine receptor (AChR) content in the soleus muscle of adult rats was examined by in vivo infusions. Internal and membrane‐inserted AChR were quantitated by the specific binding of 125I‐alpha‐bungarotoxin (a‐BuTX). Interestingly, the nerve extract had no apparent effect unless the soleus muscle was also denervated at the start of the infusion. Then, after 66 hr, substantially less (60‐80%) binding of125I‐a‐BuTX to AChR was observed compared to denervated solei that did not receive an infusion of nerve extract. However, the concentration of protein in the nerve extract had to exceed 5 mg/ml before this effect was evident. Infusions of phosphate‐buffered saline, bovine serum albumin, rat liver extract, or human transferrin had no striking effect upon AChR. The prevention of the characteristic denervation‐induced increase in nonjunctional AChR by an active component in the nerve extract may be due to a trophic signal for decreased synthesis of AChR, but it is also possible that the degradation of AChR was increased.
The β subunit of the guanine nucleotide regulatory proteins: Identification of its location on two‐dimesional gels of brain tissue and its regional and subcellular distribution in brain
Authors: Heydorn W.E. , Gierschik P. , Creed G.J. , Milligan G. , Spiegel A. , Jacobowitz D.M.
Published: January 2005

The β subunit of the guanine nucleotide regulatory proteins (also termed G proteins) has been examined in both rat and human brain, a single protein of molecular weight 36,000 daltons and pI 5.8 reacted the antisera. This protein also comigrated with one of the proteins present in a purified preparation of bovine brain G proteins. Based upon molecular weight, pI, and reactiion with specific antisera, it was concluded that this protein is the β subunit of the G proteins in brain. Using this information, the regional and subcellular distribution of the G protein β subunit was studied in rat brain. Of 25 distinct neuroanatomical areas examined, cortical regions were generally found to contain the largest amount of this protein. The subcellular distribution of the G protein β subunit revealed that large amounts are present in the synaptic membrane, crude synaptic vesicles, and microsomes. These studies serve to identify another protein visible on silver‐stained two‐dimensional electrophortograms of rat and human brain. The regional and subcellular distribution of the G protein β subunit correlate well with the proposed physiological function of this protein.
Characterization of epitopes shared by α‐melanocyte‐stimulating hormone (α‐MSH) and the 150‐kd neurofilament protein (NF150): Relationship to neurotrophic sequences
Authors: Verhaagen J. , Edwards P.M. , Schotman P. , Jennekens F.G.I. , Gispen W.H.
Published: January 2005

Immunoblot techniques and immunohistochemistry have been used to investigate epitopes shared by α‐melanocyte‐stimulating hormone (α‐MSH) and the 150‐kD neurofilament protein (NF150). Three anti‐α‐MSH antisera (namely 31H2T, 4394, and M5) from a total of 12 sera were found to react with NF150. The three crossreacting sera are different in their binding properties to peptide fragments related to α‐MSH, suggesting that at least two distinct epitopes are shared by NF150 and α‐MSH. The only known sequence common to NF150 and α‐MSH, the N‐terminal Ac‐S‐Y residues, appears to be essential for binding of sera 31H2T and 4394. However, the binding of antiserum M5 involves other, currently unknown, similarities between NF150 and α‐MSH. This is shown by the binding of M5 to peptides such as ACTH(4–10), which do not contain the N‐terminal Ac‐S‐Y sequence. Binding of M5 to tobacco mosaic virus coat protein (TMV‐coat protein), which is homologous with α‐MSH and NF150 in its Ac‐S‐Y residues, was negligible. The peptide structures that are recognized by M5 have previously been shown to exert neurotrophic activity. The data are discussed in the light of the hypothesis that similarity between NF150 and α‐MSH, as illustrated by binding to M5, may be significant in the neurotrophic activity of MSH‐related peptides.
Comparative screening for ciliary neurotrophic activity in organs of the rat and chicken
Authors: Ebendal T.
Published: January 2005

A neuron‐survival assay with dissociated ciliary ganglion neurons was used to examine the level of neurotrophic activity in organs of the rat and chicken. All tissue homogenates enhanced neuron survival at increasing protein concentrations, although 50% survival activity (1 trophic unit, TU, per ml) occurred at distinctly different levels in the different organs. Liver, spleen, T‐cells, and submandibular gland from the rat were very low in survival‐promoting activity (0.1–1.1 TU per mg of protein). Serum and lung were slightly richer (2.4–2.8 mg). Heart, brain, and skeletal muscle stimulated survival well (6.2–13 TU/mg). Unexpectedly, the highest activities were found in the rat kidney (44 TU/mg). The activity in adult chicken organs was less varied (1.6–9.1 TU/mg). Higher trophic activities were found in the chicken embryo, particularly at day 18 of development (20–30 TU/mg). The up to 100‐fold difference in trophic level between organs should make it feasible to use subtractive hybridization, differential screening, and transient expression in eukaryotic cells for molecular cloning of the ciliary neurotrophic factor(s).
Basal forebrain magnocellular neurons stain for nerve growth factor receptor: Correlation with cholinergic cell bodies and effects of axotomy
Authors: Springer J. E. , Koh S. , Tayrien M. W. , Loy R.
Published: January 2005

Recent evidence has demonstrated the presence of nerve growth factor (NGF) in areas of the central nervous system characterized by cholinergic innervation. We report that a unique population of rat basal forebrain magnocellular neurons that project to the cortex and hippocampus are immunoreactive to a monoclonal antibody to the NGF receptor. Removal of target contact results in a time‐dependent loss or shrinkage of cells in the basal forebrain that stain for NGF receptor and acetylcholinesterase, suggesting that under normal conditions, basal forebrain cholinergic neurons utilize NGF for trophic support.
Characteristics of synaptic currents in frog muscle fibers of different types
Authors: Uchitel O. D. , Miledi R.
Published: January 2005

A study was made of synaptic currents in voltageclamped muscle fibers of the frog. Fast, submaxillaris, and slow muscle fibers are innervated by nerve fibers of different conduction velocities. To avoid spatial complications, transmitter release by nerve impulses was restricted to the site of recording and reduced to single quanta (unitary endplate currents: uepc). Following nerve stimulation, the time course of transmitter release was longer and more variable in slow and submaxillaris muscle fibers than in the fast fibers. The time constant of decay of uepc in submaxillaris and slow fibers was, respectively, about 1.8 and 2.9 times slower than the decay of uepc in fast fibers. This is due mainly to differences in the lifetime of the channels opened by acetylcholine. The neuromuscular junctions in submaxillaris muscle fibers are boutonlike or longer branched contacts; and the unitary currents in the bouton junctions have a slower time course. It is concluded that the synaptic membrane in the different types of muscle fibers has synaptic acetylcholine‐operated channels that have different kinetic properties, and that these properties are determined by the type of axon that innervates the muscle fiber.
Comparison between acute and chronic effects of ammonia on branched‐chain amino acid oxidation and incorporation into protein in primary cultures of astrocytes and of neurons
Authors: Murthy C. R. K. , Hertz L.
Published: January 2005

A comparison was made of acute and chronic effects of ammonia on production of 14CO2 from the [U‐14C] labeled branched‐chain amino acids (BCAA) leucine, isoleucine, and valine as well as from [1‐14C] leucine, and on the incorporation of radioactivity from these amino acids into a perchloric‐acid‐precipitable protein fraction in astrocytes and neurons in primary cultures.

Acute exposure of astrocytes to 3mM ammonium chloride suppressed 14CO2 production from [U‐14C] BCAA and especially from [1‐14C] leucine. This inhibitory effect was abolished or even reversed ([U‐14C] leucine) after chronic exposure to ammonia. Analogously, incorporation of radioactivity into the protein fraction was inhibited after acute exposure but not after chronic exposure of astrocytes to ammonia. The total protein content per culture was increased after chronic exposure. In neurons, production of 14CO2 and incorporation of 14C into proteins were less affected than in astrocytes. These results are discussed in relation to the ability of the two cell types to synthesize glutamine.
Cyclic nucleotide phosphodiesterase isozymes in neuroblastoma cells
Authors: Walz M. A. , Holt I. L. , Howlett A. C.
Published: January 2005

Adenosine 3′,5′‐cyclic monophosphate (cAMP) content of neurons is determined not only by the rate of synthesis but also by the rate of hydrolysis by cyclic nucleotide phosphodiesterases. Multiple forms of cyclic nucleotide phosphodiesterase exist in brain and other tissues, and these may be regulated by various hormones and neuromodulators. The present study examines this regulation in a cloned line of neuroblastoma cells (N18TG2). A biphasic Lineweaver‐Burk plot of cAMP hydrolysis revealed two Kms approximating 5 and 25 μM. Lineweaver‐Burk plots of cGMP hydrolysis were linear over a range of 1 μM to 1 mM and exhibited a Km of 37 μM. Neither cAMP nor cGMP competed for hydrolysis of the alternative cyclic nucleotide. No evidence for an allosteric activation of cAMP phosphodiesterase by cGMP was found. Calcium regulation of phosphodiesterase was not found in spite of preparation of the cell extract with several protease inhibitors, and addition of exogenous calmodulin. No effect of calmodulin antagonists (calmidazolium, W7, or trifluoperazine) was observed in vitro or in situ. Growth of the cells in the presence of 200 nM 3,5,3′‐triiodothyronine (T3) resulted in an increased hydrolysis of cAMP but not of cGMP. This increase was attributed to an increase in Vmax with no change in either high or low Km. This response was blocked by cycloheximide, suggesting that the thyroid hormone effect requires protein synthesis. The thyroid hormone response in neuroblastoma cells is compared with the results of other studies of thyroid hormone effects on phosphodiesterase in other tissues in vivo.
Differential effect of mu, delta, and kappa opioid agonists on adenylate cyclase activity
Authors: de Montis G. M. , Devoto P. , Preti A. , Tagliamonte A.
Published: January 2005

D‐Ala2, D‐Leu5‐enkephalin (DADLE) and dynorphinl–13 (Dynl–13) inhibited striatal adenylate cyclase activity, both basal and dopamine‐stimulated (DA), in rats and guinea pigs. The κ‐agonists bremazocine (BRZ), U‐50,488 (trans‐3,4‐dicloro‐N‐methyl‐N‐[2‐(1‐pyrrolidiny1)‐cyclohexyl]‐benzeneacetamide), and U‐69,593 (5α, 7α 8β)‐(‐)‐N‐methyl‐N‐(7‐(l‐pyrrolidiny1)‐1‐oxaspiro (4.5) dec‐8yl) benzeneacetamide inhibited only the basal adenylate cyclase activity, and such an effect was restricted to guinea pig striatum, an area known to contain a high density of K‐binding sites. Moreover, BRZ was found to antagonize the inhibitory effect of both DADLE and Dynl–13 in rat striatum.
Cobra nerve growth factor: Structure and evolutionary comparison
Authors: Selby M. J. , Edwards R. H. , Rutter W. J.
Published: January 2005

We have cloned and sequenced an NGF cDNA from the cobra venom gland. The structure of the predicted cobra precursor resembles that of the mouse prohormone, with a highly conserved mature NGF protein at the C‐terminus. In the non‐NGF moiety, a putative signal peptide and two sets of basic residues presumably reflecting cleavage sites that are more conserved, while large interstitial regions are poorly conserved. This moiety may be involved in defining the folding of the precursor for subsequent proteolytic maturation and/or encode a bioactive polypeptide(s). Primer extension analysis indicates a single NGF transcript in the cobra venom gland.
Structure and expression of the mouse myelin proteolipid protein gene
Authors: Macklin W. B. , Campagnoni C. W. , Gardinier M. V. , Deininger P. L.
Published: January 2005

The gene for the mouse myelin proteolipid protein has been isolated and the seven exons have been sequenced. Since the sequence of a rat proteolipid protein cDNA and partial sequence of the human proteolipid protein gene have been determined, it was possible to demonstrate a very high degree of conservation for the protelipid protein gene exons among species. While there are some nucleotide changes, the protein coding region of the mouse gene encodes protein that is totally conserved relative to both rat and human prteolipid proteins. The regulatory and noncoding regions of the proteolipid protein gene are also highly conserved. The upstream regulatory and 5′‐noncoding region of the gene is 92% homologous to the comparable region of the human proteolipid protein gene, and the 3′‐noncoding region of the mouse gene is approximately 90% homologous to a rat proteolipid protein cDNA through 2,200 nucleotides of 3′‐noncoding DNA.S1 nuclease protection experiments indicated that the major 5′‐end for proteolipid protein mRNAs from mouse, rat human, ro baboon is approximately 147–160 nucleotides upstream from the initial methionine codon of the protein coding region. Other S1 nuclease protection experiments indicated the possible existence of an alernative splice site within exon 3, which may produce mRNA for DM20. This mRNA is approximately 100 nucleotides shorter than that for the protelipid protein, and it is missing the latter half of exon 3, that is, amino acids 116–150 of the proteolipid protein sequence.
Expression of myelin proteins in the developing human spinal cord: Cloning and sequencing of human proteolipid protein cDNA
Authors: Kronquist K. E. , Crandall B. F. , Macklin W. B. , Campagnoni A. T.
Published: January 2005

A full‐length clone for the human proteolipid protein (PLP) was isolated from a cDNA library constructed from poly (A)+ RNA isolated from fetal spinal cords obtained at 15–24 weeks of conceptional age. The sequence of the human PLP cDNa was determined, and the deduced amino acid sequence was found to be identical with that of rat PLP. Comparison of human and rat PLP cDNA clones indicated that the coding regions retaind 97% homology and that there were also other areas of conserved sequence. The human 5′‐untranslated region was 93% homologous to that of the rat. The 3′‐untranslated region was, overall, 73% homologous to that of the rat with areas containing >84% homology in the first 400 and last 200 nucleotides. The most variability within the 3′‐untranslated region occurred between nucleotides 2,000–2,500, where homology with the rat cDNA dropped to 55%. Expression of PLP in the human spinal cord between 11 and 23 weeks after conception was examined and compared with the expression of the myelin basic protein (MBP). RNA was isolated from pooled human spinal cords obtained at three periods of development: 11–14 weeks, 17–19 weeks, and 21–23 weeks. Northern blot analysis revealed a 3.2‐kilobase (kb) PLP mRNA that was present at higher abundance in the 21–23‐week samples. The 17–19‐week RNA than in the 17–19‐week or the 11‐14‐week samples. The 17‐19–week RNA sample also contained a PLP‐hybridizing band at 2.2 kb which may possibly have arisen by utilization of alternative polysdenylation signals. Messenger RNA for MBP was detectable at 11–14 weeks but was readily evident in both the 17–19‐ and 21–23‐week age groups. Immunoblot analysis of whole spinal cord homogenates indicated that polypeptides for MBP preceded the appearance polypeptides for PLP by 3–4 weeks.
Antisera against taurin: Quantitative characterization of the antibody specificity and its applcation to immunohistochemical study in the rat brain
Authors: Ida S. , Kuriyama K. , Tomida Y. , Kimura H.
Published: January 2005

An antiserum against taurine was generated in rabbits by immunization with taurine that was conjugated to carrier protein via glutaraldehyde. When the antiserum was applied to immunohistochemistry, structures withtaurine‐like immunoreactivity were observed in both neuronal and glial components of rat brain. The specificity of the serum was quantitatively examined using an enzyme‐linked immunoassay method, which has been newly developed for detecting such small molecules as amino acids. The best serum obtained had a high titer against taurine. It showed low cross‐reactivity with taurine metabolites and with other amino acids (less than 0.5%), while considerably higher reactivities were noted in the case of taurinecontaining peptides susch as γ‐glutamyl‐taurine and glycyl‐taurine. The results indicate that much care should be taken with taurine peptides in the evaluation of immunohistochemical results using taurine antiserum.
Methods to study changing free amino acid pools during embryonic chick development
Authors: van Gelder N. M. , Bélanger F.
Published: January 2005

During early embryogenesis of the chick, the yolk represents the only souce of free amino acids. Leucine and glutamic acid, together with valin, predominate in this nonrenewable nutrient supply; taurine and phosphoethanolamine are present in the lowest concentration. The tyrosine level is already twice as high as phenylalanine.

At embryo day 2 (E2), the not‐yet fully functioning vitelline blood plexus demonstrates an amino acid profile that rather closely resembles that of the yolk, except for three amino acids. During the transfer process from yolk to plexus; a certain amount of glutamic acid appears to be metabolized to glutamine and aspartic acid. Taurine and phosphoetanolamine are highly concentrated in the circulation, with a tenfold increase of taurine and a 50‐fold rise in phosphoethanolamine. The tyrosine‐to‐phenylalanine ratio remains 2:1, but their levels relative to valine and other essential amino acids fall by roughly 50%.

In the already contracting heart tube and the developing neural tissue on E2, taurine and phosphoethanolamine also accumulate preferentially; the levels in neural tissue are ten‐ and fivefold higher than in the circulation for the respective amino acids, whereas the heart has three time the plexus content of taurine and two times the amount of phosphoethanolamine. Tyrosine and phenylalanine, still in the same proportion, have risen to approximately twice the circulation values. The heart tube, unlike the primitive brain, concentrates leucine, whereas isoleucine accumulates in both organs.

Injections of valine (50 μmol) and taurine (200 μmol) into the yolk on E1 demonstrate that a higher yolk content of an amino acid can result in a two‐ to threefold tissue increase. This implies that the comparative amino acid profiles of early embryonic organs are to a large extent determined by the relative abundance of amino acids acids in the yolk. However, within confines of this limited supply, the organs are already able to extract preferentially certain amino acids.
Human T‐cell response to myelin basic protein in multiple sclerosis patients and healthy subjects
Authors: Tournier‐Lasserve E. , Hashim G. A. , Bach M. A.
Published: January 2005

In order to explore the T‐cell repertoire to myelin basic protein (BP) of both multiple sclerosis (MS) patients and healthy subjects (HS), we raised BP reactive T‐cell lines from blood mononuclear cells of eight MS patients and five HS. Thes lines were trigered in vitro by human BP. When analyzing their patterns of recognition of human BP versus heterologous BP, we could observe differences between healthy subjects and MS patients. Whereas T‐cell lines from healthy subjects developed a response to heterologous BP, which was in most cases equal or higher than that elicited by human BP, T‐cell lines from most MS patients displayed a low response, or no response at all, to one or several of the heterologous BP tested. A low response to bovine BP was only observed in active cases, whereas decreased responses to rat and/or monkey BP were observed both during remission and during active disease. This may indicate that T‐cell repertoire to BP in MS patients differs from that of healthy subjects.

BP‐reactive T‐cell clones were obtained by limiting dilution from two healthy subject lines. Their pattern of response to heterologous BP as compared to human BP suggest that T‐cell from the same individual can recognize different BP epitopes.
Comparative effects of morphine on leukwytic antigenic markers of monkeys and humans
Authors: Donahoe R. M. , Bueso‐Ramos C. , Falek A. , McClure H. , Nicholson J. K. A.
Published: January 2005

Knowing the in vitro effects of morphine on monkey leukocytes would be helpful in extending the utility of vestigations. Morphine effects On T1l, Leu2a, Leu3a antigenic markers on leukocytes from rhesus monkeys and humans were assessed by using single‐and two‐colour cytofluorometric analysis. Kinetics of expression Of these markers was determined after modulation of the original complement of T11 markers from the surface of T11(+) cells. Percentages of leukocytes detectable by directly staining these markers before modulation were within the expected range for monkey and human cells. Also, as expected, T11 modulation reduced the percentages of cells expressing T11 in the single‐color analysis, with reductions being greater for monkeys than human cells. Furthermore, in the single‐color analyses, the effects of morphine on kinetics of T11 expression were quite similar for both human and monkey cells. In the two‐color analyses, the simultaneous expression of T11and Leu3a markers was uniform for both monkey and human cells. These effects of morphine on kinetics of these markers varied only slightly between species. On the other hand, the distribution of Leu2 on T11 cells was markedly different for monkey and human T‐cells. Whereas human Leu2a(+) expressed Of T1l receptors' monkey high‐density Leu2a expressed fewer T1l markers than On kinetics Of Leu2a and T1l expression were at Ob‐ those with vious variance between species. Morphine primarily affected monkey Leu2a at high density and T1l at low density. Only monkeys expressed Leu2a (+)/T11 (−) cells, and morphine enhanced Leu2a expression on these cells.
Substrate specificity of cerebral cathepsin D and high‐Mr aspartic endopeptidase
Authors: Azaryan A. V. , Galoyan A. A.
Published: January 2005

The specificity of action of bovine brain cortex cathepsin D (EC 3.4.23.5) and high‐Mr aspartic endopeptidase (EC 3.4.23.‐) was studied with the vasoactive peptides renin substrate tetradecapeptide (RSTP), substance P (SP), and angiotensins I and II, and with model peptides‐Lys‐Pro‐Ala‐Glu‐Phe‐Phe (NO2)‐Ala‐Leu (I), Gly‐Gly‐His‐Phe (NO2)‐Phe‐Ala‐Leu‐NH2 (II), and Abz‐Ala‐Ala‐Phe‐Phe‐pNA (III). Cerebral aspartic peptidases show indentical substrate specificity, cleaving the Leu10‐Leu bond in RSTP and Phe‐Phe in SP and peptide I‐III, and not splitting angiotensins I and II. Because of the higher catalytic efficiency of cathepsin D (Kcat value), the specificity constants (Kcat/Km) for cathepsin D‐catalyzed hydrolysis of substrates 1–111 are much higher than those for the high‐Mr enzyme.

High‐Mr aspartic peptidase shares a number of properties with cathepsin D (sensitivity to pepstatin, substrate specificity, pH activity profile) and shows partial immunological identity; however, high‐Mr aspartic peptidase has a specific activity 7–10 times lower than that of cathepsin D. The kinetic parameters of proteolysis of model peptides presented indicate that the high‐Mr enzyme may be a complex of a single‐chain cathepsin D with another polypeptide, although the possibility that it is an independent aspartic peptidase cannot be excluded.
Transplantation of male mouse submaxillary gland increase survival of axotomized basal forebrain neurons
Authors: Springer J. E. , Collier T. J. , Sladek J. R. , Loy R.
Published: January 2005

Transaction of the fimbria‐fornix results in a loss of magnocellular neurons in the medial septum and vertical limb of the diagonal band (MS/VDB), possibly due to the deprivation of a retrogradely transported trophic substance, such as nerve growth factor (NGF), derived from the hippocampal formation. We have, utilized a transplantation model in which grafts of NGF‐rich male mouse submaxillary gland were placed in the lateral ventricle adjacent to the MS/VDB of rats with transactions of the fimbria‐fornix. At 2‐4 weeks following transection, animals with grafted submaxillary glands exhibited enhanced survival of MS/VDB neurons, which stained positive for acetycholinesterase and were immunoreactive for the NGF receptor. These experiments demonstrate that grafts of male mouse submaxillary gland can facilitate the survival of axotomized MS/VDB cholinergic neurons and may therefore prove beneficial in promoting regeneration of damaged neural systems.
Induction and quantitation of the RI cAMP‐binding protein in clonal mouse neuroblastastoma cell lines: Evidence that the increase in RI is not linked to neurite outgrowth
Authors: Liu A. Y.‐C. , Lin Z. , Kamalalannan V. , Chen K. Y.
Published: January 2005

This is a study of the regulation of expression of the RI cAMP‐binding in mouse neuroblastoma cells as it relates to neurotransmitter phenotype and neurite outgrowth. Dibutyryl cAMP was used to promote differentiation of the cholinergic NS‐20, the adrenergic N1E‐115, the neurotransmitter‐inactive N‐18, and the neurite‐minus N1A‐103 mouse neuroblastoma cells. The amount of the RI cAMP‐binding protein was quantitated by photoaffinity labeling of the 47,000‐dalton RI protein with 8‐N3‐[32P]cAMP and by Western blot, ELISA, and immunocytochemistry. Our results showed that dibutyryl cAMP inducted the RI cAMP‐binding protein by three to fivefold in each of the four neuroblastoma cell lines examined. The increased expression of the RI cAMP‐binding protein was not linked to neurite outgrowth, a parameter of morphological differentation in the neuroblastoma cells. Thus, the RI cAMP‐binding protein can be inducted in the neurite‐minus N1A‐103 neuroblastoma round cells; further, 8‐broma‐cAMP effected neurite outgrowth without inducing the RI cAMP‐binding protein in the neurite‐positive cell lines. Indirect immunocytochemistry of RI showed a cytoplasmic localization with little evidence of nuclear staining. The increase in RI cAMP‐binding protein coincided with an increase in the cAMP‐phosphodiesterase and a decrease in cAMP‐dependent phosphodiesterase activity in the mouse neuroblastoma cells. We propose that the induction of the RI cAMP‐binding protein may be a component of the homeostatic mechanism that regulates the action of cAMP in neuroblastoma calls and thet cAMP is capable of regulating its own activity through induction of cAMP‐ phosphodiesterase and RI cAMP‐binding protein and attenuation of the cAMP‐ phosphodiesterase activity.
The immunomodulatory effect of nerve growth factor
Authors: Abramchik G. V. , Yermakova S. S. , Kaliunov V. N. , Tanina R. M. , Tumilovich M. K.
Published: January 2005

A prolonged application of nerve growth factor (NGF) to neonatal prepunberal rats induced selective hypertrophy and hyperfunc tion of ganglionic sympathetic neurons. This was accompained by higher density and fluorescence intensity of varicosw adrenergic fibers and terminals in the immunocompetent organs, total increase of catecholamines in them, catecholamine diffusion into the intercellular space, and their accumulation by macrophages and mast cells. The Plaqueforming and lymphocyte blast transformation responses were activated, signs of increased lymphocyte migration to peripheral lymphoid tissues appeared, and histamine concentration in the thymus and spleen increased. All of the foregoing indicate the immkunomodulatory effect of NGF and the dependence of immune responsiveness of the organism on the functional state of the adrenergic moiety. The net effect of the increase is attributed to knon‐specific protein sensitization, changes in the sympathetic (and, probably somatic) nervous system, and direct action on immunocompetent cells.
Estrogen concentration by substance p‐immunoreactive neurons in the medial basal hypothalamus of the female rat
Authors: Akesson T. R. , Micevych P. E.
Published: January 2005

We used the combined techinque of steroid autoradiography an dimmunohistochemistry to simultaneoulsy visulaize estrogen‐concentrating and substance P‐immunoreactive (sP‐IR) cells. A substantial proportion (26.1%) of sP‐IR celss that bound estrogen was found in the anterior two thirds of the arcuate nucleus with a rapidly diminishing number of cases (8.7%) in the posterior extent of this nucleus. Highest proportions (42.9%) of sP‐Ir cells retaining estrogen were found in the ventrolateral part of the ventromedial hypothalamic nucleus. The ventral premammillary nuleus was observed to have both populations but coexistence was comparatively infrequent (2.4%). Occasional cases of coesistence were also found in the leteral hypothalamic area. These results provide anatomicat evidence that a subset of SP‐IR neurons are directly reulated by estrogen and support the hypothesis that sP‐containing cells participate in the transduction of hormonal information into a CNS cirucitry taht regulates gonadotropin secretion and sexual bhaviour.
Fetal antigen retained by mature neurons and ependyma studied with a monoclonal antibody (6B9)
Authors: Malhotra S. K. , Predy R. , Singh R. , Gupta B. L. , Bhatnagar R. , Leeuw K.
Published: January 2005

A mouse monoclonal antibody (MAb 6B9, isotype IgM) was raised against autopsy tissue sample from the central nervous system (CNS) of multiple sclerosis (MS) patients. By immunofluorescence microscopy, MAb 6B9 intensely stain most or all cell in fetal rats. However, MAb 6B9 differentially stains various cell types in adult rats. Neurons, ependymal cells, and adrenal chromaffin cells are stained intensely, whereas astrocytes and oligodendrocytes are not stained. The 6B9‐reactive antigen (6B9 antigen) is senstive to peodic acid, but insentive to treatment with protease, RNase, or hyaluronidase. Result form immunfluo‐‐microscopyon something section and cultured neuroblastoma cells indicated that 6B9 antigen is intracellular. This is supported by immunoelectron microscopy, where lableing for 6B9 antigen appear in the cytoplasm distinct from any identifiable organelle. Further studies on 6B9 antigen should reveal its chemical nature as well as the significance of developmental changes in its distribution.
Destruction by anti‐NGF of autonomic, sudomotor neurones and subsequent hyperinnervation of the foot pad by sensory fibres
Authors: Hill C. E. , Jelinek H. , Hendry I. A. , McLennan I. S. , Rush R. A.
Published: January 2005

Treatment of newborn rats with antiserum to nerve growth factor (NGF) for the first 6 postnatal days produced a loss of the sympathetic neurones that nor‐ mally project to the sweat glands of the hind paws of the rat, indicating that cholinergic sympathetic neu‐ rones require NGF postnatally for their survival. Fol‐ lowing this immunosympathectomy, there was an increase in the proportion of glands containing sen‐ sory fibres having substance‐P‐like immunoreactivity (SP‐LO. This sensory sprouting was not as extensive as that after sympathectomy using 6‐hydroxydopa‐ mine (6‐OHDA)). During normal development, fibres showing SP‐LI are associated with the glands, partic‐ ularly during the first and second postnatal weeks. Prolongation of the antibody treatment until the third postnatal week reduced the sensory fibre ingrowth from the region of the glands, suggesting that the basis of this growth is the increased availability of NGF following sympathetic denervation. Retrograde cell labelling using the fluorescent dye, fast blue, indicated that the anti‐NGF treatment did not significantly decrease the number of sensory neurones projecting to an individual foot pad. These results support the hy‐ pothesis that sympathetic and sensory neurones com‐ pete for NGF produced by target tissues.
Inhibition of the electrogenic Na,K pump and Na,K‐ATPase activity by tetraethylammonium, tetrabutylammonium, and apamin
Authors: Zemková H. , Tesinger J. , Vyskočil F.
Published: January 2005

The K+‐induced hyperploarization of of Na‐loaded mouse diaphragm muscle, enzymatic activity of Na,K‐ATpase and3H‐ouabain binding to rat brian mocro‐somes was measured in the presence of K+ channel blockers teterathylammonium (TEA), tetrabutylammomium (TBA) and apamin. TBA, and to a lesser extent TEA in millimolar conceptrations, inhibited the electrogenic of the Na,K pump, Na,K‐ATPase activity, and3H‐ouabain binging. The inhibition of3H‐ouabain binging by TEA or TBA was more evident in the presence of ATP and Na+ ions.

Apamin in nonomolar concentrations inhibited the electrogeic effect of Na,K pump and Na,K‐ATPase but not the3H‐ouabain binding. The hyperpolarizing effects of insulin and NADH, but not that of nor‐adrenaline, were also prevented by apamin.

The inhibition of Na,K pump of TEA and TBA is apparently due to both competition with K+ for a binding site on the Na,K‐ATPase is different from that of tetra‐alkylammonium compounds; it apparently decrease the turnover rate of the enzyme.