bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucose transporter localization in brain using light and electron immunocytochemistry
Authors: Gerhart D. Z. , Levasseur R. J. , Broderius M. A. , Drewes L. R.
Published: January 2005

A polyclonal antibody to a synthetic 13 amino acidpeptide found at the carboxyl‐terminal end of the glucose transporter protein was raised in rabbit and used in light and electron immunocytochemical studies of human and canine brain. This antibody identified a broad band of polypeptide of average Mr 55,000 on immunoblots (immunogold‐silver stains) of electrophoresed membrane proteins from human red blood cells. A similar polypeptide band (Mr 45,000–60,000) was identified on immunoblots of microvessel membrane proteins isolated from canine cerebrum, suggesting that this antibody is a useful tool for studying the distribution and abundance of the glucose transporter protein in mammalian nervous tissue. Peroxidase antiperoxidase stains of cerebrum using this antibody demonstrated that transporters are abundant in the intima pia, in the endothelium of blood vessels in the subarachnoid space, and in the endothelium of arterioles, venules, and capillaries of gray and white matter. In cerebellum, reaction product was localized in the vessels of the subarachnoid space and in microvessels of the molecular layer, the granular layer, and the white matter. However, transporters were not found in the intima pia of cerebellum. In medulla oblongata, transporters were found in the intima pia, the endothelium of some subarachnoid vessels, and the microvessels of gray and white matter. In pituitary, microvessels in adenohypophysis contained no reaction product, but the antigen was detected in some microvessels in neurohypophysis. Electron microscopy of cerebral cortex using a protein A‐gold technique demonstrated that glucose transporters are equally abundant on the luminal and abluminal membranes of microvessel endothelial cells. When thin sections were stained by floating grids on top of antibody and reagents (single‐sided staining), 84% of the gold particles counted were associated with endothelial plasma membranes and tight junctions, 9% were associated with endothelial cytoplasm, vesicles, or plasmalemma invaginations, and 6% were associated with nonendothelial structures (basement membrane, pericyte, smooth muscle cell, or vessel lumen).
K‐252a inhibits the increase in c‐fos transcription and the increase in intracellular calcium produced by nerve growth factor in PC12 cells
Authors: Lazarovici P. , Levi B.‐Z. , Lelkes P. I. , Koizumi S. , Fujita K. , Matsuda Y. , Ozato K. , Guroff G.
Published: January 2005

K‐252a, a kinase inhibitor isolated from the culture broth of Nocardiopsis sp., selectively inhibits, in a dose‐ and time‐dependent fashion, the increased transcription of the protooncogene c‐fos induced by nerve growth factor in PC12 cells. Induction of c‐fos by epidermal growth factor, A23187, dBcAMP, or TPA in the same cells is not affected. Pretreatment with K‐252a for 30 min results in a complete inhibition of the nerve growth factor‐induced increase in intracellular calclum. Increases in intracellular calcium induced by carbachol or by high K+ are not altered. K‐252a derivatives selective for the inhibition of various known kinases were used to inhibit the nerve growth factor‐dependent induction of c‐fos mRNA, the nerve growth factor‐dependent increase in intracellular calcium levels, and the nerve growth factor‐dependent outgrowth of neurites. K‐252a is the most effective inhibitor of all three of these actions of nerve growth factor. The possible mechanisms by which K‐252a acts on PC12 cells are considered in the light of the characteristics of the inhibitions seen here.
Human T lymphocyte response to myelin basic protein: Selection of T lymphocyte lines from mbp‐responsive donors
Authors: Vandenbark A. A. , Chou Y. K. , Bourdette D. , Whitham R. , Chilgren J. , Chou C.‐H. J. , Konat G. , Hashim G. , Vainiene M. , Offner H.
Published: January 2005

The goal of this study was to delineate the importance of blood T lymphocyte responses to several myelin basic protein (MBP) preparations in the ultimate selection of MBP‐specific T lymphocyte lines. Proliferation responses to human myelin basic protein (MBP) were assessed in blood samples from 27 multiple sclerosis (MS) patients, 20 patients with other neurologic diseases (OND), and 26 normal subjects, using five MBP preparations with different histories and electrophoretic characteristics to enhance the spectrum of epitopes represented. Substantial variations were observed in the ability of different MBP preparations to induce blood T cell proliferation in a given donor. However, four out of five of the MBPs induced modest but significant proliferation in the MS study population relative to normal individuals, with intermediate responses occurring in OND patients. Positive responses occurred more frequently in MS patients (78%) than in normal donors (31%), and were an important prerequisite for the successful selection of MBP‐specific T cell lines.
Effect of hepes on the taurine uptake by cultured glial cells
Authors: Lleu P. L. , Rebel G.
Published: January 2005

HEPES inihibited the taurine uptake in glial cells. A different kind of inhibition was observed when HEPES was present in the culture medium or in the incubation medium used for the taurine‐uptake measurement. As an example of a possible interference of HEPES in pharmacological experiments, we have studied the effect of this buffer on the modulation of taurine uptake by β agonists or ionic concentration.
Aspartate neurotoxicity on cultured cortical neurons
Authors: Choi D. W. , Viseskul V. , Amirthanayagam M. , Monyer H.
Published: January 2005

L‐aspartate neurotoxicity was quantitatively characterized in murine cortical cell cultures. Five‐minute exposure to 30 μM–3 mM L‐aspartate resulted in concentration‐dependent (ED50 about 190 μM) neuronal destruction over the next 10 hr; glia were not injured. D‐aspartate and L‐aspartate were roughly equipotent neurotoxins. Ion substitution experiments suggested that L‐aspartate neurotoxicity is comprised of both acute, sodium‐dependent “excitotoxicity” and delayed, calcium‐dependent degeneration, with the latter predominant under conditions of brief exposure. Aspartate neurotoxicity could be attenuated by D‐2‐amino‐5‐phosphonovalerate (D‐APV), dextrorphan, ketamine, and kynurenate, but not by L‐glutamate diethyl ester or gamma‐D‐glutamyl‐aminomethyl sulfonate, consistent with principal involvement of N‐methyl‐D‐aspartate receptors. DAPV and dextrorphan produced different effects on the aspartate concentration–toxicity relation; the former drug was consistent with a competitive and the latter with a noncompetitive mechanism of antagonism.
Tis gene expression in cultured rat astrocytes: Multiple pathways of induction by mitogens
Authors: Arenander A. T. , Lim R. W. , Varnum B. C. , Cole R. , de Vellis J. , Herschman H. R.
Published: January 2005

Accumulation of TIS1 and TIS11 (Lim et al.: Oncogene 1:263–270, 1987) mRNAs in secondary cultures of rat neocortical astrocytes was much greater in response to tetradecanoyl phorbol acetate (TPA) than in response to either epidermal growth factor (EGF) or fibroblast growth factor (FGF). In contrast, EGF, FGF, and TPA were equally effective in inducing accumulation of TIS8 and TIS28/c‐fos mRNAs. These data suggested that TPA and the polypeptide mitogens might induce TIS gene expression by distinct pathways. When maximally inducing concentrations of EGF and FGF were co‐administered to astrocyte cultures, TIS mRNA accumulations were no greater than those observed for the individual growth factors, suggesting that EGF and FGF saturate a common, limiting step in their induction pathways. In contrast, when either EGF or FGF was presented to astrocytes in combination with maximally inducing levels of TPA, the resulting levels of accumulation of TIS mRNAs were at least as great as the sum of the levels induced by the individual mitogens. Stimulation of [3H]‐thymidine incorporation demonstrated an identical pattern of interaction; EGF and FGF co‐administration was no more effective than either polypeptide mitogen alone, but, when presented to astrocyte cultures along with maximally inducing concentrations of TPA, either EGF or FGF was able to increase incorporation of [3H]‐thymidine.

Superinduction of all the TIS genes occurred if cycloheximide (CHX) was present during TPA exposure. Once again, two distinet classes of responses of the various TIS genes occurred; superinduction of TIS1, TIS7, TIS11, and TIS28/c‐fos mRNA accumulation ranged from 10‐ to 20‐fold, while CHX superinduction of TIS8 and TIS10 was far more modest, ranging from 2‐ to 3‐fold. Differential superinduction of TIS gene expression also occurred when astrocytes were challenged with TPA in the presence of the peripherally active benzodiazepine (BZD) Ro5‐4864, ranging from a high of fivefold (TIS28/c‐fos) to no superinduction effect (TIS8). The temporal patterns and relative efficacies of BZD and CHX superinduction of the various TIS genes were quite distinct.

Induction of TIS gene accumulation occurs in response to a wide variety of ligands by several independent pathways and can be secondarily modulated by a number of additional factors. The differential expression of this set of transient primary‐response gene products may play a major role in integrating information that impinges on cells and in directing their physiological responses.
Basic fibroblast growth factor released from synthetic guidance channels facilitates peripheral nerve regeneration across long nerve gaps
Authors: Aebischer P. , Salessiotis A. N. , Winn S. R.
Published: January 2005

Basic fibroblast growth factor (b‐FGF) has been shown to enhance the in vitro survival and neurite extension of various types of neurons including dorsal root ganglia (DRG) cells. Alpha‐1 glycoprotein (α1‐GP), an acute phase reactant, has been reported to enhance the in vitro neuritic extensions of chick DRG cells. In the present study, we investigated the ability of synthetic nerve guidance channels, which release sustained controlled amounts of b‐FGF and/or α1‐GP, to support the regeneration of a transected peripheral nerve over a 15 mm long gap, a distance that does not permit regeneration in conventional polymeric tubes. Tubes releasing bovine serum albumin (BSA), cytochrome C, BSA and b‐FGF, BSA and denatured b‐FGF, BSA and α1‐GP, or BSA, b‐FGF, and α‐1GP were fabricated by a dip‐molding technique. In vitro kinetic studies of protein release from these channels showed an initial burst during the 1st day, followed by a linear release for at least 2 weeks thereafter. In vitro studies indicated that the b‐FGF released from the polymer was biologically active as assessed by the ability of channels releasing b‐FGF to induce neurite extensions in PC12 cells. For in vivo studies, the various types of tubes were used as nerve guidance channels for the repair of a 15 mm nerve gap in the sciatic nerve of rats. Four weeks postimplantation, only the tubes releasing b‐FGF or b‐FGF and α1‐GP displayed regenerated cables bridging both nerve stumps, which contained nerve fascleles with myelinated and unmyelinated axons. We conclude that b‐FGF enhances peripheral nerve regeneration and that polymeric tubes releasing macromolecules provide a tool for the study of nerve regeneration.
Sprouting by intact Helisoma neurons: Role for glutamate
Authors: Bulloch A. G. M.
Published: January 2005

We examined the possible relationship between sprouting by intact neurons and the elevation of blood acidic amino acids that is reported to occur in Helisoma in response to osmotic stress. In the present study, buccal ganglion neurons 5 were examined following exposure of animals to conditions that induce estivation, a behavioral state exhibited by these freshwater snails in nature. Surprisingly, sprouting activity occurred in some neurons (31%) in normal animals, this being transiently elevated (to 71%) after 4 days of estivation. Blood acidic amino acids (i.e., glutamate and aspartate) were also elevated under this condition, their levels reaching a plateau on day 4. These observations give rise to two hypotheses: (1) the acidic amino acids can induce sprouting, or (2) these agents can release trophic factors, which, in turn, induce sprouting The first hypothesis was tested by exposing semi‐intact preparations in vitro to a range of concentrations of the acidic amino acids; no promotion of sprouting was observed by either glutamate or aspartate. The second hypothesis was supported, however, by the following observations. First, blood from estivated animals was tested for trophic activity. Such blood (but not that from control animals) could promote sprouting by intact neurons in vitro, a response that depended on factor(s) whose filtration behavior on Amicon membranes suggests an approximate molecular weight of 30–100 kD. Second, since medium conditioned by Helisoma ganglia is known to contain a protein(s) that is necessary for sprouting by isolated neurons, we tested the ability of such medium to promote sprouting by intact neurons. Conditioned medium promoted sprouting by intact neurons in vitro in a manner quantitatively and qualitatively similar to that observed in vivo during estivation. Additionally, sprouting by intact neurons in conditioned medium was also attributable to factor(s) that acted as 30–100 kD molecules on Amicon membranes. To test the ability of the acidic amino acids to release or activate these factors, medium was conditioned for short periods in the presence of glutamate or aspartate; glutamate, but not aspartate, was found to enhance conditioned medium. We conclude that glutamate can promote sprouting by intact neurons, but that its action is indirect and is mediated by macromolecular trophic factors.
Tyrosine phosphorylated proteins accumulate in junctional regions of the developing chick neural retina
Authors: Shores C. G. , Maness P. F.
Published: January 2005

Antibodies specific for protein phosphotyrosyl residues were used to localize sites of action of tyrosinespecific protein kinases in developing chick neural retina by immunoperoxidase staining. Phosphotyrosine‐modified proteins became prominent in growth cone‐ and process‐rich regions of embryonic retina during neuronal differentiation. Maximal levels accumulated in the synaptic layers and limiting membranes of the adult retina, where numerous junctional complexes reside. Two major phosphotyrosine‐modified proteins in adult retina (80, 42 kDal) increased markedly during maturation. In contrast, the synaptic layers of optic tectum and other brain regions exhibited low protein phosphotyrosine levels. These results suggest a specific role for protein tyrosine phosphorylation in the retina at sites of synapses and other intercellular junctions.
Phospholipid‐derived choline intermediates and acetylcholine synthesis in mouse brain synaptosomes
Authors: Yavin E. , Tanaka Y. , Ando S.
Published: January 2005

Endogenous free choline levels and acetylcholine (ACh) synthesis in nerve terminals were investigated using cerebral cortical synaptosomes of C57BL/6 mice. Endogenous choline was produced at a rate tenfold faster than ACh to provide levels adequate for the formation of the latter. The combined pool size of the water‐soluble intermediates derived from phosphatidylcholine (PhC), such as glycerophosphorylcholine (GpCh) and phosphorylcholine (PCh), increased significantly during the first 10–15 min of incubation and was always higher than that of free choline. These results most likely indicate an effective degradation of PhC by the combined action of phospholipase A2/lysophospholipase, as well as by phospholipase C in synaptosomes. ACh synthesis proceeded at a constant rate in the presence or absence of exogenous free choline (0–10 μM) and was almost entirely abolished in the presence of 10−6 M hemicholinium‐3. These results suggest that ACh is effectively synthesized by free choline generated in synaptosomes by a coupling mechanism involving the high‐affinity choline uptake system. No changes in the production rates of choline and ACh were observed between adult and aged mice.
Interferon‐gamma‐like immunoreactivity in certain neurons of the central and peripheral nervous system
Authors: Ljungdahl Å. , Olsson T. , Van der Meide P. H. , Holmdahl R. , Klareskog L. , Höjeberg B.
Published: January 2005

Interferon‐gamma (IFN‐γ) has many important immunoregulatory functions. It has previously been presumed to be produced by activated lymphoid cells alone. The present study concerning the identification of IFN‐γ in neurons was initiated against the background that nerve function influences immunological events during inflammatory diseases. Furthermore, the immune system and the nervous system may have certain signal molecules as well as certain cell surface receptors in common. Neuronal IFN‐γ‐like immunoreactivity (IFN‐γ‐LI) was studied in frozen sections of rat tissues employing both rabbit polyclonal antiserum as well as seven different mouse monoclonal antibodies (mAbs) reactive with different epitopes of rat IFN‐γ. The polyclonal antiserum and three of the mAbs (DB‐1, DB‐14, and DB‐16) showed IFN‐γ‐LI in neurons. DB‐1, the most extensively studied mAb, stained distinct networks of nerve terminal‐like profiles both in the brain and spinal cord. Also, scattered brain neuronal cell bodies showed IFN‐γ‐LI as well as a subpopulation of primary sensory ganglion cells and their intra CNS terminals. In addition, IFN‐γ‐LI was also detected in nerve terminal‐like profiles in skin, gut, and lymphoid organs. Terminals were especially prominent around blood vessels. We propose that IFN‐γ has a role in cell interactions not only in the immune system but also in the nervous system as well as in interactions between the nervous and immune systems.
Cultured neurons release an inhibitor of astroglia proliferation (astrostatine)
Authors: Rogister B. , Leprince P. , Bonhomme V. , Rigo J. M. , Delree P. , Colige A. , Moonen G.
Published: January 2005

Using in vitro techniques, we looked for a possible downregulation of rat astroglia proliferation by neuronal cells. We demonstrate that medium conditioned by 7‐day‐old rat cerebellar granule neurons or by 16‐day‐old rat embryo hippocampal neurons strongly inhibits the proliferation of cultured astroglial cells. Two neuronal cell lines, the PC12 rat pheochromocytoma and the neuro 2A (N2A) murine neuroblastoma also release such an activity. This release in N2A‐conditioned medium (CM) occurs when the cells are at high density and show a low proliferation rate. This activity is present in media conditioned by neuronal cells, but not in media conditioned by normal astrocytes, by two glioma cell lines, or by one fibroblastic cell line. This proliferation inhibitor addresses normal astrocytes: the proliferation of two glioma cell lines, of a fibroblastic cell line, and of the two neuronal cell lines (PC12, N2A) is not inhibited by N2A CM. Moreover, this activity is directed against type 1 astrocytes, but not against type 2. Using three different assays, we demonstrate that DNA synthesis by astroglial cells is inhibited. N2A CM has no cytotoxic effect on astrocytes and does not modify their overall protein synthesis.

Using affinity and gel filtration chromatography, we show that this activity is associated with a protein whose molecular weight ranges between 15 and 20 kDa. The possible relationship between this N2A cellderived astroglia proliferation inhibitor and other types of potential glial proliferation inhibitors has been investigated. A brain glycoprotein immunologically related to epidermal growth factor receptor (EGFR) was reported to inhibit astroglial cell proliferation in vitro. Using polyclonal and monoclonal antibodies against EGFR, we were unable to immunoprecipitate the astrocyte proliferation inhibitor in N2A CM or to demonstrate by immunoblotting the presence of an EGFR‐like immunoreactivity in the N2A CM or in the active chromatographic fractions of N2A CM. Transforming growth factor β (TGFβ) is a well‐known modulator of the proliferation of various cell types and was shown to be present in N2A CM. Using a polyclonal anti‐TGFβ antibody that recognizes TGFβ on Western blots of N2A CM, we were unable to immunoprecipitate the astrocyte proliferation inhibitor of N2A CM. It seems thus far that the neuronal astroglia proliferation inhibitor is a new protein for which we propose the name astrostatine.
Association of iron‐containing astrocytes with dopaminergic neurons of the arcuate nucleus
Authors: Young J. K. , McKenzie J. C. , Baker J. H.
Published: January 2005

Specialized astrocytes, identified by cytoplasmic granules that are electron‐dense and vividly stained by toluidine blue due to the presence in the granules of SH molecules and molecules of iron, have long been known to be present within the arcuate nucleus of the hypothalamus. Their function, however, is obscure. To determine whether or not these specialized astrocytes are in contact with dopaminergic neurons, rat brain sections were stained to detect tyrosine hydroxylase (TH) immunoreactive neurons by immunocytochemistry and were examined by both light and electron microscopy. Iron‐rich astrocytes were located in the same general portion of the arcuate nucleus as were TH+ neurons, and most appeared closely associated with TH + structures (somas, dendrites, and fibers) at the light‐microscopic level. At the ultrastructural level, close contact between TH + neurons and processes of iron‐rich glia was confirmed. This unique anatomical association suggests a functional relationship between the two cell types that may be related to unusual histochemical features of both cell types and/or to the location of these cells in an area with a highly permeable blood‐brain barrier.
The Alzheimer's amyloid precursor protein is produced by type I astrocytes in primary cultures of rat neuroglia
Authors: Berkenbosch F. , Refolo L. M. , Friedrich V. L. , Casper D. , Blum M. , Robakis Nikolaos K.
Published: January 2005

The production of β‐amyloid precursor protein (βAPP) by primary mixed glial cell cultures from cerebral cortex of 2–3 day postnatal rats was examined by Northern and Western blotting and by immunocytochemistry. A single 3.5 kb βAPP RNA transcript was detected using probes recognizing all forms of βAPP messenger RNA. No signal was detected with a probe specific for the alternately spliced Kuntiz protease inhibtor (KPI) region. In Western blot analysis of protein extracts, antisera specific for either the cytoplasmic or extracellular part of βAPP detected several proteins ranging from 105 to 140 kDa. None of these were recognized by an antiserum specific to the KPI insert of the βAPP. Multicolor immunofluorescence showed βAPP. immunoreactivity in type I (GFAP+A2B5−) astrocytes, distributed in a fibrillar pattern like that of glial fibrillary acidic protein (GFAP). No βAPP immunoreactivity was detected in oligodendrocytes (GC+) or in A2B5+ progenitor cells. Moreover, no cultured cells showed immunostaining with an antiserum specific for the KPI sequence of βAPP. We conclude that type I astrocytes in primary culture produce amyloid precursor, but that oligodendrocytes and their precursors do not. Moreover, type I astrocytes produce predominently the βAPP subtypes which lacks the KPI sequence.
Family of human neuronal external surface epitopes defined by torpedo monoclonal antibodies
Authors: Wright S. , Sternberg H. , Bjornskov E. K. , Stephenson D. T. , Kushner P. D.
Published: January 2005

We are employing a library of monoclonal antibodies (MAbs) that were made to torpedo cholinergic synaptosomes to identify conserved, physiologically vital epitopes of the neuronal surface. Our particular interest is in those epitopes that are present on some but not all neurons. In tjhe present study we screened this library on different cell lines, the neuronal cell lines PC12, NG108, MC‐IXC, and SY5Y, and the endocrine cell lines GH‐3 and HIT. Of these cell lines, only SY5Y cells bind MAbs that define neruonalo surface subsets. Utilizing its parent cell line, SK‐N‐SH, we verified that six MAbs, Tor 25, Tor 103, Tor 190, Tor 201, Tor 219, and Tor 233, bind the external neuronal surface. The cytolocalization of all six MAbs is very similar: the membrane of the cell body and its processes are finely outlined in a punctate distribution. Western blot analyses of Torpeco electric organ homogenates, a highly enriched source of antigenic mateial, revealed that each MAb identifies multiple polypeptides, two of which have the relative mobilities of 180 kD and 67 Kd. In a screen of peripheral nerves from cases of amyotrophic lateral scleroses (ALS), we found that all these MAbs revealed surface alteration; some displayed a decrease in binding, while others displayed an increase. The combined data provide evidence that these epitopes belong to an important, complex family of polypeptides of the external neuronal surface.
An in vivo model of varicella‐zoster virus latent infection of dorsal root ganglia
Authors: Sadzot‐Delvaux C. , Merville‐Louis M. P. , Delree P. , Marc P. , Piette J. , Moonen G. , Rentier B.
Published: January 2005

We describe here the first in vivo model of varicellazoster virus (VZV) latent infection in the adult rat peripheral nervous system. Infected Mewo cells were injected subcutaneously along the spine of healthy adult rats. No clinical sign of infection was observed even 9 months after inoculation. Humoral immune response to VZV was detected in all infected animals throughout the study (9 months). The presence of viral material in dissociated and cultured dorsal root ganglia (DRG) from inoculated animals was studied by immunoperoxidase and in situ hybridization. When DRGs from infected animals were plated in culture from 1 month and up to 9 months after inoculation, viral nucleic acids and proteins were detected in neurons. Furthermore, trypsinization and subcultivation of infected neurons in culture is needed to reactivate infectious virus at least in some of the neurons. This model provides a useful tool for studying (1) the molecular mechanisms leading to an in vivo latency, (2) the role of the immune system, in particular cellular immunity, on the establishment, maintenance, and reactivation of latency, (3) the neurotropism of mutant viruses, and (4) the effects of antiviral agents.
Prenatal alcohol exposure alters the development of sympathetic synaptic components and of nerve growth factor receptor expression selectivity in lymphoid organs
Authors: Gottesfeld Z. , Morgan B. , Perez‐Polo J. R.
Published: January 2005

Exposure to alcohol in utero has been associated with long‐term immune deficits. In addition, adult mice exposed to alcohol prenatally display altered noradrenergic synaptic transmission selectively in lymphoid organs. This is consistent with the hypothesis that sympathetic neurons play an important role in immunomodulation. The development and maintenance of sympathetic neurons are critically dependent on nerve growth factor (NGF).Furthermore, NGF has been shown to modulate immune responses and NGF receptor expression has been localized to lymphoid organs. The present work examined the effects of prenatal alcohol exposure on the development and maturation of pre‐and postsynaptic sympathetic components, including norepinephrine and β‐adrenoceptors, respectively, as well as the early expression of NGF receptors in lymphoid and other organs of the C57BL/6 mouse.

Infant mice that were exposed to alcohol in utero displayed reduced levels of norepinephrine and β‐adrenoceptor density, as well as increased NGF receptor expression in the thymus and spleen, but not the heart. These selective changes may account, in part, for the persistent immune incompetence characteristic of fetal alcohol syndrome.
Cerebral energy metabolism and intracellular pH during severe hypoxia and recovery: A study using 1H, 31P, and 1H [13C] nuclear magnetic resonance spectroscopy in the guinea pig cerebral cortex in vitro
Authors: Kauppinen R. A. , Williams S. R.
Published: January 2005

1H and 31P nuclear magnetic resonance spectroscopy was used to study intracellular pH (pH1), high‐energy phosphates, lactate, and amino acids in cortical brain slices superfused in Krebs‐Henseleit bicarbonate buffer during and after severe hypoxia at 0, 10, and 50 mM glucose. An extensive drop in phosphocreatine (PCr) and a rapid build‐up of intracellular lactate and H+ were the first signs of hypoxia. Adenosine triphosphate (ATP) was significantly more resistant to hypoxia provided that glucose was present. In the preparations that had been exposed to hypoxia in the presence of glucose, PCr became detectable within 2 min of reoxygenation, and both PCr and ATP concentrations were restored to 72–80% of normoxic levels within 30 min. Lactate was washed out, and pHi returned to normal within 4–8 min. Using 1‐[13C]glucose as a tracer, we demonstrated that the rate of lactate production in the immediate posthypoxic period was at the prehypoxic level, indicating that hte elevated lactate during this period was due solely to that produced during hypoxia. During reoxygenation of the preparations that were denied glucose during hypoxia, only 30% of total creatine + PCr and 18% of PCr were restored, and ATP was not detectable. The lactate concentration rose twofold in this period, and pHi became significantly more alkaline than before the hypoxic insult. Thus acute metabolic damage was considerably greater if glucose was absent during the insult, suggesting that either anaerobic ATP production or low pH may exert some protective effect against acute cell damage.
N‐linked glycoprotein synthesis and transport during G1 are necessary for astrocytic proliferation
Authors: Ishii S. , Volpe J. J.
Published: January 2005

The proliferation of astrocytes, purified by a selective detachment technique from mixed glial primary cultures derived from newborn rat cerebrum, was studied. The cells were synchronized by first inducing a quiescent state by removing fetal calf serum (FCS) from the culture medium for 2 days; reversal of the quiescent state by return of serum to the culture medium caused a marked increase in DNA synthesis 12‐24 hr later. 2‐Deoxyglucose, an inhibitor of dolichol‐linked oligosaccharide and thereby N‐linked glycoprotein biosynthesis, prevented not only an increase in glycoprotein biosynthesis in G1 phase of the cell cycle but also the burst of DNA synthesis that followed during S phase. Addition of mannose to the culture medium prevented the inhibitions by deoxyglucose of both glycoprotein and DNA synthesis. These data indicated an obligatory relationship in astrocytes between dolichol‐linked glycoprotein synthesis and DNA synthesis. To determine whether transport of the newly synthesized glycoproteins to the plasma membrane for incorporation therein or for secretion were necessary for DNA synthesis and astrocytic proliferation, we studied cells treated with monensin, an ionophore for monovalent cations, and an inhibitor of intracellular transport of glycoproteins. The presence of monensin in the first 12 hr after repletion of serum to synchronized astrocytes prevented progression to the S phase and cell proliferation; addition of monensin after the first 12 hr, at the onset of the S phase, had no effect on progression through S phase. Lectin‐staining methods combined with fluorescence microscopy demonstrated in monensin‐treated cells failure of intracellular glycoproteins to be transported to the plasma membrane. These data suggest that the glycoproteins synthesized during G1 and required for traverse of astrocytes through G1 and the S phase of the cell cycle must be transported to the cell surface. The nature of these proteins, e.g., whether plasma membrane components or secretory glycoproteins, and their specific functions remain to be elucidated.
Intracellular Na+ activity in cultured mouse oligodendrocytes
Authors: Ballanyi K. , Kettenamann H.
Published: January 2005

Na+‐selective double‐barrelled microelectrodes were used to measure the intracellular Na+ activity (aiNa) and membrane potential (Em) in oligodendrocytes from cultures of embryonic mouse spinal cord. In Na+‐free solutions aiNa rapidly fell from its baseline of about 15 mM to values below 1 mM. Elevation of the K+ concentration in the bath ([K+]o) from 5.4 to 15 or 50 mM elicited an aiNa decrease of 4.7 or 9.0 mM, respectively. Ouabain blocked the aiNa decrease in response to 50 mM K+ by 37%. Bath application of 1 mM glutamate resulted in a membrane depolarization of 4.5 mV and a concomitant rise of aiNa by 8.6 mM. aiNa increased by approximately 11 mM after washout of a solution containing 20 mM NH4+. This aiNa increase was not blocked by amiloride, excluding a major contribution of a Na+/H+ antiporter. We conclude that, in cultured oligodendrocytes, transmembraneous Na+ movements are involved in pH regulation, glutamate causes an influx of Na+, and that the Na+/K+ pump and passive KCI uptake contribute to K+ accumulation.
Microtubule associated protein (MAP1B) is present in cultured oligodendrocytes and co‐localizes with tubulin
Authors: Fischer Itzhak , Konola J. , Cochary E.
Published: January 2005

Differentiation of Oligodendrocytes is accompanied by the extension of processes and the assembly of the myelin membrane. It is likely that the cytoskeleton plays an important role in this process in terms of changes in cell shape, transport of myelin components, and organization of the myelin membrane. Oligodendrocytes contain microtubules (MT) which associate with other components of the cytoskeleton, and microtubule associated proteins (MAPs) may mediate some of these interactions. In this study we haves shown the presence of MAP1B in Oligodendrocytes grown in primary glial cultures by double‐label im‐munofluorescence using antibodies to galactocerebro‐side (GC) and MAP1B. The staining of the cultures showed that GC‐positive Oligodendrocytes were also stained with MAP1B antibodies. However, MAP 1B stain was limited to ceil bodies and processes, whereas GC stain was also seen in flattened membrane sheets and punctate staining in processes. MAP1B staining was also compared with that of myelin proteolipid (PLP), myelin basic protein (MBP) and beta‐tubulin in secondary glial cultures that were enriched for Oligodendrocytes. The results showed a typical staining of cell bodies and membranous profiles using PLP antibodies, and the staining of cell bodies and flattened regions of membranous sheets by MBP antibodies. In contrast, both polyclonal and monoclonal antibodies to MAP1B showed a uniform diffuse staining of cell bodies, major processes, and fine interconnected processes. Double‐labeling of the cells showed that MAP1B was co‐localized with tubulin, but was not present in glial fibrillary acidic protein (GFAP)‐positive astrocytes. Western and Northern blot analyses of primary glial cultures showed that MAP1B had a molecular mass of 320 kDa and a mRNA of 10 kb. These values are identical to those previously reported for brain MA1MB (Safaei and Fischer, 1989) and demonstrate the presence of MAP1B in oligodendrocytes.
Amino acid content of rat cerebral astrocytes adapted to hyperosmotic medium in vitro
Authors: Olson J. E. , Goldfinger M. D.
Published: January 2005

Rat cerebral astrocytes from confluent primary cultures were grown for two weeks in medium made hyperosmotic with additional NaCl. At the time the cells were harvested (four weeks in culture), the medium osmolality of experimental cultures was approximately 600 mOsm. Amino acid, protein, and potassium contents and the cell volume were measured. Compared to cells maintained in control medium (approximately 300 mOsm), cells grown in hyperosmotic conditions had over two times the content of taurine and five times the content of glutamine. Alanine, aspartate, glutamate, glycine, and tyrosine contents also were elevated in these hyperosmotic‐treated cells, while asparagine contents were unchanged relative to control cells. Cell volume and potassium contant were decreased to approximately 50% of control levels by the hyperosmotic treatment while total protein content per cell was unchanged relative to cells from control cultures. Seven min after hyperosmotic‐exposed cells were rapidly diluted into PBS with osmolality equal to about 330 mOsm, cell contents of alanine, asparagine, glutamine, glutamate, glycine, taurine, and tyrosine fell toward control levels. The data indicate that significant alterations in intracellular osmolytes occur in astrocytes adapted to hyperosmotic conditions. We suggest that a loss of intracellular potassium is at least partially compensated by accumulation of taurine, glutamine, and perhaps other amino acids acting as intracellular osmolytes.
Production of colony‐stimulating factor‐1 (CSF‐1) by mouse astroglia in vitro
Authors: Hao C. , Guilbert L. J. , Fedoroff S.
Published: January 2005

We investigated whether astroglia produce any of the known macrophage growth factors, CSF‐1, GM‐CSF, and IL‐3, and if so, whether any of these cytokines stimulate the growth of CNS macrophages. In this work we used highly enriched cell cultures of C3H/HeJ mouse neopallium: cultures of astroglia and cultures of macrophage‐like cells derived from nutritionally deprived astroglia cultures.

We found that astroglia in cultures accumulate in the medium, an activity that stimulates the proliferation of macrophage‐like cells. The activity has been identified as CSF‐1 by using growth assays of cells dependent and nondependent on CSF‐1, and by radioreceptor analysis which is highly specific for CSF‐1. Northern blot analysis demonstrated the presence in astroglia of CSF‐1 mRNA and the presence of CSF‐1 receptor (c‐fms) mRNA in macrophage‐like cells but not in astroglia. The astroglia did not produce GM‐CSF or IL‐3. We concluded that a paracrine relationship exists between astroglia production of CSF‐1 and the response of macrophage‐like cells to the cytokine in culture.
Glial cells of the human developing brain and B cells of the immune system share a common DNA binding factor for recognition of the regulatory sequences of the human polyomavirus, JCV
Authors: Major E. O. , Amemiya K. , Elder G. , Houff S. A.
Published: January 2005

Cells of the nervous system and the immune system perform phighly specialized functions which reflect the tissue‐specific regulation of their genes. However there are some functions between these two cell systems such as antigen presentation, cytokine release, and expression of MHC molecules which suggest a common mechanism for regulation of certain genes. We present data that extend this observation to in clude the recognition of specific neurotropic viral DNA sequences by glial cells and B cells. The experiments here provide evidence that both human glial cells and B cells possess nuclear DNA binding proteins that interact with nucleotide sequences on the regulatory region of the JC viral genome. These DNA binding proteins are present in human lymphoma B cell lines and fetal glial cultures. The fetal glial cultures are characterized as astrocytes by unique cDNA expression and the presence of GFAP. Data are also presented that demonstrate the presence of JCV infected B cells in brain tissue derived from progressive multifocal leucoehcephalopathy, the demyelinating disease caused by JCV infection. The possibility that the glial and B cell protein factor(s) responsible for recognition of the JCV genome belong to a family of proteins similar to known transcriptional control elements such as the Octamer binding proteins or Nuclear Factor‐1 is discussed.
PET demonstrates different behaviour of striatal dopamine D‐1 and D‐2 receptors in early Parkinson's disease
Authors: Rinne J. O. , Laihinen A. , Någren K. , Bergman J. , Solin O. , Haaparanta M. , Ruotsalainen U. , Rinne U. K.
Published: January 2005

Striatal dopamine D‐1 receptor binding was investigated in vivo with positron emission tomography (PET) in five patients with early. Parkinson's disease using [11C]‐SCH 23390. All patients had predominantly unilateral symptoms and showed a significant reduction in the accumulation of [18F]‐6‐F‐DOPA in the striatum contralateral to the symptoms. None of the patients had received any antiparkinsonian medication. The striatal and cerebellar radioactivity was measured and corresponding striatum/cerebellum ratios were counted. The mean striatum/cerebellum ratio of [11C]‐SCH 23390 binding was symmetric be‐tween the hemispheres. By contrast, the striatum/cerebellum ratio of [11C]raclopride binding, labelling dopamine D‐2 receptors, was increased significantly in the hemisphere contralateral to the symptoms as compared with the opposite hemisphere. Thus, the present results show that the behaviour of striatal D‐1 and D‐2 receptors is different in early Parkinson's disease.
Neurochemical aspects on aging and diseases with cognitive impairment
Authors: Gottfries C. G.
Published: January 2005

The concentrations of monoamines and cholineacetyl transferase (CAT) are reduced in brains from patients with normal aging. According to findings about the dopamine (DA) system, the age‐sensitive decrease has its onset in the 60s and after that has a continuous progress. In patients with Alzheimer's disease (AD/SDAT), multiple changes indicate disturbed metabolism in the acetylcholine (ACh), DA, noradrenaline (NA), and 5‐hydroxytryptamine (5‐HT) systems. The activity of monoamine oxidase B (MAO‐B) increases significantly with age and a further‐increase has been reported to occur in Alzheimer‐afflicted brains. In the platelets, too, there is increased MAO‐B activity in AD/SDAT patients. Increased enzyme activity is considered a marker of gliosis in the brain and a marker of macrocytosis in platelets. Gangliosides are reduced in several areas of the brain in AD/SDAT patients, which indicates a reduction of nerve terminals. Significantly reduced concentrations of myelin components have also been recorded in brains from patients with AD/SDAT, which is in agreement with histopathological findings.

The neurochemical changes recorded in brains from normally aged individuals are very similar to those found in AD/SDAT brains, although the latter changes are more‐severe. Thus, it has been suggested that the pathological process in AD/SDAT may be an exacerbation of the process taking place in normal aging.

The multiple neurochemical changes recorded in grey, as well as in white matter must still be considered changes of secondary nature. At the present level of knowledge, it is not possible to single out any one of these changes as of special etiological importance. In the brains of patients with AD/SDAT, more fundamental disturbances must be considered, the nature of which we still do not know. It should also be emphasized that the pathological changes in the central nervous system reported in patients with AD/SDAT have, to some extent, also been found outside the brain, indicating that the pathological process in AD/SDAT is a general process to which the central nervous system is possibly especially sensitive.
Cholinergic receptors in human brain: Effects of aging and alzheimer disease
Authors: Giacobini E.
Published: January 2005

A general review of cholinergic receptors in human brain is presented. The paper focuses upon changes in normal aging brain and in Alzheimer disease. Studies from five different approaches are reported: (1) molecular biology; (2) receptor binding studies; (3) studies with specific neurotoxins; (4) immunocyto‐chemistry; and (5) PET scan. These studies document profound and characteristic differences between the normal aging and the pathological Alzheimer brain with regard to cholinergic receptor localization, distribution, and function.
Kinesin heavy chain from bovine brain and Drosophila appear to be highly homologous molecules
Authors: Green L. A. D. , Kaplan M. P. , Liem Ronald K. H.
Published: January 2005

A microtubule‐enriched fraction was prepared from bovine white matter, and kinesin and other microtubule‐associated proteins were extracted from taxol‐stabilized microtubules by homogenization and ultracentrifugation in the presence of nucleotides (guanosine triphosphate and adenosine triphosphate). The kinesin‐enriched fractions were subjected to preparative SDS‐PAGE, and the band representing the kinesin heavy chain was excised, homogenized, and subjected to partial enzymatic digestion with Staphylococcus aureus V8 protease. Four peptides were selected for sequence analysis and compared to the previously published sequence for the Drosophila kinesin heavy chain (Yang JT, Laymon RA, Goldstein LSB, Cell 56:879–889, 1989). All four peptides matched closely with portions of the Drosophila sequence corresponding to the central, α‐helical domain. Total amino acid composition analysis of bovine kinesin heavy chain also reveals a high degree of homology to the Drosophila sequence.
Specificity of human T cell clones reactive to immunodominant epitopes of myelin basic protein
Authors: Chou Y. K. , Henderikx P. , Vainiene M. , Whitham R. , Bourdette D. , Chou C. H.‐J. , Hashim G. , Offner H. , Vandenbark A. A.
Published: January 2005

Several recently discovered lines of evidence support the involvement of myelin basic protein (BP)‐specific T cells in multiple sclerosis (MS). To identify potentially relevant immunodominant T cell epitopes, human BP (Hu‐BP)‐reactive T cell lines were selected from MS and normal donors and tested for reactivity to cleavage fragments and synthetic peptides of Hu‐BP. The MS T cell lines responded to more Hu‐BP epitopes than did normal lines, showing biased recognition of the N terminal half of the molecule, and one region in the C terminal half, suggesting increased sensitization to BP. The MS lines also differed from normal lines in their decreased percentage of CD8 + cells. One hundred nine T cell clones isolated from these lines confirmed the reactivity pattern of the lines but did not reflect the mixed phenotype, since all but three clones tested were CD4+. T cell clones from HLA‐DR2 homozygous donors responded to a variety of epitopes, indicating that this molecule was permissive in its ability to restrict T cell responses. Other epitopes, including the immunodominant 149‐170 sequence, were restricted by several different major histocompatibility complex (MHC) molecules from both MS and normal donors. T cell receptor (TCR) V gene products could be identified on six of 38 clones tested using monoclonal antibodies. From one HLA‐DR2 homozygous donor, four of eight clones utilized Vβ5.2 in response to different BP epitopes, providing initial support for the preferential use of a limited set of V region genes in the human response io BP. Preferential TCR V gene use in MS patients would provide the rationale to regulate selectively BP‐reactive T cells through immunity directed at the TCR and thus test for the first time the hypothesis that BP‐reactive T cells play a critical role in the pathogenesis of MS.
Regulation of heat shock protein synthesis in rat astroeytes
Authors: Dwyer B. E. , Nishimura R. N. , De Vellis J. , Clegg K. B.
Published: January 2005

Rat forebrain astrocytes synthesize heat shock proteins with molecular weights 97,89,70,68, and 30‐34 kilodaltons. The stress inducible 68‐kDa heat shock protein (HSP‐68) was vigorously expressed by astro‐cytes in culture after a 45°C, 20 min heat shock. HSP‐68 synthesis was poorly inducible by a second heat shock given 16 hr after the initial heat shock. Decreased [35S]methionine incorporation into HSP‐68 correlated with low levels of HSP‐68 mRNA present after the second heat shock. The data suggest that control of HSP‐68 mRNA levels by transcriptional/ posttranscriptional mechanisms is a major site for regulation of HSP‐68 synthesis.