bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative image analysis with densitometry for immunohistochemistry and autoradiography of receptor binding sites—methodological considerations
Authors: Peretti‐Renucci R. , Feuerstein C. , Manier M. , Lorimier P. , Savasta M. , Thibault J. , Mons N. , Geffard M.
Published: January 2005

Major technical progress in the development of computer‐based image analysis has made possible the entry of autoradiography and immunohistochemistry into a new era where quantification by densitometry has become easily accessible. Autoradiography could become quantitative and displayed adequate reproducibility with the help of emulsion‐coated films and the use of scales of standards of known radioactivity exposed and analyzed in parallel to the tissue sections.

Immunohistochemistry after revelation by a color‐based enzymatic technique can also become quantitative, providing that standardization of the crucial steps of the procedure and calibration through a parallel treatment ofa scale of antigen standards can be ensured. Such an approach is described here in the rat with reference to tyrosine hydroxylase (TH), the main synthesizing enzyme for catecholamines, and with dopamine (DA) itself, a catecholaminergic neurotransmitter. The different parts of the procedure, which can influence the results, such as the fixation of the animals by perfusion and the evaluation of the fluctuations via the calibration curve, are discussed in detail.

Biological validation of the proposed procedure is described by reference to experiments already well documented biochemically, such as the induction effect of reserpine on TH in the rat locus coeruleus and the depleting effect of α‐methyltyrosine (AMPT), a well‐known blocker of TH activity, on rat striatal DA content. Finally the importance of restricting the measurements to the (pseudo)linear portion of the calibration curve is illustrated by the autoradiographic identification of the differential intrastriatal repartition of the dopaminergic D1 and D2 receptorsites, particularly the dual patch‐matrix compartments.
Expression of protein kinase C isozymes in primary neuronal cultures of the rat cerebellum
Authors: Shimohama Shun , Uehara‐Kunugi Y. , Terai K. , Taniguchi T. , Kimura J. , Saitoh T.
Published: January 2005

Protein kinase C (PKC), a family of closely related enzymes, has been implicated in molecular processes involved in differentiation in a variety of cells, including neuronal cells. We studied the presence and distribution of four PKC isozymes immunocytochemi‐cally in primary neuronal cultures of the rat cerebellum. We employed four anti‐PKC antisera raised against synthetic peptides predicted from the cDNA sequence of the C‐terminal portion of four PKC isozymes, a, βI, βII, and γ‐ The majority of neurons were PKC(βII) immunoreactive both in the early and late (14 days) stage of culture, whereas PKC(α)‐, (βI)‐, and (γ)‐immunoreactive neurons were most abundant in the late stage of culture. Im‐munoreactivity of each PKC was high in the cytoplasm, processes, and growth cones. Prominent nuclear staining was observed with anti‐PKC(γ) antibody. These results are in contrast with in vivo results where each PKC isozyme is localized in a distinct population of neurons and subcellular compartment, suggesting the presence of regulatory mechanisms for PKC expression and compartmentalization in vivo.
Glutamine stimulates growth in rat cerebral endothelial cell culture
Authors: Dux E. , Noble L. , Chan P. H.
Published: January 2005

Endothelial cells were isolated from rat cerebral cortices using combined enzymatic digestions and Percoll gradient centrifugation. Primary cultures were subsequently grown on collagen‐covered dishes in a medium containing 20% fetal calf serum and 0.6 mmol glutamine. The majority of cultures became confluent by day 7 or 8, but some could not reach confluence. The cells were fusiform in shape and exhibited immunoreactivity to factor VIII‐related antigen and binding to the lectin Griffonia simplicifolia. Exposure of cultures to media containing 2.6 mmol glutamine resulted in accelerated growth (in cultures were confluent at days 3–4) and change in culture morphology, namely the formation of circular, cell‐free areas. However, this treatment did not restore γ‐glutamyl transpeptidase activity that was lost during cultivation. As for other amino acids, asparagine was less potent, glycine and phenylalanine failed to mimic the glutamine effect. In summary, glutamine stimulates growth of cerebral endothelial cells in vitro and so it may supplement for other growth factors in the culture media.
Actin and actin‐binding proteins in differentiating astroglia in tissue culture
Authors: Abd‐El‐Basset E. M. , Ahmed I. , Fedoroff S.
Published: January 2005

In this paper we have described the organization of F‐actin and actin‐binding proteins (ABP): α‐actinin, myosin, tropomyosin, caldesmon, vinculin, talin, and spectrin, in differentiating astroglia in colony cultures. We observed that the microfilament (MF) network arrangements differ at various stages of astroglia development, but the composition of MF bundles and stress fibers is the same at all developmental stages. F‐actin is closely colocalized with myosin, tropomyosin, caldesmon, and α‐actinin. The striated pattern of myosin, tropomyosin, and caldesmon are superimposable. Tropomyosin and caldesmon extend along F‐actin but are interrupted for short periods, whereas myosin is interrupted for longer periods. α‐actinin colocalizes with tropomyosin and caldesmon but not with myosin. In astroglia at different stages of development spectrin is arranged in the form of fine networks spreading through the cell and does not follow the arrangement of MF bundles. Only F‐actin, α‐actinin, and vinculin can be detected at cell‐cell junctions. In the areas of the focal contacts, F‐actin, α‐actinin, vinculin, and talin are present. They overlap each other, although talin and vinculin extend toward the cell membrane beyond F‐actin and α‐actinin.

Astroglia undergo well‐defined states of nonmotility, motility, and nonmotility again during differentiation. The changes in motility are paralled by changes in the organization of F‐actin and ABP: as GFAP‐containing intermediate filaments increase in differentiating astroglia, the F‐actin and ABP are down‐regulated, leading to non motility.
Association of ezrin isoforms with the neuronal cytoskeleton
Authors: Brigbauer E. , Dinsmore J. H. , Winckler B. , Lander A. D. , Solomon F.
Published: January 2005

We are studying the changes in the organization of the cytoskeleton which accompany expression of differentiated neuronal morphology. Of particular interest is the elaboration of growth cones, the motile domains of the neuronal plasma membrane, and the cytoskeletal structures that underlie them. A candidate for a component of the growth cone cytoskeleton of cultured hippocampal neurons is the antigen recognized by the monoclonal antibody, 13H9 (Birgbauer and Solomon, J Cell Biol 109:1609–1620, 1989; Goslin et al., J Cell Biol 109:1621–1631, 1989). That antibody binds strongly to growth cones, but barely stains neurites. The characterization of the antigen, both biochemical and microscopic, suggests that it may interact with microfilaments and microtubules. We have established that 13H9 recognizes a subset of the isoforms of ezrin (unpublished results). Here, we describe the properties and localization of ezrin isoforms in differentiating neuronal cells, using two in vitro systems and developing spinal cord. In embryonal carcinoma cells, both the abundance of ezrin and the proportion of ezrin associated with the cytoskeletal fraction increase upon induction of neuronal differentiation with retinoic acid. In the neuronal cells within such cultures, the 13H9‐positive forms of ezrin are enriched in the growth cone, while the bulk of ezrin identified by a polyclonal antibody shows no specific localization. In mouse DRG neurons, 13H9 staining is asymmetrically distributed along the edges of the complex growth cones of these cells. Staining of developing spinal cord in rat embryos also demonstrates that the 13H9‐positive forms of ezrin do not colocalize with the majority of ezrin. These results support the conclusion that ezrin is a component of the neuronal cytoskeleton, and that the localization of covalently distinct isoforms of the protein may be significant for growth cone function.
Tyrosine phosphorylation and immunodetection of vinculin in growth cone particle (GCP) fraction and in GCP‐cytoskeletal subfractions
Authors: Igarashi M. , Komiya Y.
Published: January 2005

The growth cone, the motile tip of developing neuronal processes, is considered responsible for the exact guidance of axons and synaptogenesis. High activity of tyrosine kinases in growth cones may contribute to the functions of growth cones. Our previous work revealed that vinculin is one of the endogenous substrates for intrinsic tyrosine kinases in the growth cone particle (GCP) fraction isolated from fetal rat brain. In the present study, we examined tyrosine phosphorylation and immunoblot analysis of vinculin in various fractions from fetal rat brains and adult synaptosomal fraction. Tyrosine phosphorylation of vinculin in the GCP fraction was more prominent than in any other fraction from fetal brain or synaptosomes from adult Compared to other fractions, however, the enrichment of vinculin in the GCP fraction was not observed. Tyrosine phosphorylation of vinculin in the fraction was inhibited by genistein, a specific tyrosine kinase inhibitor. Although vineulin was also phosphorylated by protein kinase C in the GCP fraction, it incorporated a much smaller amount of 32P than MARCKS protein or GAP‐43. The cytoskeletal subfraction from the GCP fraction contained a considerable amount of vineulin and it was one of the major substrates for tyrosine kinases in the GCP cytoskeleton. The membrane skeleton from the GCP fraction contained a low amount of vinculin but showed high kinase activity that phosphorylated vineulin. Taken together, our results suggest that tyrosine phosphorylation of vinculin contributes to the cytoskeletal organization of growth cones.
Establishment and characterization of a human leptomeningeal cell line
Authors: Murphy M. , Chen J‐N. , George Donna L.
Published: January 2005

Human leptomeningeal cells grown in culture were immortalized via transfection with an SV40 large T antigen gene construct under the control of the Rous sarcoma virus promoter. This cell line, designated LTAg2B, maintained the polygonal morphology characteristic of primary cultures, and stained positively in early passage for cytokeratin, a specific marker for arachnoid cells of the leptomeninges. Additional immunofluorescent staining revealed that these cells express vimentin and desmoplakin as well; these antigens have been found together only in normal arachnoid tissue and in meningiomas, which are the neoplastic derivatives of leptomeningeal cells. Significantly, LTAg2B cells demonstrate a greatly increased life span and growth rate relative to primary cultures. The establishment of this cell line should thus facilitate studies on the cellular and molecular biology of leptomeningeal cells, as well as elucidate their roles in certain pathological situations involving the leptomeninges, such as meningitis and meningioma tumor formation.
Establishment of highly enriched type‐2 astrocyte cultures and quantitative determination of intense glutamine synthetase activity in these cells
Authors: Juurlink B. H. J. , Hertz L.
Published: January 2005

We report procedures that allow one to develop and maintain cultures highly enriched in rat neopallial type‐2 astrocytes. Even after four weeks such cultures consist of more than 90% type‐2 astrocytes, ˜ −5% O‐2A progenitors and fewer than 2% type‐1 astrocytes. Their survival for more than 5 days requires the addition of conditioned medium from type‐1 astrocyte cultures. The type‐2 astrocytes have an intense glutamine synthetase activity whose basal level is sevenfold higher than in type‐1 astrocytes. The glutamine synthetase activities of both the type‐2 and type‐1 astrocytes are increased after exposure to cortisol. Thus, type‐2 astrocytes express the two quint‐essential astrocytic features: glial fibrillary acidic protein (previously reported by others) and glu‐tamine synthetase.
Redox modulatory site of the NMDA receptor–channel complex: Regulation by oxidized glutathione
Authors: Sucher N. J. , Lipton S. A.
Published: January 2005

We monitored increases in both intracellular calcium concentration ([Ca2+]i) and whole‐cell current responses induced by N‐methyl‐D‐aspartate (NMDA), applied with co‐agonist glycine, using fura‐2 digital imaging and patch‐clamp recording techniques. Extracellular application of oxidized glutathione (GSSG), but not reduced glutathione (GSH), inhibited responses mediated by activation of the NMDA subtype of glutamate receptor in cultures of rat cortical and retinal ganglion cell neurons. The NMDA responses were persistently inhibited by GSSG (500 μM to 10 mM) until introduction of a selective sulfhydryl reducing agent, dithiothreitol, which resulted in complete recovery of the responses. Exposure of the neurons to 5,5‐dithio‐bis‐2‐nitrobenzoic acid (DTNB), an efficacious oxidizing agent, also resulted in persistently smaller responses to NMDA. The addition of GSSG following exposure to DTNB, however, did not result in a further decrement in NMDA responses in our experimental paradigm. These findings suggest that a predominant action of GSSG is oxidation of vicinal thiol groups to form a peptide disulfide bond(s) comprising the redox modulatory site of the NMDA receptor–channel complex. Evidence for such regulatory sulfhydryl centers associated with the NMDA receptor has been presented previously. Moreover, the fact that DTNB produced little if any additional attenuation of the NMDA [Ca2+]i response when administered alter GSSG implies that GSSG is also an efficacious oxidant at this site. GSSG displayed little or no effect on [Ca2+]i responses elicited by high extracellular K+ or by kainate, suggesting that, at least under the conditions of the present experiments, GSSG was somewhat selective for the NMDA redox modulatory site. Based on these observations, we suggest that GSSG exerts its NMDA‐specific redox effects in a novel extracellular manner.
Calbindin‐D28K and ischemic damage of pyramidal cells in rat hippocampus
Authors: Rami A. , Rabié A. , Thomasset M. , Krieglstein J.
Published: January 2005

An antibody against rat calbindin‐D28K, a calcium‐binding protein present at high concentration in certain neurons of the central and peripheral nervous systems, was used to determine the progression of the pathological events in the rat hippocampus following experimental cerebral ischemia. Calbindin‐D28K immunoreactivity is present in dentate granule cells and in the CA1–CA2 pyramidal cells. CA1 subfield contains a higher proportion of calbindin‐D28K‐positive pyramidal cells than does the CA2 subfield and CA1 cells are more immunoreactive than the CA2 cells. The pyramidal cells of the CA1 and CA2 subfields are vulnerable to ischemia. The cells in the C A 1 became necrotic within 3–4 days after ischemia while those of the CA2 became necrotic within 2 days. There was a concomitant decrease in calbindin‐D28K immunoreactivity in the whole hippocampal regio superior after ischemia which peaked 3 days postischemia. The difference in CA2 and CA1 vulnerability seemed to be inversely correlated with the calbindin‐D28K contents of the CA2 and CA1 pyramidal cells. The decrease in the calbindin‐D28K contents of these neurons was accompanied by cell damage. We therefore suggest that calbindin‐D28K is an important factor for the survival of pyramidal cells in the hippocampal formation after ischemia.
Phenotypic plasticity of avian embryonic sympathetic neurons grown in a chemically defined medium: Direct evidence for noradrenergic and cholinergic properties in the same neurons
Authors: Barbu M. , Pourquié O. , Vaigot P. , Gateau G. , Smith J.
Published: January 2005

Avian embryonic sympathetic ganglia possess both catecholaminergic and cholinergic features and can synthesize noradrenaline (NAd) and acetylcholine (ACh) simultaneously. In the present study we sought to determine (1) whether or not this coproduction of NAd and ACh corresponds to the existence of two non‐overlapping populations, and (2) to what extent the levels of synthesis are influenced by non‐neuronal ganglion cells. We have focused on the correlation between the immunocytochemically demonstrable presence of the noradrenergic and cholinergic enzymes tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT), respectively, and the synthesis of the corresponding neurotransmitters in embryonic quail sympathetic neuronal and non‐neuronal cells purified by fluorescence‐activated cell sorting. We show that (1) freshly sorted neurons synthesize both NAd and ACh, whereas non‐neuronal cells produce neither; (2) the overwhelming majority of the sympathetic neurons display TH immunoreactivity; (3) about half of the TH‐positive neurons are recognized by an anti‐ChAT antibody in an artificial medium that selectively enhances synthesis and/or accumulation of ACh; (4) the non‐neuronal cells are important for survival of the neurons and potentiate their synthesis of ACh in this medium, and (5) finally, we present evidence that expression of TH in noradrenergic neurons and in small intensely fluorescent cells of sympathetic ganglia is differentially regulated. © 1992 Wiley‐Liss, Inc.
Time lapse study of neurite growth in hypothalamic dissociated neurons in culture: Sex differences and estrogen effects
Authors: Díaz H. , Lorenzo A. , Carrer H. F. , Cáceres A.
Published: January 2005

Cultures of dissociated hypothalamic cells taken from rat fetuses of 19 days of gestation were studied using time‐lapse recording and sequential microphotography from 1 to 5 days in vitro (DIV) and at 7 and 21 DIV. Cultures were seeded with cells taken from fetuses grouped by sex or sexually mixed; experimental cultures were raised in medium containing 17‐β‐estradiol 100 nM (E2). Cells were plated on poly‐D‐lysine‐coated coverslips at a culture density of approximately 4,000 cells/cm2. Immunocytochemistry of cell cultures was performed using a Tau monoclonal antibody (clone Tau‐1 PC1C6) and a monoclonal antibody against MAP‐2 (clone AP‐20). Cells started to produce lamellipodia and neuritic processes approximately 4 hr after plating. Forty‐eight hours later a few neurons had defined their morphological polarity by the differentiation of an axon‐like process that grows faster than the others; at 5 DIV almost all neurons had defined their axons. At this time, monoclonal antibody against MAP‐2 clearly stained soma and dendrites, but not axons. Tau immunoreactivity (lots CCA101 and CCA101N from Boeringher Mannheim) was differentially distributed, with a clear predominance in axon and soma. Results on the morphometric analysis of control and E2 treated neurons provide direct evidence for the existence of sex related differences in the neurite out‐growth response of hypothalamic neurons, since cultured neurons taken from female fetuses differentiated axons later and had fewer primary neurites and shorter dendrites than neurons taken from male fetuses or sexually mixed cultures. Also, it was demonstrated in living neurons that E2 effectively enhances outgrowth and elongation in axons. The frequency distribution curves of axonal length for control and E2 treated cultures was unimodal, suggesting that the effect of E2 was a uniform increase in the axonal length of all neurons. The structural differences between neurons from both sexes and the changes induced by E2 may contribute to explain the differences in brain function found between the sexes. © Wiley‐Liss, Inc.
Biosynthesis and expression of the myelin‐associated glycoprotein in cultured oligodendrocytes from adult bovine brain
Authors: Yim S. H. , Quarles R. H.
Published: January 2005

The biosynthesis and expression of myelin‐associated glycoprotein (MAG) were investigated in cultured oligodendrocytes isolated from adult bovine brain. Western blotting revealed two prominent MAG bands that were present in comparable amounts; the larger component electrophoresed above the 97 kD standard but was slightly smaller than the MAG band in purified bovine myelin, and the smaller component electrophoresed below the 97 kD standard. In comparison to other precursors of oligosaccharides, inorganic [35S]sulfate was a relatively specific isotope for labeling MAG relative to other glycoproteins in the cells. Sulfate labeled only the larger of the two MAG components, which contains complex N‐linked oligosaccharides, but which appears to be glycosylated to a lesser extent than MAG in vivo. The smaller MAG band in the cells is a form with high‐mannose oligosaccharides and was not detected in purified bovine myelin. Both the large and small MAG components were expressed on the oligodendrocyte surface as indicated by their sensitivity to neuraminidase and/or trypsin treatment of live cells. MAG was also released by the oligodendrocytes into the culture medium. The MAG in the medium was slightly smaller than that in the cells, suggesting that it may be released from the cell surface by limited proteolysis. The release of MAG by myelin‐forming cells could be relevant to physiological roles that have been postulated for soluble forms of MAG and other adhesion proteins. Published 1992 by Wiley‐Liss, Inc.
Specific binding of cytotactin to sulfated glycolipids
Authors: Crossin K. L. , Edelman G. M.
Published: January 2005

The binding of the glial glycoprotein, cytotactin, to a variety of purified glycolipids was examined. Clear‐cut evidence was found for binding of radiolabeled cytotactin to sulfatides purified from bovine brain, but the molecule did not bind to gangliosides or cerebrosides. The sulfatide binding was sensitive to pH and ionic strength and was dependent on the presence of divalent cations. Binding was inhibited by purified unlabeled cytotactin, by polyclonal antibodies to cytotactin, and by several monosaccharides and polysaccharides. It was not inhibited by fibronectin, a chondroitin sulfate proteoglycan, or the HNK‐1 monoclonal antibody, all of which are known to bind to cytotactin. These findings raise the possibilities that sulfated glycolipids may function as cellular receptors for cytotactin and that binding by sulfatides may modulate the varied effects of cytotactin on cellular processes. © 1992 Wiley‐Liss, Inc.
L2/HNK‐1 carbohydrate and protein‐protein interactions mediate the homophilic binding of the neural adhesion molecule P0
Authors: Griffith L. S. , Schmitz B. , Schachner M.
Published: January 2005

The neural adhesion molecule P0, the most abundant glycoprotein in peripheral myelin of mammals, is a member of the immunoglobulin superfamily and expresses the L2/HNK‐1 and L3 oligosaccharides at a single N‐glycosylation site. It acts in both homophilic and heterophilic binding mechanisms. To investigate the molecular requirements for homophilic interaction, we have used P0 from human sciatic nerve and the extracellular domain of P0 expressed in bacteria to determine binding of P0 to P0 in solid phase and bead aggregation assays. The binding of P0 to P0 could be partially inhibited in both assays by antibodies to the L2/HNK‐1 epitope and by the L2/HNK‐1 carbohydrate, but not by L3 antibodies or other carbohydrates. Inhibition of binding was also seen with polyclonal antibodies reacting with the protein backbone of P0. These observations indicate that both carbohydrate and protein structures are involved in the binding of P0 to P0 and that P0 acts as a presenter of and a receptor for a functionally important carbohydrate. © 1992 Wiley‐Liss, Inc.
Distinct hypomyelinated phenotypes in MBP‐SV40 large T transgenic mice
Authors: Jensen Niels A. , Smith G. M. , Shine H. D. , Garvey J. S. , Hood L.
Published: January 2005

To study the effect of SV40 large T‐antigen expression in myelin‐forming cells of both the central and peripheral nervous system, a series of transgenic mice were generated expressing the SV40 large T‐antigen under control of the myelin basic protein (MBP) promoter. Two neurologic phenotypes, designated A and B, appeared among individual transgenic founders and their progeny. The A mice developed a severe action tremor at about 10 days of age that progressed into periods of convulsions and early death by three to four weeks of age. In contrast, the B mice exhibited a progressive hindlimb ataxia and had a more normal lifespan. The A mice displayed hypomyelinating lesions in the central nervous system (CNS), whereas the B mice had lesions in either the peripheral nervous system (PNS) alone or in both the PNS and CNS. Immunohistochemical staining of spinal cord sections of a type A mouse showed a substantial depletion in MBP. Moreover, T‐antigen‐positive cells appeared prodominantly in white matter tracts as randomly distributed single cells. Double labeling immunocytochemistry demonstrated that some of these T‐antigen‐positive cells were positive for oligodendrocyte differentiation markers MBP and O4. Thus, T‐antigen expression appeared to coincide with a terminal stage of oligodendrocyte differentiation. © 1993 Wiley‐Liss, Inc.
Microtubule‐associated protein MAP1B expression precedes the morphological differentiation of oligodendrocytes
Authors: Vouyioukiis D. A. , Brophy P. J.
Published: January 2005

The microtubule‐associated protein MAPIB is belieyed to play an important role in the outgrowth of neurites from neurons (Tucker and Matus, Dev Biol 130:423–434, 1988). We have investigated the possibility that MAPIB might participate in the formation of processes incultured oligodendrocytes by an anal‐ ysis of the expression of MAPIB during oligodendrocyte progenitor development. The appearance of the antigens recognized by the monoclonal antibodies A2B5, O4, and O1 which define distinct stages in the maiuration of progenitors, was compared with the developmental expression of MAPIB. MAPIB is first detectable in O4 + preoligodendrocytes prior to the acquisition ofgalactocerebroside and immediately before they develop the complex process‐bearing mor‐ phology characteristic of terminally differentiated myelin‐forming oligodendrocytes in the CNS. In contrast, astrocytes have negligible amounts of MAP1B. These results demonstrate that the expression of MAP1B precedes the development of the mature oligodendrocyte phenotype and suggest that interactions between microtubules and MAP1B might have a role in the formation and stabilization of myelinforming processes. © 1993 Wiley‐Liss, Inc.
Secretion of amyloid precursor protein and laminin by cultured astrocytes is influenced by culture conditions
Authors: Shea T. B. , Beermann M. L. , Honda T. , Nixon R. A.
Published: January 2005

Although normally quiescent, astrocytes in the adult brain respond to various types of brain injury by rapidly dividing, swelling, extending cellular processes, and expressing increased amounts of glial fibrillary acidic protein (GFAP). These phenomena are collectively referred to as “astrogliosis.” Similarly, astroglia in primary culture stop dividing when they attain confluency, yet, as seen in situ, they retain their proliferative capacity for extended periods and resume rapid division when subcultured. To examine the impact of glial division on secretion of neuritepromoting factors, conditioned medium (CM) was removed from subconfluent, newly confluent, and longterm confluent (“aged”) neonatal rat astrocyte cultures, and from aged confluent cultures that had been repassaged, “Lesioned” (scraping with a rubber policeman), or triturated 3 days before harvest. Secretion of neurite‐promoting factor(s) by glial cells into these CM was then assayed by treating neuroblastoma cultures with these various CM and quantitating neurite elaboration. Extensive neurite sprouting was elicited by CM from cultures just reaching confluency and from repassaged, lesioned, or triturated cultures. CM from aged confluent cultures did not induce sprouting. These results indicate that secretion of neurite‐promoting factors(s) is regulated by glial division, and suggest that gliosis in situ may contribute to neurite sprouting by similar mechanisms. Immunoblot analysis demonstrated the presence in CM of varying amounts of laminin and amyloid precursor protein (APP), including isoforms containing the Kunitz‐type protease inhibitor domain. CM from subconfluent cultures contained trace amounts of these protiens, but CM from cultures just reaching confluency contained significant amounts. Although CM from aged cultures contained barely detectable levels of either protein, trituration or repassage of aged cultures dramatically increased secretion of these proteins. APP‐and laminin‐enriched CM fractions promoted neuritogenesis to a similar level as respective unfractionated CM; anti‐APP and antilaminin antisera blocked this effect. Purified human brain APP promoted neuritogenesis when added to non‐conditioned medium and aged CM. Increased secretion of APP and laminin therefore mediates at least a portion of CM‐induced neuronal sprouting; these proteins may perform analogous functions during astrogliosis in situ. © 1994 Wiley‐Liss, Inc.
Autocrine regulation of proliferation of cerebellar granule neurons by nerve growth factor
Authors: Muller Y. , Duperray C. , Caruso F. , Clos J.
Published: January 2005

Premigratory cerebellar granule neurons, which highly express nerve growth factor (NGF), low (gp75NGFR) and high (gp140trkA) affinity NGF receptors, were used as a physiological model to investigate the effects of NGF on neuronal replication. Studies in vivo and on cultures showed that NGF stimulates DNA synthesis, mitotic activity and related cell acquisition by initiating the entry of cells into the S phase and regulating their time in the G1 and S phases. The NGF‐induced effects were blocked in vivo and in vitro by both monoclonal anti‐NGF and anti‐gp75NGFR antibodies. These results clearly demonstrate that NGF is essential for the crucial first step of cerebellar ontogenesis and support the idea that low affinity receptors are involved in the biological response, possibly by interacting with gp140trkA. By comparison with a number of well known mitogens, the high affinity form could be the main transducer of the mitogenic signal pathway. The early developing cerebellum appears therefore to be the first autocrine (and/or paracrine) model of NGF action on neurogenesis in the CNS. © 1994 Wiley‐Liss, Inc.
Brain macrophages stimulate neurite growth and regeneration by secreting thrombospondin
Authors: Chamak B. , Morandi V. , Mallat M.
Published: January 2005

The presence of macrophages in the developing or lesioned central nervous system (CNS) led us to study the influence of these cells on neuronal growth. Macrophages were isolated from embryonic rat brain and we observed that factors released in vitro by these cells stimulate neurite growth and regeneration of cultured CNS neurons. This effect was inhibited by antibodies directed against thrombospondin, an extracellular matrix protein that we found to be synthesized and released by brain macrophages. Immunodetection of thrombospondin in the adult rat brain lesioned by kainic acid confirmed the production of this protein by brain macrophages and indicated an early intraparenchymal accumulation of thrombospondin following injury. These results suggest that brain macrophages contribute actively to neurite growth or regeneration during the development or in pathological contexts. © 1994 Wiley‐Liss, Inc.
Retrograde axonal transport of locally synthesized proteins, e.g., actin and heat shock protein 70, in regenerating adult frog sciatic sensory axons
Authors: Edbladh M. , Ekström P. A. R. , Edström A.
Published: January 2005

The local synthesis and subsequent retrograde axonal transport of [35S]methionine‐labelled proteins was studied in the in vitro regenerating adult frog sciatic sensory axons. By the use of a three compartment culture system, proteins in the outgrowth region were selectively labelled. After 2 days in culture a rise in TCA‐insoluble radioactivity was detected in the dorsal root ganglia, which could be prevented by the addition of viblastine or 2,4‐dinitrophenol to the nerve proximal to the crush site. Two‐dimensional polyacrylamide gel electrophoresis of ganglionic proteins revealed a pattern of 35 labelled polypeptides with apparent molecular masses (Mm) ranging from < 15 to 95 kDa and with isoelectric points (pI) ranging from 4.5 to 6.5. The major ones, representing about 75% of the activity in a gel, were of Mm/pI 47/5.4, 48/6.1, ©57/6.0, 62/5.2, 65/4.9–5.0, 65/5.2, and 81/5.4 respectively. One of these polypeptides (47/5.4) was identified as actin and another (81/5.4) as a member of the heat shock protein 70 family. The spots at 65/4.9‐‐5.0 were tubulin isoforms. There was a striking similarity between transported proteins on one hand, and proteins synthesized in the injured nerve on the other, with respect to the Mm/pI of at least 14 protein species. The results suggest that selected set of proteins, synthesized by non‐neuronal cells, e.g., Schwann cells, is transferred to the ganglionic cell bodies by retrograde axonal transport.© 1994 WiIey‐Liss, Inc.
Amphiphilic properties of molecular forms of acetylcholinesterase in normal and dystrophic muscle
Authors: Cabezas‐Herrera J. , Campoy F. J. , Vidal C. J.
Published: January 2005

Acetylcholinesterase (AChE) molecular forms were studied in normal (NM) and in dystrophic (DM) 129B6F1/J mouse muscle. Successive extractions of the tissue with saline and saline‐Triton X‐100 buffers yielded two soluble fractions, S1 and S2. Forty percent of the AChE in NM was measured in S1 and 60% in S2, and 65% and 35%, respectively, in extracts from DM. A12, A8, G4, G2, and G1 forms of AChE were found in S1 and S2 from NM and DM. A similar content of asymmetric molecules was noticed between NM and DM. G4 AChE was a minor species in DM, and G1 and G2 AChE were more abundant in DM than in NM. The amphiphilic properties of the several molecules were assessed by Triton X‐114 phase‐partitioning and hydrophobic chromatography. Thirty and 70% of the enzyme in a mixture of S1 and S2 partitioned in the detergent‐rich and in the detergent‐poor phases, respectively, whether the extracts were obtained from NM or DM. Asymmetric and G4 AChE predominated in the aqueous phase and G1 and G2 in the detergent phase. Ten and 25% of the enzyme in S1 from NM or DM, respectively, was adsorbed to the phenyl‐agarose. Elution of the retained enzyme followed by sedimentation analysis revealed that a certain amount of asymmetric and most of the G1 and G2 forms were associated with the matrix. The content of amphiphilic asymmetric and light globular forms was notably higher in DM than in NM. The results suggest that dystrophic muscle produces a specific pattern of molecular forms of AChE. © 1994 Wiley‐Liss, Inc.
Mutations in demyelinating peripheral neuropathies support molecular model of myelin PO‐glycoprotein extracellular domain
Authors: Kirschner D. A. , Saavedra R. A.
Published: January 2005

Homophilic interactions of the major integral membrane protein of peripheral nerve myelin, P0‐glycoprotein, are thought to mediate membrane adhesion and compaction. Molecular modeling of its extracellular domain (P0‐ED), based on its resemblance to an immunoglobulin variable domain and on X‐ray diffraction measuements of inter‐membrane spacings of myelin, has suggested which amino acid sidechains may be involved in the homophilic adhesion. Recently identified point‐mutations in the human P0 gene result in amino acid substitutions in P0 protein and correlate with demyelinating motor and sensory neuropathies. The molecular model explains how these changes result in disrupted P0‐P0 interactions; indicates how compensatory changes in amino acids, as occur in P0‐ED of other species, preserve normal homophilic interactions; and predicts what other residue substitutions might underlie additional cases of demyelinating neuropathies. Copyright © 1994 Wiley‐Liss, Inc.
Protection by an ACTH4–9 analogue against the toxic effects of cisplatin and taxol on sensory neurons and glial cells in vitro
Authors: Hol E. M. , Mandys V. , Sodaar P. , Gispen W. H. , Bär P. R.
Published: January 2005

Sensory neuropathy is a serious side effect of antitumour drugs such as cisplatin and taxol. There are indications that an analogue of the adrenocorticotrophic hormone 4–9 fragment (ACTH4–9: Met(O2)‐Glu‐His‐Phe‐D‐Lys‐Phe) can prevent these neurotoxic effects. We studied the potential protective effects of this analogue in cultures of chick dorsal root ganglia and rat Schwann cells treated with cisplatin or taxol to gain insight into the mode of action and characteristics of this neuroprotection. Neurite outgrowth of sensory neurons in vitro was dose‐dependently inhibited by cisplatin and taxol; after 48 hr, 10 μg/ml cisplatin reduced outgrowth from 431 ± 17 μm to 220 ± 6 μm and 0.01 μg/ml taxol from 344 ± 3 μm to 200 ± 43 μm. Co‐treatment of 10 μg/ml cisplatin with the ACTH4–9 analogue (0.1 nM–1 nM) resulted in about 35% more outgrowth than cisplatin alone. In contrast, the analogue could not prevent taxol neurotoxicity. Migration of neurons and satellite cells from the DRG‐body is completely inhibited by 10 μg/ml cisplatin. Taxol had no effect on the migration of these cells. In addition, cisplatin was more toxic to Schwann cells than taxol; 3–10 μg/ml cisplatin significantly reduced their laminin content, total protein, 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase activity, and cell division. The ACTH4–9 analogue (0.01 nM–100 nM) had no effect on the migration of cells out of the DRGs and could not prevent the toxic effect on the Schwann cells. These data support our hypothesis that the neuroprotective effect of ACTH4–9 analogue is brought about by a direct action on neurons, possibly by replacing a Schwann‐/satellite‐cell derived trophic factor. Copyright © 1994 Wiley‐Liss, Inc.
Nerve growth factor induces the Oct‐2 transcription factor in sensory neurons with the kinetics of an immediate‐early gene
Authors: Kendall G. , Brar‐Rai A. , Ensor E. , Winter J. , Wood J. N. , Latchman D. S.
Published: January 2005

The Oct‐2 transcription factor has a predominantly inhibitory effect on gene expression in neuronal cell lines. This factor and its corresponding mRNA have previously been shown to be elevated in adult rat dorsal root ganglion (DRG) neurons chronically exposed to nerve growth factor (NGF). Here we show that the Oct‐2 mRNA is rapidly induced in DRG cells exposed to NGF and that such induction still occurs to a lesser extent in the presence of the protein synthesis inhibitor cycloheximide. These findings characterize Oct‐2 as a novel member of the immediate‐early class of NGF‐induced transcription factors whose previously defined members have a predominantly stimulatory effect on the expression of other genes. Induction of the Oct‐2 mRNA was also observed in DRG neurons treated with acidic fibroblast growth factor or epidermal growth factor but not with a range of other growth factors and neurotrophins. The role of the Oct‐2 transcription factor in mediating the response of DRG neurons to NGF is discussed. © 1995 Wiley‐Liss, Inc.
Altered processing of a mutant amyloid precursor protein in neuronal and endothelial cells
Authors: Zhao Boyu , Sisodia S. S. , Kusiak J. W.
Published: January 2005

Altered proteolysis of the amyloid precursor protein (APP) may play an important role in Alzheimer disease (AD). To better understand the role of mutant APP in the pathogenesis of the disease, we stably overexpressed the mutant APP717F approximately twofold vs. the endogenous wild‐type gene in several cell types. The processing of APP was examined by Western blot analysis and immunoprecipitation. We observed distinctive patterns of APP metabolites among various cell lines. Neuronal and endothelial cells expressing mutant APP717F generated higher levels of large, potentially amyloidogenic carboxyl terminal fragments, which were enhanced upon treatment of the cells with leupeptin. These results suggest that mutations in the APP gene shift the protein processing towards the amyloidogenic pathway in neuronal and endothelial cells possibly involving the endosomal‐lysosomal system. © 1995 Wiley‐Liss, Inc.
Characterization of sialidase activity in mouse synaptic plasma membranes and its age‐related changes
Authors: Saito M. , Tanaka Y. , Tang C.‐P. , Yu R. K. , Ando S.
Published: January 2005

Sialidase activity in synaptic plasma membranes (SPM) isolated from C57BL/6 mouse brain was examined using exogenous ganglioside substrates. The enzyme activity directed toward GM3 showed sharp pH dependency with optimal pH of 4.0, and was greatly enhanced by Triton CF‐54, Nonidet P‐40 or CHAPS. The apparent Km and Vmax values for enzyme activity in SPM were 11 μM and 164 pmol/mg protein/min, respectively. Examination of sialidase activities in subcellular fractions of brain tissues showed the enrichment of enzyme activity in SPM prepared from either young adult or senescent mice. Substrate specificity of SPM sialidase was compared with that of myelin sialidase using delipidated, solubilized enzyme preparations. The SPM sialidase hydrolyzed GD1a more effectively as compared with the myelin enzyme. While SPM sialidase could hydrolyze GM1, the hydrolytic rate by the SPM enzyme was significantly lower than that by the myelin enzyme. The sialidase activity in SPM decreased with increasing age; activity was highest between the ages of 4–7 months, decreased to a relatively constant level between 13–25 months, and reached its lowest level at 31 months. These results demonstrate that SPM contain a distinct sialidase activity which is regulated in an age‐dependent manner. © 1995 Wiley‐Liss, Inc.
Endotoxin administration stimulates cerebral catecholamine release in freely moving rats as assessed by microdialysis
Authors: Lavicky J. , Dunn Adrian J.
Published: January 2005

In vivo microdialysis was used to measure changes in extracellular concentrations of catecholamines and indolamines in freely moving rats in response to administration of endotoxin (lipopolysaccharide, LPS). Dialysis probes were placed stereotaxically in either the medial hypothalamus or the medial prefrontal cortex. We used a repeated‐measures design in which each rat received LPS or saline, and each subject was retested with the other treatment one week later. With the dialysis probes in the medial hypothalamus, intraperitoneal (ip) administration of LPS (5 μg) increased dialysate concentrations of norepinephrine (NE, 187%), dopamine (DA, 119%), and all their measured catabolites, except normetanephrine. Dialysate concentrations of NE and DA were elevated significantly in the fourth or fifth (20 min) collection period with a peak response at around 2 hr. They returned to baseline by about 4 hr. When the dialysis probes were placed in the medial prefrontal cortex, the same dose of LPS also elevated dialysate concentrations of NE and DA, but the increases were much smaller (ca. 20%). However, a dose of 100 μg LPS increased dialysate concentrations of NE and DA from the medial prefrontal cortex to an extent comparable to that of the 5 μg dose in the hypothalamus, and the response was more prolonged. Dialysate concentrations of serotonin could not be measured reliably, but those of its catabolite, 5‐hydroxyindoleacetic acid (5‐HIAA), were also elevated in both regions. The peak of 5‐HIAA occurred at around 4 hr. Pretreatment of the rats with indomethacin (10 mg/kg ip) completely prevented the changes due to 100 μg LPS in the medial prefrontal cortex. These results support earlier neurochemical data suggesting that LPS stimulates the release of both DA and NE in the brain, and probably also release of serotonin. © 1995 Wiley‐Liss, Inc.
CNTF or (−)‐deprenyl in immature rats: Survival of axotomized facial motoneurons and weight loss
Authors: Zhang F. , Richardson P. M. , Holland D. P. , Guo Q. , Tatton William G.
Published: January 2005

The application of ciliary neurotrophic factor (CNTF) to the cut ends of transected facial nerves in newborn rats has been reported to reduce the death of facial motoneurons (FMns) axotomized by the transection. Systemically delivered CNTF has been found to cause cachexia in adult mice. We compared the influence of dosage of CNTF and (−)‐deprenyl on FMn death, weight loss, and animal survival in rat pups that underwent facial nerve transection at the 14th postnatal day (P14). CNTF was administered by osmotic mini‐pumps connected to tubing ending either intrathecally or extrathecally near the craniocer‐vical junction. CNTF caused weight loss and animal death that was similar to the cachexia reported in mice if administered in amounts of 1.1 μg/day or greater. At the same doses, intrathecal CNTF was more effective than extrathecal CNTF in inducing the cachexia. (−)‐Deprenyl did not alter animal survival or weight gain, even at high doses (10 mg/kg every 2 days). Intrathecal CNTF and intraperitoneal (−)‐deprenyl, but not extrathecal CNTF, significantly increased the survival of the axotomized FMns. (−)‐Deprenyl administered twice daily at 0.01 mg/kg was considerably more effective than CNTF in increasing FMn survival due to the limitation on CNTF dosage caused by the animal death. © 1995 Wiley‐Liss, Inc.
Bradykinin induces rise of free calcium in nuclei of neuroblastoma × glioma hybrid NG 108‐15 cells
Authors: Beckmann R. , Lindschau C. , Haller H. , Buchner Klaus
Published: January 2005

Confocal fluorescence microscopy was used to study the bradykinin‐induced calcium signals in the neuro‐blastoma × glioma cell line NG 108–15. We found that bradykinin induced a rise in free calcium, not only in the cytoplasm but also in the nucleus. The nuclear and cytosolic calcium concentrations were not significantly different and rose to about 1.2 :μM. The signal was mediated by the B2‐receptor subtype as confirmed using the specific antagonist Hoe 140. Both the onset and the intensity of the calcium signals were concentration‐dependent. The rise of nuclear calcium level was independent of extracellular calcium and suppressed by thapsigargin which is known to deplete inositol 1,4,5‐trisphosphate‐sensitive calcium stores. Bradykinin‐induced calcium increase desensitizes rapidly. This desensitization was shown not to involve activation of protein kinase C. © 1995 Wiley‐Liss, Inc.
Preparation of a monoclonal antibody to a glycidic epitope of the epidermal growth factor receptor that recognizes inhibitors of astrocyte proliferation and reactive microglia
Authors: Río C. , Pérez‐Cerdá F. , Matute C. , Nieto‐Sampedro M.
Published: January 2005

A mouse monoclonal antibody (5B9), directed against a carbohydrate epitope of human epidermal growth factor receptor (EGFR), recognized an 81‐kDalton glycoprotein in buffer‐soluble and detergent‐solubilized rat brain extracts (BE). The glycoprotein was more abundant in extracts prepared from injured brain than in those from normal tissue. Removal from BE of the antigens recognized by 5B9 increased their astrocyte mitogenic activity. Sections of injured rat brain and cultures derived from damaged brain, enriched in microglia, showed 5B9 immunoreactivity in ED1‐positive cells. The abundance of the glycoprotein recognized by 5B9 in injured, relative to normal, tissue, suggested that molecules with EGFR immunoreactivity may be expressed in reactive microglial cells and released after injury. © 1995 Wiley‐Liss, Inc.