bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective expression of foreign genes in glioma cells: Use of the mouse myelin basic protein gene promoter to direct toxic gene expression
Authors: Miyao Y. , Shimizu K. , Moriuchi S. , Yamada M. , Nakahira K. , Nakajima K. , Nakao J. , Kuriyama S. , Tsujii T. , Mikoshiba K. , Hayakawa T. , Ikenaka K.
Published: January 2005

We have previously demonstrated that retrovirusmediated genes were transferred to mouse glioma cells in a meningeal gliomatosis model (Yamada et al.: Japanese Journal of Cancer Research 83:1244–1247, 1992). This retrovirus vector contains the Escherichia coli. β‐galactosidase (β‐gal) gene as a marker for intergration of the lacZ gene, which is controlled by the SV40 early promoter. We investigated whether lacZ genes could be specifically controlled in mouse glioma cells by glial‐specific promoters, including the 2.5 kb 5′ flanking region of the mouse glial fibrillary acidic protein (GFAP) gene, the 1.3 kb 5′ flanking region of the myelin basic protein (MBP) gene, and the 1.5 kb 5′ flanking region of the myelin proteolipid protein (PLP) gene. Psi‐2 packaging cells were transfected with each retrovirus vector (GFAP promoter‐, MBP promoter‐, and PLP promoter‐lacZ) and the infectious virus particles were recovered from the supernatants. Blue staining for β‐gal was detected in various fibroblast, myeloma, and glioma cell lines transduced with the retrovirus BAAG vector. On the other hand, blue staining was only detected in glioma cells after transduction with the lacZ gene‐bearing retrovirus controlled by glial‐specific promoters. The strongest promoter activity was detected after transduction with the retrovirus in which the MBP promoter controlled the lacZ gene. Mouse glioma cells transduced with retrovirus containing the MBP promoter directing the herpes simplex virus type 1 thymidine kinase (HTK) gene wre extremely sensitive to ganciclovir, while the parental cells and cells transduced with retrovirus containing the lacZ gene were not sensitive to ganciclovir. Furthermore, none of the nonglioma cells tested was sensitive to ganciclovir, even when transduced with the MBP promoter‐HTK gene‐containing retrovirus. In conclusion, retrovirus targeted gene therapy of malignant glioma may be applicable with a low risk of side effects such as bone marrow suppression, in constrast to chemotherapy or the use of retroviruses bearing toxic genes controlled by nonspecific promoters. © 1993 Wiley‐Liss, Inc.
Cranin interacts specifically with the sulfatide‐binding domain of laminin
Authors: Smalheiser N. R.
Published: January 2005

Cranin is a 120 kDa integral membrane glycoprotein which binds laminin under conditions of physiologic ionic strength in a calcium‐dependent manner. Here, binding of cranin to laminin has been characterized using both ligand‐blotting assays and laminin affinity bead assays. Binding was specifically inhibited by anti‐laminin antibodies against the A chain terminal domain G, but not by several other region‐specific antibodies. Dextran sulfate, fucoidin, and sulfatide were potent inhibitors of binding (50% inhibition at 0.03, 0.5, and 1.7 μg/ml, respectively); heparin was a weaker inhibitor (50% inhibition ∼5 μg/ml), and mannan and chondroitin sulfate were not inhibitory at 100 μ/ml. Binding was not inhibited by lactose or the A chain peptide PA22‐2. The mobility of the broad, fuzzy cranin band was shifted after digestion with neuraminidase, N‐glycanase, and O‐glycanase, though none of these treatments decreased band heterogeneity nor destroyed the ability to bind laminin. Cranin bound to Jacalin lectin, which recognizes the Galβ1‐3GalNAc linkage expressed in certain classes of mucins. These findings indicate that cranin binds at or near the high affinity sulfatide‐binding site previously mapped to the E3 domain of laminin, which is known to exhibit bioactivity for neural cells. In view of the extremely low abundance of cranin in brain membranes (∼0.005%), its avid laminin‐binding activity is remarkable, and strongly suggests that cranin may play a physiologic role in regulating specific neural cell interactions. © 1993 Wiley‐Liss, Inc.
K252a potentiates epidermal growth factor‐induced differentiation of PC12 cells
Authors: Wu C.‐F. , Zhang M. , Howard B. D.
Published: January 2005

Epidermal growth factor (EFG) induced short neurites in two different strains of PC12 cells. The length of the EGF‐induced neurites was markedly increased in the presence of the protein kinase inhibitor K252a, which is known to inhibit differentiation induced by nerve growth factor (NGF). EGF‐induced differentiation of PC12 required RNA synthesis and activity of the ras proto‐oncogene product. EGF increased the levels of three neurofilament proteins and the mRNA level of two late response genes (SCG10 and 63) known to be induced by NGF. Together, EGF and K252a caused a greater increase in these mRNAs than did either agent alone. K252a did not alter the extent of EGF‐induced autophosphorylation of the EGF receptor, but it did decrease the extent of receptor phosphorylation in the absence of added EGF. Thus, the ability of the EGF receptor to trigger neuronal differentiation may depend on the state of its phosphorylation at serine and/or threonine residues. Two other strains of PC12 did not extend neurites when exposed to EGF, even when K252a was also present. Thus the differentiating effect of EGF on PC12 is PC12 strain‐specific. © 1993 Wiley‐Liss, Inc.
Immunolocalization of proteolipid protein peptide 103–116 in myelin
Authors: Sobel R. A. , Greer J. M. , Isaac J. , Fondren G. , Lees M. B.
Published: January 2005

Determination of the topographic orientation of proteolipid protein (PLP) within myelin is part of an overall understanding of the functions of PLP and the roles of its multiple domains in diseases that primarily affect central nervous system (CNS) myelin. As part of an analysis of PLP orientation, two mouse monoclonal antibodies (mAb) and a rabbit antiserum against a synthetic peptide corresponding to PLP residues 103–116 (YKTTICGKGLSATV) were tested for their reactivity on compact CNS myelin. By ELISA, the antibodies react with intact PLP and PLP residues 103–116, but not with other PLP peptides. Ultrathin cryosections of adult rat optic nerve were immunostained and antibody binding was localized using appropriate second antibodies coupled to 1 nm gold particles that were visualized by silver enhancement. Localization of the particles on the major or intermediate dense lines was determined by three in dependent observers. Using the PLP peptide mAb and the polyclonal antibody, we demonstrated that ≥71% of the particles were localized on the major dense line. At least 66% of particles directed against myelin basic protein, which is known to occur on the major dense line, were also found in that location. These semiquantitative morphologic observations suggest that PLP residues 103–116 occur on the cytoplasmic face of the myelin membrane. © 1994 Wiley‐Liss, Inc.
Postnatal decrease of acetate concentration in rat cerebellum
Authors: Martin M. , Merele M. , Labouesse J. , Canioni P.
Published: January 2005

We examined the evolution of 1H‐NMR detectable metabolites in rat cerebellum fro the postnatal day 1–25, a period associated with intense metabolic changes as well as physiological and morphological modifications. The unexpected result reported here is the existence of a high concentration of acetate in neonatal rat cerebellum, progressively decreasing with age and maturation. In contrast, the cerebellum content of various metabolites such as N‐acetyl‐L‐as‐partate (NAA), glutamate, and aspartate increases from the first day onward. © 1994 Wiley‐Liss, Inc.
In vitro and in vivo characterisation of glial cells immortalised with a temperature sensitive SV40 T antigen‐containing retrovirus
Authors: Jung M. , Crang A. J. , Blackemore W. F. , Hoppe D. , Kettenmann H. , Trotter J.
Published: January 2005

An oncogene‐carrying repiication‐defective retrovirus was used to establish immortalised lines of murine glial cells. Primary cultures of early postnatal cerebellar cells were infected with a retrovirus based on the Murine Moloney Leukemia Virus containing a temperature‐sensitive mutant of the Simian Virus 40 large T antigen (SV40 T) oncogene and a gene coding for resistance to the antibiotic G418. Infected cells were selected in G418 and after several in vitro passages cells expressing the O4 antigen were established as a cell line. At a later time point O4‐positive single‐cell clones were established. Two different types of clones were obtained: (1) “plastic” clones consisting of cells which initially had a morphological and antigenic phenotype of young glial precursor cells but which gradually lost these features, and (2) “stable” cell clones including a clone with the immunological and electrophysiological characteristics of Schwann cells. Culture of the latter cells in the presence of 1 mM dibutyryl cyclic adenosine monophosphate for a period of at least 10 days induced a change in shape and a shift in antigen expression towards a more “diferentiated” maturation stage. When the SV40 T O4‐positive immortalised cell line isolated on the cell sorter was transplanted into demyelinated lesions in adult rats, cells were observed ensheathing axons and forming limited amounts of PNS‐type myelin.

Glial cells immortalised with a temperature‐sensitive mutant of the SV40 T oncogene thus retain many physiological properties of their primary culture counterparts and can be induced to undergo limited differentiation in vitro and in vivo. These cell lines, which represent immature CNS glia or Schwann cells, are providing useful tools for investigating the role of cell surface antigens involved in neuron‐glial interactions. © 1994 Wiley‐Liss, Inc.
Cholinergic markers are expressed in developing and mature neurons of chick dorsal root ganglia
Authors: Tata A. M. , Plateroti M. , Cibati M. , Biagioni S. , Augusti‐Tocco G.
Published: January 2005

The presence of acetylcholinesterase has been reported in chick dorsal root ganglia at early developmental stages although acetylcholine is not known to play a role in these ganglia. Recently, we reported that during development the level of acetylcholinesterase increases continuously and the enzyme becomes gradually expressed in all sensory neurons. These observations prompted the study of the developmental pattern of expression of other cholinergic markers, such as choline acetyltransferase (ChAT) and the high affinity transport mechanism for choline. ChAT activity is barely detectable at early developmental stages (E7) and increases markedly thereafter, with an activity profile similar to that described for acetylcholinesterase. A similar increase in enzyme activity is also observed when ChAT is measured in dorsal root ganglia explants and in dissociated cells in culture. The study of ChAT activity in cultured cells shows an increase over a period of 3 days, thus ruling out the hypothesis that motor fibers, still associated to the ganglia, may represent a possible source of the enzyme. Immunostaining of whole ganglia or cultured cells shows that ChAT immunoreactivity is not restricted to a specific neuronal subpopulation but appears as a common marker of sensory neurons. High affinity choline uptake, blocked by hemicholinium, is present in sensory neurons cultured from E7 dorsal root ganglia. Observations on cultured neurons from later stages (E18) indicate that choline transport is not a transient property of sensory neurons. These observations show a similar pattern of expression of several cholinergic markers during development. Such a pattern is maintained at significant levels also in mature ganglia. © 1994 Wiley‐Liss, Inc.
Induction of glioma cell death by 1,25 (OH)2 vitamin D3: Towards an endocrine therapy of brain tumors?
Authors: Naveilhan P. , Berger F. , Haddad K. , Barbot N. , Benabid A.‐L. , Brachet P. , Wion D.
Published: January 2005

The secosteroid 1,25‐dihydroxyvitamin D3 (1,25 (OH)D2DD3 is the major biologically active metabolite of vitamin D. Antitumor activity of this hormone has been observed on several cell lines and on breast cancer in vivo. The purpose of this in vitro study was to determine the possible effect of 1,25(OH)2D3 on glioma cells. Two glioma cell lines from rat (C6) or human (GHD) origin were cultured in the presence of 1,25(OH)2D3. The sensitivity of these cells to 1,259OH)2D3 was assessed with a colorimetric MTT assay. A cytotoxic effect of 1,25(OH)2D3 was detected at concentrations around 10−8 M. A lag period of 3 days was required between the onset of the treatment and the observation of the effects. However, the continuous presence of 1,25(OH)2D3 is not required since cell death occurred even when C6 cells were challenged for 24 hr with 1,25(OH)2D3and then cultured in the absence of the hormone. In addition, 1,25(OH)D2D3 regulates the expression of its own recepors in C6 glioma. These results provide to our knowledge the first evidence for a cytotoxic effect of 1,25(OH)2D3 on rat and human glioma cells and could offer both an experimental model to study a programmed cell death in a brain‐derived cell line and a new strategy for the inhibition of glioma growth in vivo. © 1994 Wiley‐Liss, Inc.
Increased CNTF gene expression in process‐bearing astrocytes following injury is augmented by R(−)‐deprenyl
Authors: Seniuk N. A. , Henderson J. T. , Tatton W. G. , Roder J. C.
Published: January 2005

R(−)‐deprenyl has been shown to rescue axotomixed immature facial motoneurons with an efficacy comparable to that of the neurotrophic factors CNTF and BDNF (Salo and Tatton, J Neurosci Res 31:394–400, 1992; Ansari et al., J Neurosci 13:4042–4053, 1393). Recent work has suggested that some of the actions of (−)‐deprenyl may be mediated through reactive astrocytes (Biagini et al., NeuroReport 4:955–958, 1993). To test this proposal we have developed an in vitro model of reactive gloisis consisting of a mixed astrocyte population of flat and process‐bearing (PB) astroglia taken from postnatal day (PD) 2 or PD5 rat cerebral cortex. After mechanical wounding, PB astrocytes preferentially migrate into the wound zone while flat astrocytes maintain their position at the wound edge. CNTF mRNA was localized to PB astrocytes, but not flat astrocytes, as determined by in situ hybridization using biotin‐labelled riboprobes. Following “wounding,” there was an increase in CNTF mRNA in PB astrocytes only, which could be further enhanced by a single pulse of (−)‐deprenyl (10−8−10−11 M) 48 hr after injury. (−)‐Deprenyl also increased the total process length of PB astrocytes after wounding by an average of 50%. The stereoisomer (+)‐deprenyl (10−9 M) had no effect on either astrocyte process length or CNTF mRNA content. This is the first report to our knowledge of an agent which can upregulate CNTF gene expression in astroglial cell culture as well as influence glial cell process length. We propose that some of the trophic‐like action of (−)‐deprenyl may be mediated through a specific subpopulation of astroglia. © Wiley‐Liss, Inc.
Cellular distribution of calbindin D28K mRNAs in the adult mouse brain
Authors: Frantz G. D. , Tobin A. J.
Published: January 2005

We have determined the cellular distribution of calbindin D28K mRNAs throughout the mouse brain by in situ hybridization. While these studies identified neuronal populations similar to those previously identified in rat brain by immunohistochemistry, some discrepanicies exist. These may derive from species differences or from the immunological cross‐reactivity of calbindin D28K antiserum with other proteins. We note an intriguing association between the distribution of neurons containing calbindin D28K mRNA and those reported by others to contain the inositol 1,4,5‐triphosphate (InsP3) receptor. © 1994 Wiley‐Liss, Inc.
Protein synthesis and mRNA in isolated growth cones from differentiating SH‐SY5Y neuroblastoma cells
Authors: Meyerson G. , Parrow V. , Gestblom C. , Johansson I. , Påhlman S.
Published: January 2005

The human neuroblastoma cell line, SH‐SY5Y, differentiates into a neuronal, sympathetic phenotype in the presence of phorbol ester and serum. Growth cones prepared from differentiating SH‐SY5Y cells have characteristics similar to those of growth cones from embryonic rat brain. In addition, SH‐SY5Y growth cones contain ribosomes. In this study we show, by metabolic labeling of isolated growth cones, that local protein syntheisis occurred in these structures. The pattern of labeled proteins was very similar to that of the corresponding cell body fraction. RNA was shown to be transported to the growth cone compartment, and by in situ hybridization. β‐actin mRNA could be visualized in intact neuritic growth cones. Comparison by Northern blot hybridizations of RNA prepared from growth cones and cell bodies, respectively, showed that mRNAs coding for growth‐associated protein 43, microtubule‐associated protein 2, actin, neuropeptide tyrosine, and glyceraldehyde‐3‐phosphate dehydrogenase were present in both fractions. In contrast, mRNAs coding for the nuclear proteins c‐jun and N‐myc were virtually absent in the growth cone, but readily detectable in the cell body preparation. The selective distribution of mRNAs to the growth cones was not restricted to stable, abundant mRNA species, since mRNA coding for the insulin‐like growth factor I receptor was stable, but not present in growth cones. Thus, differentiating SH‐SY5Y cells can sort and transport RNA to the growth cone compartment, suggesting that this system of clonal cells could be useful to unravel mechanisms involved in the compartmentalization of mRNA. © 1994 Wiley‐Liss, Inc.
Tenascin mRNA isoforms in the developing mouse brain
Authors: Dörries U. , Schachner M.
Published: January 2005

The extracellular matrix glycoprotein tenascin is expressed in the developing mouse cerebellum as a group of four protein species of different molecular weights. The difference is most likely due to alternative splicing which is known to occurr in tenascin mRNA isoforms that would account for this heterogeneity, tenascin splice variants were isolated from mouse brain by the polymerase chain reaction (PCR). In agreement with Northern blot analysis, amplification by PCR revealed a general decrease in tenascin mRNA expression during development from embryonic and early postnatal to adult stages. This decrease was more pronounced for isoforms of high molecular weight compared to those of low molecular weight. In accord with the observations at the protein level, four splice variants were found to be predominantly expressed, containing insertions of either six, five, or one fibronection type III repeat, or comprising no insertion. In addition, a minor splice variant with an insertion of four fibronectin type III repeats was isolated. Three of the isolated mRNA splice variants have not yet been described for mouse tenascin. Among them, an isoform containing six alternatively spliced repeats was found to include a novel fibronectin type III repeat. The sequence of this repeat displays 96.7% similarity to a corresponding type III repeat in human tenascin, revealing a strict evolutionary conservation between tenascin molecules from different species in the region of alternative splicing. Southern blot analysis of the amplified mRNA isoforms showed that the novel mouse type III repeat is confined to splice variants with an insertion of six fibronectin type III repeats. Furthermore, in situ hybridization on sections from mouse embryos indicated that tenascin‐specific mRNAs containing the novel type III repeat are predominantly expressed in the central nervous system. © 1994 Wiley‐Liss, Inc.
In vitro differentiation of E‐N‐CAM expressing rat neural precursor cells isolated by FACS during prenatal development
Authors: Blass‐Kampmann S. , Reinhardt‐Maelicke S. , Kindler‐Röhrborn A. , Cleeves V. , Rajewsky M. F.
Published: January 2005

Most fetal rat brain cells expressing the embryonal, highly sialyated form of the cell adhesion molecule N‐CAM (E‐N‐CAM) are precursor cells, as jugded from the absence of marker molecules specific for mature neural cell types. However, the detection of E‐N‐CAM+ cells in frozen sections does not provide information on the lineage‐specific differentiation of these cells during development. To investigate their differentiation behaviour in vitro, E‐N‐CAM+ cells were isolated at different times of brain development by fluorescence‐activated cell sorting (FACS), using a monoclonal antibody (Mab RB21‐7) which specifically recognizes polysialic acid (PSA) residues on E‐N‐CAM. Double‐immunofluorescence analyses showed that ht emajority of E‐N‐CAM+ cells isolated on prenatal days 15 to 18 differentiated into neurons while a small subset of Mab RB21‐7 binding cells proved to be astrocytic precursors and/or bipotential. The proportion of E‐N‐CAM+ astrocytic precursors increased during later development (prenatal day 22) concomitantly with the onset of gliogenesis. While conversion of E‐N‐CAM to mature forms of N‐CAM was never observed in neurons during cultivation, E‐N‐CAM+ cells of the astrocyte lineage switched to N‐CAM soon after the onset of GFAP expression. A lineage‐specific transition of E‐N‐CAM to mature N‐CAM expression is, therefore, suggested for these astrocytic progenitor cells during rat brain development. © 1994 Wiley‐Liss, Inc.
In vitro modulation of changes in ganglioside patterns of differentiating neurons in the presence of an anti‐GM1 antibody
Authors: Allende M. L. , Panzetta P.
Published: January 2005

Retinal cells from 7‐day‐old chicken embryos were cultured in the presence of a polyclonal anti‐GMI antibody, at low and high density in a “sandwich cell culture”. Cells that were about 80% neurofilament positive at all times, changed their morphology and emitted processes as controls. By examining immunocytochemical expression of gangliosides, cells cultured in the presence of the antibody maintained GD3 expression longer than controls, albeit the expression of the gangliotetraosylgangliosides (GTOG) was not considerably affected. This leads to an extension of the transient period in which differentiating cells coexpressed both types of gangliosides (GD3 and GTOG). At 3–4 days in vitro the relative synthesis of GD3 was about 30% higher and that of GD1a about 40% lower than in controls, indicating a delay in the shift of the synthesis pattern. Nevertheless, the pattern of ganglioside composition resembled at 4 days in vitro.

Results indicate that the anti‐GMI antibody may modulate the expression and synthesis of gangliosides without a detectable decrease in neuritogenesis. Considering that the emission of neurites occurs in coexpressing GD3 and GTOG neurons, it is suggested that neuritogenesis could be irrespective of losing the GD3 expression.
Many naturally occurring mutations of myelin proteolipid protein impair its intracellular transport
Authors: Gow A. , Friedrich V. L. , Lazzarini R. A.
Published: January 2005

The primary structure of the proteolipid protein (PLP) from the central nervous system (CNS) myelin of mammals is highly conserved with only three amino acid differences between the mouse, rat, dog, bovine, and human proteins. Furthermore, within a particular species no polymorphisms in the protein have been identified. Recent interest has focused on the targeting of PLP in oligodendrocytes and the role that mutant forms of this protein play in generating dysmyelinating or hypomyelinating diseases. We previously expressed the human cDNA encoding PLP in transiently transfected Cos‐7 cells and characterized the subcellular distribution of the protein in this simple heterologous system. In the current study we have used the same paradigm to examine the effect of five missense mutations in the PLP gene on processing of the encoded protein. The mutations chosen span the carboxy‐terminal half of PLP and encompass that part of the protein in which most mutations have been identified. Our results show that transport of all mutations examined was arrested in the secretory pathway at an early stage, causing the mutant proteins to accumulate in the endoplasmic reticulum. Thus, a common mechanism of protein misfolding and failure of PLP to reach the cell surface of oligodendrocytes rather than the inability of the mutant protein to perform some crucial function at the cell surface may be responsible for the diseases caused by many PLP mutations. Our results, together with those of others, prompt us to speculate that the pathobiology observed in PLP mutants may result from oligodendrocyte cell death caused by the accumulation of misfolded protein in the endoplasmic reticulum. This speculation is consistent with the observations that oligodendrocytes bearing misfolded PLP, as in the jimpy mutant, proliferate but die rapidly while oligodendrocytes from PLP deletion survive and produce a myelin‐like membrane which lacks PLP. © 1994 Wiley‐Liss, Inc.
Effects of nerve segment supernatants on cultured Schwann cell proliferation and laminin production
Authors: Zhang Q.‐L. , Lin P.‐X. , Chang Y. , Webster H. deF.
Published: January 2005

Mouse sciatic nerves were transected and 3 hr to 16 days later proximal segments were removed and homogenized. Supernatants of these segments or of normal sciatic nerves were added to Schwann cells maintained in Dulbecco's modified Eagle's medium (DMEM) + 15% fetal calf serum (FCS). After 6 days, Schwann cells were solubilized and the protein content was measured using a Bio‐Rad (Melville, NY) protein assay. Samples containing the same amounts of protein were then applied to microtiter plates and the laminin content was determined by enzymelinked immunosorbent assay (ELISA). Lysates of cultures treated with 24 hr proximal segment supernatants contained significantly higher levels of laminin than those prepared from other intervals, from distal segments, or from control nerves. Increased surface and cytoplasmic anti‐laminin immunoreactivity also was found in Schwann cells treated with 24 hr supernatants. To identify the source(s) of this effect, proximal segments removed 24 hr after transection were bisected; supernatants were prepared from each half and tested. Significant increases in laminin production were produced by supernatants from both halves. When supernatants from proximal and distal halves were compared, the latter produced significantly higher laminin levels. Electron microscopic examination of both halves showed that distal halves contained sprouting neurites and growth cones ensheathed by Schwann cells which had a basal lamina and resembled those seen during development and regeneration. Proximal halves appeared normal. Schwann cell proliferation also was compared in supernatant‐treated cultures by using a bromodeoxyuridine (BrdU) ELISA. The 24 hr and 2 day supernatants increased Schwann cell proliferation significantly; 12 hr, 4 day, and 8 day supernatants produced smaller increases. Our observations suggest that axons undergoing early regenerative changes are one of several possible sources of substance(s) in our proximal segment supernatants which increased Schwann cell proliferation and laminin production. © 1994 Wiley‐Liss, Inc.
In vivo acidosis reduces extracellular concentrations of taurine and glutamate in the rat hippocampus
Authors: Puka M. , Lehmann A.
Published: January 2005

Microdialysis‐perfusion of the urethane‐anesthetized rat hippocampus was performed to assess the effects of acidosis on extracellular amino acids. Perfusion with Krebs‐Ringer bicarbonate buffer at pH 6.9 produced a selective decrease in taurine. A further reduction of pH to 6.4 induced diminished glutamate levels. The CI−/HCO3− exchange inhibitor 4,4′‐diisothiocyano‐2,2′‐disulfonic acid stilbene (DIDS) did not affect interstitial taurine during perfusion, but there was a rebound increase in taurine levels upon withdrawal of the agent. In contrast, glutamate concentrations were elevated during DIDS administration, and decreased upon reperfusion with standard buffer. The reduction of extracellular taurine and glutamate concentrations caused by low pH was inhibited by DIDS. The results suggest that taurine and glutamate uptake and/or release in vivo is pH‐dependent, and that the effects of acidosis possibly are mediated by the CI−/HCO3− antiporter. The decrease in extracellular glutamate brought about by low pH may have pathophysiologic implications in conditions associated with disturbed pH homeostasis such as cerebral ischemia and spreading depression. © 1994 Wiley‐Liss, Inc.
Transcriptional regulation of myelin associated glycoprotein gene expression by cyclic AMP
Authors: Ye P. , Laszkiewicz I. , Wiggins R. C. , Konat G. W.
Published: January 2005

The treatment of rat glioma C6 cells with 10μ isoproterenol (Ipt) for 4 days upregulated the expression of the myelin‐associated glycoprotein (MAG) gene by approximately 55‐fold over the control value. The constant presence of Ipt in the medium was required for the maximal upregulation, as time‐restricted exposures to the drug produced only partial, or no upregulation of the gene. No difference in the MAG mRNA stability could be detected in Ipt‐treated vs untreated cells indicating that the drug upregulates the MAG gene at transcriptional level. Serum (FCS) strongly attenuated the response of the MAG gene to Ipt.

The stimulatory effect of Ipt was profoundly reduced by supermine and H‐89, indicating that protein kinase A‐dependent protein phosphorylation is involved in the MAG gene activation. Within 30 min after Ipt administration, the c‐fos gene was upregulated by 10‐fold, and thereafter, its message level decreased and stabilized at approximately 3‐fold over control. In contrast, the c‐jun gene was downregulated to approximately 20% of control within 30 min after Ipt administration. Subsequently, its message level rose and fell once again within 12 h to approximately half of control, and returned to control level within 72 h.

Transfection of C6 cells with progressively deleted pCAT constructs containing up to 861 bp of the MAG gene upstream region demonstrated that active core promoter of the gene is located within 138 bp upstream from the initiation start site. Additional sequences contain both activating and repressive cis‐elements. Practically no upregulation of the CAT activity could be elicited by challenging the transfected cells with Ipt, indicating that AP‐1 sites (14 within the 861 bp region) are not solely responsible for the Ipt‐induced upregulation of the MAG gene, and that the responsible cis‐elements map further upstream from the transcriptional unit. Wiley‐Liss, Inc.
Properties of osmolyte fluxes activated during regulatory volume decrease in cultured cerebellar granule neurons
Authors: Pasantes‐Morales H. , Chacón E. , Murray R. A. , Morán J.
Published: January 2005

Efflux pathways for amino acids, K, and CI activated during regulatory volume decrease (RVD) were characterized in cultured cerebellar granule neurons exposed to hyposmotic conditions. Results of this study favor diffusion pores (presumably channels) over energy‐dependent transporters as the mechanisms responsible for the efflux of these osmolytes. The selectivity of osmolyte pathways activated by RVD was assessed by increasing the extracellular concentrations of cations, anions, and amino acids to such an extent that upon opening of the pathway, a permeable compound will enter the cell and block RVD by reducing the efflux of water carried by the exit of intracellular osmolytes. The cationic pathway was found selective for K (and Rb), whereas the anionic pathway was rather unselective being permeable to CI, nitrate, iodine, benzoate, thiocyanate, and sulfate but impermeable to gluconate. Glutamate and aspartate as K but not as Na salts were permeable through the anion channel. RVD was slightly inhibited by quinidine but otherwise was insensitive to known K channel blockers. RVD was inhibited by 4,4′‐ diisothiocyanostilbene‐2‐2′‐disulfonic acid (DIDS), niflumic acid, and dipridamole. Gramicidin did not affect cell volume in isosmotic conditions but greatly accelerated RVD, suggesting that cell permeability to CI is low in isosmotic conditions but increase markedly during RVD making K permeability the rate limit of the process. The permeability pathway for amino acids activated during RVD was permeable to short chain α‐ and β‐amino acids, but excluded glutamine and basic amino acids. Wiley‐Liss, Inc.
Met5‐enkephalin is localized within axon terminals in the subfornical organ: Vascular contacts and interactions with neurons containing gamma‐aminobutyric acid
Authors: Pickel V. M. , Chan J.
Published: January 2005

Met5‐enkephalin inhibits sodium and water excretion and antagonzes the central actions of angiotensin II in subfornical organ of rat brain. We examined the ultrastructural basis for enkephalin modulation in this circumventricular region. Additionally, we examined the possibility that there might be cellular sites for functional interactions involving Met5‐enkephalin and gamma‐aminobutyric acid (GABA), a known inhibitory transmitter throughout the central nervous system. Met5‐enkephalin and GABA were indentified in single coronal sections through the subfornical organ using immunoperoxidase and silver‐enhanced immunogold labeling methods, repectively, Enkephalin‐like immunoreactivity was most prominently localized within axon terminals. These were distributed primarily in the central, highly vascular, regions of the subfornical organ. Enkephalinlabeled terminals were apposed to the basement membranes of fenestrated capillaries and also formed symmetric, inhibitory type synapses with neurons. In terminals associated with either blood vessels or neurons, the enkephalin immunoreactivity was enriched in large (80–150nm) dense core vesicles. The immunoreactive vesicles were usually located within portions of the axon in close proximity to astrocytic processes. In contrast, smaller vesicles in the same terminals were more often aggregated near the basement membrane of the capillaries and the active zone of the synape. The targets of enkephalin‐immunoreactive terminals were either unlabeled or GABA‐labeled dendrites of local neurons. Enkephalin was also co‐localized with GABA in perikarya and in axons terminals. Terminals containing only GABA were far more abundant than those containing enkephalin or enkephalin and GABA. GABA‐immunoreactive terminals formed symmetric synapses on unlabeled dendrites some of which also received convergent input from terminals containing enkephalin. Additionally, the enkephalin‐immunoreactive terminals were closely apposed to GABA‐labeled and unlabedled terminals. These results suggest sites for nonsynaptic release of Met5‐enkephalin from dense core vesicles in contact with astrocyes near blood vessels and synaptic complexes in the rat subfornical organ. Moreover, the observed dual localization and pre‐and postsynaptic associations between neurons containing Met5‐enkephalin and GABA indicate that inhibitory effects of opioids in the subfornical organ may be mediated or potentiated by GABA. Wiley‐Liss, Inc.
Isolation and characterization of a soluble lactose‐binding lectin from postnatal chicken retina
Authors: Castagna L. F. , Landa C. A.
Published: January 2005

We investigated the presence of endogenous lectins in postnatal chicken retinal tissue assaying the hemagglutinating activity of crude soluble extracts of the tissue that was homogenized in a buffer supplemented with different sugars. Lactose was the most effective sugar to extract an hemagglutinating activity. Using similar extraction conditions, other sugars, such as glucose, N‐acetylglucosamine, mannose, fucose, glucuronic and sialic acid, were ineffective to extract any significant hemagglutinating activity. The lectin was purified by affinity chromatography on lactosyl‐Sepharose. SDS‐PAGE and isoelectric focusing analyses showed that it has a subunit molecular weight of 16 kDa and a pl about 4.5. The retinal lectin crossreacted immunologically with a rabbit antiserum raised against a lectin purified from adult chicken liver, which is a CLL‐I (Beyer et al.: J Biol Chem 255:4236–4239, 1980) or C‐16 (Sakakura et al.: J Biol Chem 265:21573–21579, 1990) form of chicken endogenous soluble lactose‐binding lectins. Gel filtration studies showed that the oligomeric structure of the retinal lectin is dependent on the ionic strength of the elution buffer. The lectin hemagglutinating activity and the amount of lectin protein reached their highest levels at late developmental stages of the retinal tissue, suggesting that retinal lectin might have a functional role during terminal differentiation of retinal cells. Wiley‐Liss, Inc.
Abnormal phosphorylation of tau proteins associated with bovine brain microtubules: Activation by excess ATP and tyrosine dephosphorylation
Authors: Lu Q. , Kanumury R. , Wood J. G.
Published: January 2005

Phosphorylation of tau protein has been suggested as a major mechanism regulating its functions. In assembled brain microtubules, tau is phosphorylated, but additional phosphorylation can be induced in vitro. Supply of excess ATP alone was sufficient to reduce migration of tau on SDS gels, diminish Tau‐1 immunostaining, and induce the expression of epitopes recognized by the PHF‐1 antibody, suggesting that Alzheimer‐type phosphorylation may have occurred. Okadaic acid had no further effect. However, treatment with tyrosine kinase inhibitors modifies the phosphorylation profiles of tau proteins. Most evidently, migration of the largest tau isoform was further retarded on SDS gels and PHF‐1 immunostaining was enhanced. The profound effect of tyrosine kinase inhibitors on tau phosphorylation was also demonstrated in living cells following microinjection of cultured hippocampal neurons. Identification of proline‐directed protein kinases and their regulatory factors associated with assembled microtubules indicated the presence of multiple phosphorylation pathways in microtubules. Our results are consistent with the hypothesis that sequential phosphorylation of tau proteins is at least partially mediated through tyrosine phosphorylation/dephosphorylation mechanisms. Wiley‐Liss, Inc.
Heat shock alters Alzheimer's β amyloid precursor protein expression in human endothelial cells
Authors: Ciallella J. R. , Rangnekar V. V. , McGillis J. P.
Published: January 2005

One of the pathological lesions in Alzheimer's disease (AD) is the amyloid or senile plaque. The plaque core is predominantly made up of amyloid beta peptide (Aβ), a 42–43 amino acid peptide derived from amyloid precursor protein (APP). APP is a membrane bound glycoprotein which is expressed ubiquitously is many cells. Although normal or pathological functions for APP are not well understood, several observations suggest that APP may play a role in cellular stress and inflammation at the endothelial cell/vascular barrier. APP is found in platelets and endothelial cells, it can inhibit a blood coagulation factor, and secreted APP can be neuroprotective. Changes in expression of APP during cellular stress or inflammation may contribute to pathological deposition of Aβ. In the present studies, expression of APP in human endothelial cells was examined following heat shock. In human umbilical vein endothelial cells (HUVECs) exposed to 42°C for 30 min, there was a five‐to eight‐fold increase in APP mRNA levels which peaked at 4 hr. The increase in APP mRNA was followed by an increase in APP protein immunoreactivity in the cytoplasm in a perinuclear Golgi‐like region, and in discrete granular cytoplasmic structures. Immunoblot analysis of APP in the cell media found a transient increase in APP which peaked at 1 hr after heat shock. These results suggest that cellular stress induces the secretion of APP from endothelial cells followed by a subsequent increase in APP mRNA and protein synthesis. The upregulation of APP mRNA and protein supports a cellular stress role for APP. Wiley‐Liss, Inc.
Tacrine alters the secretion of the beta‐amyloid precursor protein in cell lines
Authors: Lahiri D. K. , Lewis S. , Farlow M. R.
Published: January 2005

The characteristic features of Alzheimer's disease (AD) include a high density of β‐amyloid‐containing plaques in the cerebral cortex and the loss of basal forebrain cholinergic neurons. Amyloid β‐protein (Aβ, Mr. ∼4.5 kDa) is derived from a family of large (Mr. ∼110–140 kDa) β‐amyloid precursor proteins (APP) which are integral membrane glycoproteins consisting of a large extracytoplasmic domain, a transmembrane domain, and a short cytoplasmic tail. Secreted derivatives of APP lacking the cytoplasmic tail, transmembrane domain, and a small portion of the extracellular domain are generated by the proteolytic processing of full length APP by a family of proteolytic enzymes known as APP secretases. Using cell cultures, we investigated the possibility that APP processing can be regulated by a centrally active cholinesterase inhibitor, tacrine, which has recently been shown to improve memory and cognitive functions in patients with AD. We analyzed the level of APP in glial, fibroblast, pheochromocytoma (PC12), and neuroblastoma cells by immunoblotting cell lysates and conditioned media. Normal levels of secretion of soluble APP derivatives by cells into conditioned media were severely inhibited by treating cells with tacrine. A similar decrease after treatment with tacrine was observed when neuroblastoma and PC12 cells were pretreated with either growth factors, phorbol ester, or retinoic acid. To determine whether the effect of tacrine on APP levels was specific or a more general phenomenon affecting other proteins, we measured the level of heat shock protein‐70 (HSP‐70) and another secretory protein, protease nexin‐1 (PN‐1). Tacrine treatment did not alter the level of HSP‐70 in cell extracts and tacrine affected mildly the secretion of PN‐1. Thus, the processing of HSP and PN‐1, unlike APP, was not severely affected by treating cells with tacrine. Our results suggest that tacrine may inhibit an acetylcholinesterase‐associated proteolytic activity involved in the secretion of APP, which results in less secretion of soluble APP into the conditioned media from tacrine treated cells. These results demonstrate that tacrine regulates APP secretion in cell cultures and suggest the possibility that tacrine therapy of Alzheimer's disease may, in the longer term, have effects on the process of Aβ deposition. Wiley‐Liss, Inc.
Extracellular ATP‐induced currents in astrocytes: Involvement of a cation channel
Authors: Walz W. , Gimpl G. , Ohlemeyer C. , Kettenmann H.
Published: January 2005

Whole‐cell currents were measured with the perforated patch clamp technique in cultured rat astrocytes to analyze the underlying ionic mechanism for a P2‐purinoceptor‐mediated depolarization. ATP (100 μM) induced an inward current with a mean amplitude of 130 pA and an EC50 of 17 μM. The response desensitized during a 1 min application. Replacement of extracellular Na+ with NMDG or K+ abolished the ATP‐evoked inward current. Replacement of Na+ with choline, however, resulted in an ATP‐evoked response of one‐third the amplitude in normal solution. This is indicative of a cation rather than Na+ channel. However, due to difficulties in voltage‐clamping these gap junction‐coupled cells at voltages different from the membrane resting potential, the current reversal potential could not be determined. Measurements with K+‐sensitive microelectrodes showed that 100 μM ATP lowered the intracellular K+ concentration. Replacement of extracellular Ca2+ or Cl− did not alter the ATP‐induced inward currents. Fura‐2 imaging experiments revealed a transient rise of the intracellular Ca2+ concentration during ATP application. Removal of extracellular Ca2+ did not influence the peak response; it did, however, shorten the time course. These results and previous observations that the permeability changes are caused by a P2× receptor are indicative of an ATP‐sensitive cation conductance. In addition, cytoplasmic Ca2+ is increased by mobilization from intracellular stores, and by additional influx across the cell membrane. Extracellular ATP released by neurons could evoke K+ release from astrocytes as well as be a mediator for cation changes that signal cell activation processes when released by damaged cells. © 1994 Wiley‐Liss, Inc.
5′‐flanking sequences of the human HPRT gene direct neuronal expression in the brain of transgenic mice
Authors: Rincón‐Limas D. E. , Geske R. S. , Xue J.‐J. , Hsu C. Y. , Overbeek P. A. , Patel P. I.
Published: January 2005

Total deficiency of hypoxanthine phosphoribosyl‐transferase (HPRT) in humans causes the neurological disorder Lesch‐Nyhan syndrome. The HPRT gene is expressed at basal levels in all tissues but at higher levels in the brain, the relevance and mechanism of which is unknown. To determine if cis‐acting DNA elements play a role in the tissue‐differential pattern of expression, we generated transgenic mice carrying different sequences of the human HPRT (hHPRT) promoter fused to the bacterial lacZ gene. We show that a 1.6 kb fragment of the hHPRT promoter contains essential information to direct β‐galactosidase expression preferentially to the basal ganglia, cerebral cortex, hippocampus, and several other areas of the forebrain. At least two elements within the 1.6 kb fragment appear to be required for neuronal expression. A 182 bp element (hHPRT‐NE) represents one of those sequences and is involved not only in confering neuronal specificity but also in repressing transgene expression in non‐neuronal tissues. These studies provide molecular insight into the mechanism of increased HPRT expression in the brain. © 1994 Wiley‐Liss, Inc.
Glucocorticoids enhance the potency of Schwann cell mitogens
Authors: Neuberger T. J. , Kalimi O. , Regelson W. , Kalimi M. , De Vries G. H.
Published: January 2005

Previous studies have documented that cultured Schwann cells require serum‐containing medium to respond maximally to mitogens. We now report that Schwann cells are able to proliferate to a mitogenic response in a serum‐free defined medium termed oligodendrocyte defined media (ODM). Glucocorticoids are the essential component of ODM which allow Schwann cell proliferation in the serum‐free medium. Charcoal treatment of the fetal calf serum decreases the mitogenic potency of the axolemma‐enriched fraction (AEF) by 50%. The addition of 2 μM hydrocortisone to charcoal‐treated fetal calf serum restores 75% of the lost mitogenicity. These observations are consistent with the view that glucocorticoids present in fetal calf serum are potent co‐mitogens essential for AEF‐induced Schwann cell proliferation. The synthetic glucocorticoid, dexamethasone, is a more potent co‐mitogen than hydrocortisone, with a maximal effect at concentrations less than 10 nM. In contrast, other steroids including aldosterone, progesterone, testosterone, and 17β‐estradiol have no effect on enhancing the mitogenic response of Schwann cells to the AEF. The glucocorticoid antagonists RU 486 and dehydroepiandrosterone (DHEA), but not the antiestrogenic compound tamoxifen, block AEF‐induced Schwann cell proliferation. These results suggest that glucocorticoid‐induced Schwann cell proliferation is mediated through a glucocorticoid receptor (GR) mechanism. We detected immunoreactivity to the GR in the cytoplasm, but not in the nuclei of Schwann cells grown in ODM lacking dexamethasone. The addition of 100 nM dexamethasone to these cultures resulted in immunoreactivity in the nucleus. This data suggests that glucocorticoids working through the GR are potent co‐mitogens for Schwann cell proliferation. © 1994 Wiley‐Liss, Inc.