bluuebunny/crossref_metadata_2025 huggingface dataset:
Histamine effects on the 5‐HT1c receptor expressed in Xenopus oocytes
Authors: Shichijo S. , Payan D. G. , Harrowe G. , Mitsuhashi M.
Published: January 2005

To analyze the cross‐reactivity between serotonin (5‐HT) and histamine, the in vitro transcribed RNA for the 5‐HT1c receptor was functionally expressed in Xenopus oocytes. 5‐HT significantly increased 45Ca2+ efflux in RNA‐injected oocytes, but not in uninjected and water‐injected control oocytes. Furthermore, histamine and the H1 receptor agonists, but not the H2 and H3 agonists, significantly induced 45Ca2+ efflux in 5‐HT1c receptor RNA‐injected oocytes, but not in uninjected and water‐injected oocytes. However, the H1, H2, and H3 antagonists failed to inhibit histamine‐induced 45Ca2+ efflux at 10−6M. This finding suggests that the 5‐HT1c receptor can be activated by both 5‐HT and histamine, although the action of histamine is different from classic histamine pharmacology.
Changes in β‐adrenergic receptor distribution on immunocytes during differentiation: An analysis of T cells and macrpphages
Authors: Radojcic Tricia , Baird Stephen , Darko Denis , Smith Dennis , Bulloch Karen
Published: January 2005

The ability of cells of the immune system, immunocytes, to receive signals from the nervous as well as the endocrine system is dependent on the cells' expression of receptors for neurotransmitters and neurohormones. The number of receptors, especially beta adrenergic receptors (BARs), varies with the functional subset of immunocytes. However, little is known about how receptor number may vary during the life span (stem cell to mature activated immunocyte) of a given cell type. In this study, we have addressed this question by examining the affinities (Kd's) and the changes in receptor number (Bmax) on (1) clones of T cells and macrophages, (2) fresh thymocytes and splenocytes before and after activation with Concanavalin A (Con A) and phorbol myristate acetate (PMA)/ Ca+2 ionophore (A23187), and (3) the cloned human monocyte U937 and the murine thymic lymphoma cell BW5147, in the presence and absence of PMA and A23187 ionophore.

Drug treatments of fresh and cloned immunocytes alter the number but not the affinity of β‐receptors. Con A increases the number of β‐adrenergic receptors per cell, whereas PMA/ionophore decreases it. Similar decreases in BAR number are induced by PMA on cell lines BW5147 and U937. These data indicate that changes in receptor number can be regulated with different states of cell maturation and function. Thus, the immunological status of a given cell can influence the expression of BARs, thereby modulating its ability to respond to signals from the nervous and endocrine systems.
Insulin‐like growth factor I stimulates oligodendrocyte development and myelination in rat brain aggregate cultures
Authors: Mozell R. L. , McMorris F. A.
Published: January 2005

Insulin‐like growth factor I (IGF‐I) and high concentrations of insulin have been shown to stimulate an increase in the number of oligodendrocytes that appear in developing monolayer cultures of rat brain cells (McMorris et al., Proc Natl Acad Sci USA 83:822–826, 1986; McMorris et al., Ann NY Acad Sci 605:101–109, 1990; McMorris and Dubois‐Dalcq, J Neurosci Res 21:199–209, 1988). In the present study, we investigated whether IGF‐I or insulin treatment induces a corresponding increase in the synthesis and accumulation of myelin. Aggregate cultures, established from 16‐day‐old fetal rat brains, were treated with either 100 ng/ml IGF‐I or 5,000 ng/ml insulin and analyzed for the number of oligodendrocytes, activity of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP), total amount of myelin, and synthesis rate of myelin proteins. Cultures treated with IGF‐I beginning on day 2 after explanation contained 35–80% more oligodendrocytes and had 60–160% higher CNP activity than controls when tested on day 13, 20, or 27. By day 27, treated cultures had 35–90% more myelin than controls. Similar results were observed in response to 5,000 ng/ml insulin, a concentration at which insulin binds to IGF receptors and acts as an analogue of IGF‐I. The synthesis rate of myelin proteins was measured in experiments using 5,000 mg/ml insulin. When treatment was begun at day 20 rather than day 2, cultures did not exhibit an increased number of oligodendrocytes over control during the following 4–6 days. Therefore, this paradigm was used to separate the effects of insulin‐like peptides on the number of oligodendrocytes from the effects on the synthesis rate of myelin proteins and the accumulation of myelin. Cultures treated from day 20 and controls showed no difference in either the myelin yield or the synthesis rate of myelin proteins. These data show that IGF‐I (and insulin at high concentrations) increases the amount of myelin produced in aggregate cultures; and that the increase appears to be due primarily to an increase in the number of oligodendrocytes in the cultures rather than to a direct increase in the rate of myelin protein synthesis or an increase in the amount of myelin synthesized per oligodendrocyte.
Brain cysteine proteinase inhibitors II: Evidence that a 21‐kDa papain‐binding component resembles ras p21
Authors: Berg M. J. , Marks N.
Published: January 2005

A 21‐kDa protein extracted from rat or bovine brain at high pH was purified on alkylated‐papain and shown to have dual ras like and cysteine proteinase inhibitory (CPI) properties. This was demonstrated by its GTP‐binding activity, cross‐reactivity toward pan‐reactive ras p21 monoclonal antibody, and inhibition of papain. The material eluted earlier than cystatins or kininogens on the alkylated papain‐affinity column and was devoid of other CPIs based on immunoblot analysis. In a second procedure, ras p21s isolated from rat or bovine brain membranes by cholate extraction and purified by gel‐permeation and hydrophobic interaction were shown to act also as potent CPIs, inhibiting rat brain cathepsin L, pa‐pain, or rat brain cathepsin B with K1 values of 3, 11, and 167 nM, respectively. This component cross‐reacted with the monospecific anti‐ras, but not with other anti‐CPIs, and represented 3–4% of total GTP binding present in homogenates. The specific activity of the purified 21 kDa component was 4.7 nmol GTP‐γ‐S bound per mg protein. The data support the notion that brain ras p21s constitute a separate group of CPIs and are available for regulating some aspects of brain protein turnover.
Rat retina has an active and stable ubiquitin–protein conjugating system
Authors: Naash M. , Izbicka E. , Anderson R. E.
Published: January 2005

We describe here the presence of ubiquitin and its conjugation system in the rat retina. Retinal homogenates and supernatants conjugate [125I] human ubiquitin with either endogenous or exogenous proteins. The conjugating activity is relatively stable over time, requires ATP, and has a pH optimum of ∼8. The most prominent [125I]ubiquitin conjugates formed are larger than 130 kDa. Several other minor conjugates are also formed between the molecular weights 17 and 75 kDa. The endogenous levels of free and conjugated forms of ubiquitin have been determined in the rat retina. More than 50% of retinal ubiquitin is covalently bound to target proteins. In addition, activities responsible for the ATP‐dependent degradation and disassembly of both endogenous and exogenous ubiquitin conjugates have been detected in vitro. These results provide evidence that the retina contains active and stable ubiquitin‐conjugating enzymes that recognize retinal proteins and have ATP‐dependent proteolytic activity.
Phosphorylation of MARCKS (80‐KDA) protein, a major substrate for protein kinase C in oligodendroglial progenitors
Authors: Bhat N. R.
Published: January 2005

We have recently reported a potent mitogenic stimulation of oligodendroglial (OL) progenitors by the protein kinase C (PKC) activating phorobol ester, i.e., phorbol 12‐myristate 13‐acetate (PMA) (Bhat NR, J Neurosci Res 22:20–27, 1989). The present study deals with PMA‐induced protein phosphorylation reactions in cultured OL progenitors. The phorbol ester induced the phosphorylation of several cytosol and membrane‐associated proteins, including a major protein with an apparent molecular weight of 80 kDa. In both control and PMA‐treated cultures, phosphorylation level of the 80‐kDa protein in cytosol was higher than that in the particulate fraction. Okadaic acid, an inhibitor of protein phosphatases, also increased the phosphorylation of several proteins and substantially enhanced protein phosphorylation induced by PMA. In vitro incubation of the cell membranes with phosphatidylserine and diacylglycerol (a physiological activator, of PKC) in the presence of [γ32p]‐ATP resulted in an increased phosphorylation of the 80‐kDa protein. The induction of phosphorylation of the 80‐kDa protein under both in situ and in vitro conditions was subject to inhibition by l‐[5]isoquinolinyl sulfonyl)‐3‐methylpiperazine (H‐7), a potent inhibitor of PKC. The 80‐kDa phosphoprotein was identified as the prominent PKC substrate, i.e., myristoylated alanine‐rich C‐kinase substrate (MARCKS) protein by immunoprecipitation with anti‐MARCKS antibodies.
Temporal changes in edema, Na+, K+, and Ca++ in focal cortical stroke: GM1 ganglioside reduces ischemic injury
Authors: Karpiak S. E. , Wakade C. G. , Tagliavia A. , Mahadik S. P.
Published: January 2005

Cortical focal ischemia in the rat was induced by middle cerebral artery occlusion (MCAo) together with permanent occlusion of the ipsilateral common carotid artery (CCAo) and a temporary (1 hr) occlusion of the contralateral CCA. By using a defined cortical tissue sampling procedure at 3, 6, 24, 72, 96, and 120 hr after the MCAo + CCAo, patterns of edema and ion (Na+, K+, and Ca++) changes in a primary and three peri‐ischemic cortical areas are described. Ionic imbalances and edema formation have distinct patterns, are time dependent, and are different when comparing primary and peri‐ischemic areas. Calcium increases to “neurotoxic” levels appear temporally independent of edema formation, reaching magnitudes 20 times greater than basal levels in the pri‐mary infarct area. Na+ increases correlate with increases in water, while K+ losses do not appear to be directly related to edema formation or Na+ and Ca++ increases. K+ losses are only significant in the primary infarct area. Rats treated with GM1 ganglioside (10 mg/kg, i.m.) daily showed significant reductions in edema, Na + and Ca ++ increases. These ganglioside effects were evident as early as 24 hr after the ischemic injury. Ca++ increases, which was maximal at 72 hr after the ischemic injury, was reduced by greater than 50% in GM1‐treated animals. The mechanism by which GM1 is an effective neuroprotective agent may be evidenced by its effects on Ca++ influx/efflux processes in injury.
GABA‐ and glutamate‐activated currents in glial cells of the mouse corpus callosum slice
Authors: Berger T. , Walz W. , Schnitzer J. , Kettenmann H.
Published: January 2005

Whole‐cell transmitter‐activated currents were recorded with the patch‐clamp technique from glial cells in thin frontal brain slices of the corpus callosum. In slices from 6‐ to 8‐day‐old mice, glioblasts were predominantly found, while oligodendrocytes were predominant in slices from 10‐ to 13‐day‐old mice. These developmental stages could be readily distinguished by their K+ channel pattern and their morphology and ultrastructural features. Both cell types expressed GABA and glutamate receptors in this in situ preparation. GABA responses showed similarities to those described for GABAA receptors, i.e., they were mimicked by muscimol, blocked by bicuculline, and enhanced by pentobarbital. Glutamate responses showed similarities to those of the kainate/quisqualate receptor subtype. The amplitude of GABA‐activated currents recorded in oligodendrocytes was significantly smaller than that from glioblasts, while glutamate responses did not show marked differences in either cell type.
Effect of dexamethasone on the nerve growth factor‐induced increase in the NILE glycoprotein in PC12 cells
Authors: Tocco M. D. , Fujita K. , Guroff G.
Published: January 2005

The long‐known and well‐documented increase in the NILE glycoprotein produced by nerve growth factor in PC12 cells is prevented by simultaneous treatment with dexamethasone. The absence of this surface marker on the fully differentiated cells has been demonstrated by both glucosamine incorporation and immunohistofluorescence.
Dexamethasone blocks nerve growth factor induction of nerve growth factor receptor mRNA in PC12 cells
Authors: Foreman P. J. , Taglialatela G. , Jackson G. R. , Perez‐Polo J. R.
Published: January 2005

Glucocorticoids and nerve growth factor (NGF) have been shown to have antagonistic effects on chromaffin cells in vivo. Here we determined the effect of the synthetic glucocorticoid, dexamethasone, on levels of mRNA for the nerve growth factor receptor (NGFR) in rat PC12 pheochromocytoma cells. Following administration of dexamethasone (1 μM) there is a decline in NGFR mRNA expression. More importantly, administration of dexamethasone appears to block the NGF‐mediated induction of NGFR when both agents are administered simultaneously. These data support the hypothesis that glucocorticoids and NGF act in opposition in determination of the phenotype of chromaffin cells.
Expression of plasmolipin in the developing rat brain
Authors: Sapirstein V. S. , Nolan C. E. , Stadler I. I. , Fischer I.
Published: January 2005

Plasmolipin is an hydrophobic plasma membrane proteolipid present in both kidney and brain. The protein consists of two subunits of 17–18.5 kD, which together form K+ selective voltage‐dependent channels. In this report, we define the embryonic and postnatal expression of plasmolipin in the developing rat brain. Plasmolipin was found to be essentially restricted to the postnatal period increasing eight‐fold between the first to fourth week after birth. A fetal plasmolipin immunoreactive protein (FPIP) was identified in embryonic brain and also during the early postnatal development of the cerebellum. The expression of FPIP was biphasic with an initial transient increase between E15‐E20 followed by a decrease in its levels. FPIP was not detected in the developed rat CNS. FPIP was found in a variety of dividing and immature cells including cultured astrdcytes and embryonic neurons, neuroblastoma cells, and rat thymus. In contrast, plasmolipin was restricted to oligodendrocytes of the neural cells tested and to renal tubular epithelial cells.
MPTP‐induced parkinsonism: Acceleration of biochemical and behavioral recovery by GM1 ganglioside treatment
Authors: Schneider J. S.
Published: January 2005

The effects of GM1 ganglioside administration on functional recovery and recovery of caudate nucleus dopamine levels have been assessed in cats made parkinsonian by administration of the dopaminergic neurotoxin l‐methyl‐4‐phenyl‐ 1,2,3, 6‐tetrahydropyridine (MPTP). Cats made severely parkinsonian by MPTP administration began to show spontaneous functional recovery by the third week after MPTP, as had been observed in previous studies with this model. In contrast, cats with similar initial impairment but which received 3 weeks of GM1 ganglioside treatment (30 mg/kg, i.p. daily) showed an accelerated behavioral recovery, showing significant functional improvement after the first week of GM1 treatment and almost normal function by the end of the third week of treatment. The GM1‐treated cats had caudate nucleus dopamine, 3,4‐dihydroxyphenylacetic acid (DOPAC), and HVA levels significantly increased above levels measured in saline‐treated MPTP control cats. A second group of cats received MPTP only until the first signs of parkinsonism were observed and thus overall had a less severe initial syndrome than the cats described previously. Again, while all cats showed functional recovery over time, the recovery process was accelerated in GM1 ‐treated cats. GM1 treatment also caused a significant increase in caudate dopamine levels in these cats. These results suggest that GM1 ganglioside administration can result in increased dopamine levels even in the heavily denervated striatum and accelerate functional recovery after an MPTP‐induced lesion of the nigrostriatal dopamine system in the cat. This suggests that GM1 or other trophic factor therapies may be fruitful treatment strategies for a disorder of nigrostriatal function such as Parkinson's disease.
Interaction of multiple nuclear proteins with the promoter region of the mouse 68‐kDa neurofilament gene
Authors: Ivanov T. R. , Brown Ian R.
Published: January 2005

Four brain‐specific, DNase I hypersensitive sites (HSS) have been mapped to the 5′ flanking region of the mouse 68‐kDa neurofilament gene. These sites are contained within a 1.7‐kb sequence that confers neuronal specificity of expression on this gene in transgenic mice. To identify DNA sequences that might be involved in gene regulation, the HSS situated near the promoter region has been analyzed by gel mobility shift assays and DNase I footprinting to investigate protein binding sequences. Of particular interest are two footprints localized to a 9‐nucleotide sequence that flanks both the light and medium neurofilament gene in mouse and to a sequence that demonstrates partial homology to several promoter regions, including element‐1, a motif required for neuron specificity in Drosophila. A prominent footprint was also detected at a sequence that contains a near‐perfect palindrome centered at a PstI restriction site. © 1992 Wiley‐Liss, Inc.
Molecular cloning of a novel mRNA using an antibody directed against synaptic glycoproteins
Authors: Johnston I. G. , Rush S. J. , Gurd J. W. , Brown Ian R.
Published: January 2005

It has been suggested by a number of investigators that glycoproteins may play a role in the development and/or maintenance of synapses in the mammalian CNS. For many synaptic glycoproteins, however, little precise structural or functional information is available. In an effort to isolate probes specific to individual glycoproteins, we have screened a rat brain cDNA expression library with a mixed polyclonal antibody directed against concanavalin A‐binding synaptic junctional glycoproteins. Using this approach, we have previously reported the cloning of SC1, a putative extracellular matrix glycoprotein found in adult brain (Johnston et al., Neuron 2:165–176, 1990). We now report the cloning and characterization of a second novel cDNA, which has been designated SC2. Northern blots show that this cDNA recognizes a 1.2‐kb mRNA that is present throughout postnatal development in the rat. It is expressed at high levels in brain and is also found at lower levels in several other tissues. In situ hybridization suggests that the SC2 mRNA is strongly expressed by many types of neurons. Sequence data reveals a single open reading frame in the cDNA, encoding a putative hydrophobic protein with a calculated molecular weight of 36.1 kDa. Sequence analysis reveals some similarity between SC2 and 5α‐reductase, a microsomal membrane protein important in testosterone metabolism. © 1992 Wiley‐Liss, Inc.
Expression of p75NGFR trkA, and trkB mRNA in rat C6 glioma and type I astrocyte cultures
Authors: Hutton L. A. , DeVellis J. , Perez‐Polo J. R.
Published: January 2005

Using a quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) we have investigated the expression of the neurotrophin receptors p75NGFR, trkA, and trkB, mRNAs in cultures of rat pup type I astrocytes and in the C6 rat glioma cell line. All three neurotrophin receptor mRNAs are expressed in both C6 cells and in type I astrocytic cultures. p75NGFR mRNA levels are increased by either cycloheximide or nerve growth factor (NGF) treatment of C6 cells as measured using RT‐PCR. Type I astrocyte cultures also expressed p75NGFR mRNA and NGF treatment increased p75NGFR mRNA levels in these cultures. TrkB mRNA levels were increased by cycloheximide treatment of type I astrocyte cultures but not by NGF treatment. Using RT‐PCR, trkA mRNA was detected in astrocytic cultures as well as in the rat C6 and PC‐12 cell lines. We conclude that cultures of type I astrocytes express active NGF receptors and that glia can elicit a response to NGF as seen by an increase in p75NGFR mRNA levels following exposure to NGF. © 1992 Wiley‐Liss, Inc.
Differential expression of cytochrome oxidase (COX) genes in different regions of monkey brain
Authors: Chandrasekaran K. , Stoll J. , Giordano T. , Atack J. R. , Matocha M. F. , Brady D. R. , Rapoport S. I.
Published: January 2005

A frontal pole cDNA library from monkey (Macaca mulatta) brain was screened to identify mRNAs that are expressed more in frontal pole as compared to primary visual cortex. Three cDNA clones, whose greater expression was confirmed by Northern blot analysis, were identified as cytochrome oxidase (COX) subunits I, II, and III (COX I, II, and III). Each clone showed higher levels of mRNA in the frontal pole, dorsal lateral prefrontal cortex, and hippocampus than in the primary visual or somatosensory cortices. COX histochemistry of prefrontal, visual, and somatosensory cortical regions demonstrated heterogeneous distributions, with highest activity in dendrite‐rich neuropil of the cortex. A laminar distribution of COX mRNA expression also was demonstrated with in situ hybridization. mRNA was detected in cell bodies and in apical dendrites. These results indicate region specific differences in the distribution of COX activity and in the corresponding mRNA for three of its subunits within the monkey brain. Such differences may be related to differences in the distribution of neuropil as compared with cell bodies among the brain regions studied, and may be relevant to selective vulnerability in Alzheimer's disease. Published 1992 by Wiley‐Liss, Inc. © 1992 Wiley‐Liss, Inc.
Multiple intracellular signaling pathways of the neuropeptide substance P receptor
Authors: Mitsuhashi Masato , Ohashi Y. , Shichijo S. , Christian C. , Sudduth‐Klinger J. , Harrowe G. , Payan D. G.
Published: January 2005

The rat substance P (SP) receptor cDNA has been transfected into cultured rat KNRK cells, and a stable cell line expressing functional SP receptors established. Upon stimulation with SP, these cells responded by simultaneously activating two signaling pathways: the mobilization of intracellular Ca2+ and the raising of cyclic adenosine triphosphate (cAMP) levels. Both Ca2+ and cAMP responses were elicited in a similar dose‐dependent manner with half maximal concentrations of approximately 5 × 10−10 M. Following ionomycin treatment SP‐dependent Ca2+ responses were abolished, whereas cAMP responses were preserved. Forskolin eliminated the SP‐dependent cAMP elevation, however, the SP‐induced Ca2+ mobilization remained unchanged. Furthermore, treatment with phorbol esters had no significant effect on either of the two SP‐induced responses. Thus it appears that the SP receptor is capable of independently activating Ca2+ mobilization and cAMP pathways. These results may provide new insights for further understanding the diverse activities of SP in various systems in vitro and in vivo. © 1992 Wiley‐Liss, Inc.
Myelin/oligodendrocyte glycoprotein is a unique member of the immunoglobulin superfamily
Authors: Gardinier M. V. , Amiguet P. , Linington C. , Matthieu J.‐M.
Published: January 2005

Myelin/oligodendrocyte glycoprotein (MOG) is a primary target autoantigen in experimental autoimmune encephalomyelitis, a widely used animal model for autoimmune demyelinating diseases such as multiple sclerosis. We have isolated several rat MOG cDNAs and confirmed their identity by comparision with MOG N‐terminal peptide sequence. As expected, MOG mRNA expression is CNS‐specific and peaks during active myelination. Our studies show that full length MOG mRNA is ≈︁ 1.6 kb and encodes a signal peptide of 27 amino acids, followed by 218 residues for mature MOG (24,962 MW). A single site for N‐glycosylation is found at Asn‐31. Rather than the ubiquitous AAUAAA polyadenylation signal, a series of three overlapping, rare poly A signals were identified. The N‐terminal half of mature MOG shares 52% identity with bovine butyrophilin, a possible lipid receptor. This same region has 39% identity with chicken B‐G antigen, a major histocompatibility complex antigen involved in B cell selection and immune repertoire development. We show that both MOG and butyrophilin, each exhibiting a single Ig like variable region domain, meet criteria for inclusion in the immunoglobulin superfamily. Moreover, MOG appears to represent a unique member of this superfamily in that it possesses two potential transmembrane domains, in contrast to a single membrane‐spanning domain or glycophospholipid anchor found in all other members of Ig superfamily members. © 1992 Wiley‐Liss, Inc.
Evidence for intracellular cleavage of the Alzheimer's amyloid precursor in PC12 cells
Authors: Sambamurti K. , Shioi J. , Anderson J. P. , Pappolla M. A. , Robakis N. K.
Published: January 2005

The Alzheimer's amyloid precursor (APP) is cleaved by an unidentified enzyme (APP secretase) to produce soluble APP. Fractionation of PC12 cell homogenates in a detergent‐free buffer showed the presence of the Kunitz protease inhibitor (KPI)‐containing soluble APP (nexin II) in the particulate fraction. Digitonin or sodium carbonate treatment of this fraction solubilized nexin II suggesting that it is contained in the lumen of vesicles. Nexin II production was not affected by lysosomotropic agents, suggesting that APP secretase is not a lysosomal enzyme. Labelling of cell surface proteins by iodination failed to detect full‐length APP on the surface of PC12 cells, suggesting that most of this protein is located intracellularly. Furthermore, pulse‐chase experiments showed that nexin II is detected in cell extracts before it appears in the culture medium. Cellular nexin II was detected at zero time of chase after only 5 min of pulse labelling with 35S‐sulfate, indicated that APP secretase cleavage takes place immediately after APP is sulfated. Temperature block, pulse‐chase, and 35S‐sulfate‐labelling experiments suggested that APP is cleaved by APP secretase intracellularly in the trans‐Golgi network (TGN) or in a post‐Golgi compartment. © Wiley‐Liss, Inc.
β‐Oxidation of [1‐14C]palmitic acid by mouse astrocytes in primary culture: Effects of agents implicated in the encephalopathy of Reye's syndrome
Authors: Murphy M. G. , Jollimore C. , Crocker J. F. S. , Her H.
Published: January 2005

β‐Oxidation of [1‐14C]palmitic acid was examined in homogenates of astrocytes cultured from neonatal mouse brain. Under optimal reaction conditions (≤50 μg protein, 10 min at 37°C), oxidation increased as a function of palmitate concentration (15 μM to 2 mM) and reached a maximum rate of 1.98 ± 0.29 nmol/min/mg protein (mean ± SEM) at 0.2 mM substrate. Eadie‐Hofstee analysis of data from four experiments yielded apparent values for Vmax of 1.87 nmol/min/mg protein, and for Km, 35–40 μM. There were no dramatic changes in the oxidation rate in cells between 10 and 36 days in culture. During the 10‐min assays, less than 0.05% of the radioactivity was converted to 14CO2 by the astrocytes; water‐soluble products accounted for 1–2% of the total substrate added. Studies with KCN indicated that 60–70% of the total activity occurred in the mitochondria.

We have been studying the structural and functional changes associated with the cerebral encephalopathy of Reye's syndrome (RS). Three‐week‐old astrocytes exposed to serum from RS children for the final 7 days of culture exhibited minor mitochondrial pleomorphism and had increased numbers of other intracellular organelles. Examination of the effects of agents implicated in RS indicated that oxidation of [1‐14C]palmitate was not altered by Na+ salicylate (1–3 mM), but was inhibited by the industrial surfactant, Toximul MP‐8 (≥10 μg/ml), 4‐pentenoic acid (≥01 μM), or with 4 days' exposure to ammonia (10 mM). The latter treatment also resulted in an increase in protein synthesis, cell volume, and malondialdehyde formation. These results suggest that some of the “toxins” implicated in RS inhibit fattyacid oxidation in the astrocytes and produce other lipid‐related abnormalitities that could be related to encephalopathy. © Wiley‐Liss, Inc.
The neural cell adhesion molecule (NCAM) heparin binding domain binds to cell surface heparan sulfate proteoglycans
Authors: Kallapur S. G. , Akeson R. A.
Published: January 2005

The neural cell adhesion molecule (NCAM) has been strongly implicated in several aspects of neural development. NCAM mediated adhesion has been proposed to involve a homophilic interaction between NCAMs on adjacent cells. The heparin binding domain (HBD) is an amino acid sequence within NCAM and has been shown to be involved in NCAM mediated adhesion but the relationship of this domain to NCAM segments mediating homophilic adhesion has not been defined. In the present study, a synthetic peptide corresponding to the HBD has been used as a substrate to determine its role in NCAM mediated adhesion. A neural cell line expressing NCAM (B35) and its derived clone which does not express NCAM (B35 clone 3) adhered similarly to plates coated with HBD peptide. A polyclonal antiserum to NCAM inhibited B35 cell‐HBD peptide adhesion by only 10%, a value not statistically different from inhibition caused by preimmune serum. Both these experiments suggested no direct NCAM‐HBD interactions. To test whether the HBD peptide bound to cell surface heparan sulfate proteoglycans (HSPG), HSPG synthesis was inhibited using β‐D‐xyloside. After treatment, B35 cell adhesion to the HBD peptide, but not to control substrates, was significantly decreased. B35 cell adhesion to the HBD peptide could be inhibited by 10−7 M heparin but not chondroitin sulfate. Preincubation of the substrate (HBD peptide) with heparin caused dramatic reduction of B35 cell‐HBD peptide adhesion whereas preincubation of B35 cells with heparin caused only modest reductions in cell‐HBD adhesion. Furthermore, inhibition of HSPG sulfation with sodium chlorate also decreased the adhesion of B35 cells to the HBD peptide. These results strongly suggest that, within the assay system, the NCAM HBD does not participate in homophilic interactions but binds to cell surface heparan sulfate proteoglycan. This interaction potentially represents an important mechanism of NCAM adhesion and further supports the view that NCAM has multiple structurally independent binding sites. © 1992 Wiley‐Liss, Inc.
Interaction of AChE with Lens culinaris agglutinin reveals differences in glycosylation of molecular forms in sarcoplasmic reticulum membrane subfractions
Authors: Campoy F. J. , Cabezas‐Herrera J. , Vidal C. J.
Published: January 2005

Fractionation of muscle microsomes rich in sarcoplasmic reticulum (SR) by isopicnic centrifugation yielded three types of membranes. Heavy (HM), intermediate (IM), and light membranes (LM), with isopicnic points of 38, 33, and 25% w/w sucrose, were rich in terminal cisternae/triads, longitudinal SR, and T‐tubules, respectively. All membrane subfractions displayed acetylcholinesterase (AChE) activity. About 60, 80, and 50% of total AChE in HM, IM, and LM was extracted with a Tris‐saline‐Triton buffer. AChE molecular forms of 4.5 S (G1), 10.5 S (G4), and 16 S (A12) were found in all membranes but their relative proportion varied among the several membranes. Asymmetric and tetrameric forms were partly sedimented with Lens culinaris agglutinin (LCA), but most of the monomeric AChE failed to interact with the lectin. However, some of the monomers, exclusively found in LM, reacted with LCA. The data suggest that monomeric AChE is classified in rough endoplasmic reticulum. A subset is destined to SR, a second one converted into oligomeric forms, and a third one is associated to external membrane after passing through the Golgi system. © 1992 Wiley‐Liss, Inc.
In search of the central respiratory neurons: II. Electrophysiologic studies of medullary fetal cells inherently sensitive to CO2 and low pH
Authors: Rigatto H. , Fitzgerald S. C. , Willis M. A. , Yu C.
Published: January 2005

Although extensively pursued, the central respiratory neurons have remained elusive. We departed from the more conventional physiologic and morphologic methods of system and tissue examination and cultured dissociated fetal rat cells (Fitzgerald et al., J Neurosci Res 33:579–589, 1992) from the area of the nucleus ambiguus and the nucleus tractus solitarius located within the 2 mm rostral to the obex. Pacemaker‐like cells, with a regular single or bursting activity, studied at 3–5 weeks of age, responded to very small pulses of CO2 (50 ms) and low pH with an increase in spike frequency and a decrease in spike amplitude. Other irregularly beating or silent cells did not respond or else required very large pulses (>200 ms) to do so. The pacemaker cells also responded to hypoxia induced by administration of sodium hydrosulfite with an increase in spike frequency and amplitude; high oxygen (>600 torr) and adenosine produced a decrease in electrical activity. Most of these cells were multipolar after staining with antibodies to neuron‐specific enolase (NSE) and Fragment C of tetanus toxin. They did not stain for choline acetyltransferase (ChAT). The results suggest that these cultured cells, expressing a phenotype inherently responsive to CO2 and low pH, have the characteristics of central respiratory chemoreceptors, and may be involved in the generation of the respiratory rhythm. © 1992 Wiley‐Liss, Inc.
Lack of effect of interleukins 1α and 1β, during in vitro perifusion, on anterior pituitary release of adrenocorticotropic hormone and β endorphin in the male rat
Authors: Parsadaniantz S. Mélik , Lenoir V. , Terlain B. , Kerdelhué B.
Published: January 2005

It has been demonstrated that interleukin 1 (IL1) injection provokes a great variety of biological effects, notably an activation of the corticotropic axis, increasing plasma adrenocorticotropic hormone (ACTH) and corticosterone. However, the primary site of action of IL1 is still controversial. In the present study, we first verified the in vivo capability of human interleukins 1α (hIL1α) and 1β (hIL1β) to release ACTH and β endorphin (β EP) in the normal male rat, before investigating, through an anterior pituitary (AP) perifusion system, the hIL1α and hIL1β effects on basal and corticotropin‐releasing factor (CRF)‐induced ACTH and β EP secretions. This system enabled the examination of a dynamic profile of hormones secretion, avoiding the possibility of feedback mechanisms, as is the case with the use of regular but very often longtime incubations.

The results showed that in a perifusion system, with a short duration treatment (below 2 hr) compatible with the kinetics of action observed in vivo, basal and CRF‐induced ACTH and β EP release were not modified in the presence of a broad range of concentrations (from 10−12 to 10−9 M) of hIL1α or hIL1β. Taken together, these results clearly show that in an in vitro situation close to physiological conditions, the primary site of action of hIL1α and hIL1β on ACTH and β EP release is not located at the AP level in the male rat. © 1993 Wiley‐Liss, Inc.
Regulation of phospholipase D activity in a human oligodendroglioma cell line (HOG)
Authors: Dawson G. , Dawson S. A. , Post G. R.
Published: January 2005

Oligodendroglial cells express many specific proteins, such as myelin basic protein (MBP), which are physiologically phosphorylated by protein kinase C (PKC). Diacylglycerols are physiological activators of PKC and can be liberated from phospholipids by the direct receptor‐mediated activation of phospholipase C (PL‐C) or indirectly via the activation of phospholipase D (PL‐D). In a well‐characterized human oligodendroglioma (HOG) cell line, PL‐C (measured by release of [3H]inositol phosphates) and PL‐D (formation of [3H]myristoylated or palmitoylated phosphatidylethanol) were activated by both carbachol (blocked by pirenzepine, suggesting an M1 receptor) and histamine (H1 receptor) but not glutamate, bradykinin, or phenylephrine. PL‐C stimulation by carbachol or histamine was completely inhibited by short‐term treatment (<30 min) with phorbol ester (TPA), a PKC activator. In contrast, PL‐D activation by either carbachol or histamine was stimulated in additive fashion by TPA, suggesting at least two distinct mechanisms for PL‐D activation. Down regulation of PKC by prolonged (24 hr) treatment with TPA reversed the inhibitory effects of TPA on PL‐C and the stimulatory effects on PL‐D. However, the PKC inhibitors H‐7 and galactosylsphingosine did not inhibit the TPA‐mediated stimulation of PLD while the less‐specific PKC inhibitor, staurosporine, was only partially inhibitory. Preexposure of cells to carbachol, greatly reduced both PL‐C and PL‐D activation by carbachol, suggesting homologous desensitization. Time‐course studies indicated that PL‐D activation (10 sec or less) was at least as fast as PL‐C activation, and the affinity of carbachol and histamine for the receptor coupled to either phospholipase (EC50 = 5–10 μM) was about the same. We conclude that in this oligodendroglioma, and by inference in oligodendroglial cells, the receptor‐coupled PL‐D pathway, is at least as important as the PL‐C pathway as a source of DAG and that its relationship to PKC is complex. © 1993 Wiley‐Liss, Inc.
Plasminogen activator inhibitor 1, the primary regulator of fibrinolysis, in normal human cerebrospinal fluid
Authors: Rao Jasti S. , Chen M. , Festoff Barry W.
Published: January 2005

The occurrence of type‐1 plasminogen activator inhibitor (PAI‐1) in human cerebrospinal fluid (CSF) has not previously been reported. As a member of the serpin superfamily of serine protease inhibitors and an acute phase response component, PAI‐1 has powerful potential roles in nervous system homeostasis. We have detected this serpin antigen using a polyclonal anti‐PAI‐1 antibody in normal human CSF. In Western blotting, PAI‐1 in several CSF samples appears as a two‐band antigen of Mr = 54 and 35 kDa, presumably the intact and proteolytic fragment, respectively. In vitro complex formation studies confirm that the 54 kDa form of PAI‐1 interacts with 125I‐urokinase after activation with SDS, but the 35 kDa form does not. Quantification of total PAI‐1 antigen in 18 normal human CSF samples by ELISA reveals a mean value of 1.0 ± 0.07 (SEM) μg/dL, indicating that a relatively low concentration of the inhibitor occurs in normal human CSF. This information should now allow comparison of PAI‐1 levels and activity in various neurologic disorders. © 1993 Wiley‐Liss, Inc.
Microglial conditioned medium promotes survival and development of cultured mesencephalic neurons from embryonic rat brain
Authors: Nagata K. , Takei N. , Nakajima K. , Saito H. , Kohsaka Shinichi
Published: January 2005

We previously reported that microglial conditioned medium (Mic‐CM) has a neurotrophic effect on cultured rat neocortical neurons [Nakajima et al. (1989): Biomed Res 10:411–423]. In order to investigate the interaction between microglia and neurons in more detail, we determined the effects of Mic‐CM on the primary cultured mesencephalic neurons from 16‐day embryonic rats. The addition of Mic‐CM to the culture medium significantly enhanced the survivability of neurons and promoted neurite extension in a low cell‐density culture condition. In a high cell‐density culture condition, Mic‐CM markedly increased dopamine uptake, which was quantified by assessing the specific [3H]dopamine uptake, and also increased the dopamine content of cultured cells. Furthermore, the number of mesencephalic dopaminergic neurons, which was determined by quantitative analysis of tyrosine hydroxylase (TH)‐immunoreactive cells, increased significantly in the presence of Mic‐CM. These results suggest that Mic‐CM enhances survival or maturation of TH‐positive neurons present in cultures of the embryonic mesencephalon and that these neurotrophic effects may be due to a diffusible factor(s) from microglia. © 1993 Wiley‐Liss, Inc.
Detection of choline‐acetyltransferase activity in lymphocytes
Authors: Rinner I. , Schauenstein K.
Published: January 2005

The cholinergic system takes part in the immune‐neuroendocrine interation. Lymphocytes have been found to express muscarinic and nicotinic cholinergic receptors, as well as acetylcholine‐esterase on their surface, and cholinergic agents modulate immune functions in vitro and in vivo. In the present study we provide evidence that purified organ resident and circulating lymphocytes, as well as various lymphoid cell lines derived from different species, exhibit choline‐acetyltransferase activity and, therefore, have the potential to produce the neurotransmitter acetylcholine. © 1993 Wiley‐Liss, Inc.
110/140 laminin‐binding protein immunoreactivity in spinal dorsal root ganglia: A capsaicin‐insensitive reduction induced by constriction injury of the sciatic nerve in rats
Authors: Ren K. , Kibbey M. C. , Kleinman H. K. , Ruda M. A.
Published: January 2005

The distribution of 110/140 laminin‐binding protein (110/140 LBP) in the spinal dorsal root ganglia (DRG) and its regulation by partial constriction of the sciatic nerve was studied in adult rats. The cross‐sectional area of neurons with 110/140 LBP‐immunoreactivity (−I) showed an approximately normal frequency distribution. The 110/140 LBP‐I was observed in neuronal cell bodies exclusive of the nucleus. Following sciatic nerve constriction, the 110/140 LBP‐I was downregulated in the ipsilateral L4‐5 DRG. DRG neurons with a cross‐sectional area ≥ 1600 μm2 were preferentially affected. Neonatal capsaicin‐treatment, a procedure that selectively destroys a subpopulation of DRG neurons with fine unmyelinated axons, had no effect on the reduction of 110/140 LBP in the DRG induced by sciatic nerve constriction. Western immunoblot analysis confirmed a reduction of 110/140 LBP on the side ipsilateral to the constriction. These results demonstrate a LBP within primary sensory neurons and its suppression by peripheral nerve injury. The data support a role for LBP in the adult nervous system.© 1993 WiIey‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the Unitcd States oC America.
Evidence for hemi‐gap junctional channels in isolated horizontal cells of the skate retina
Authors: Malchow R. P. , Qian H. , Ripps H.
Published: January 2005

Prolonged depolarization of isolated, voltageclamped skate retinal horizontal cells produces an outward current that exhibits a late onset and develops slowly with time. This current, which we refer to as the Q‐current, is associated with an increase in membrane conductance, and is present when other voltage‐gated conductances have been pharmacologically blocked. The reversal potential for the Q‐current, obtained using tail current analysis, was close to 0 mV. The magnitude of the current was greatly reduced by superfusion with 25 mM acetate, and by 4 mM cobalt chloride, 2 mM 1‐octanol, and a saturated solution of the general anesthetic halothane. In addition, the low‐molecular weight fluorescent dye Lucifer yellow, applied extracellularly, entered the cells during activation of the Q‐current, whereas a 3 kD dextran‐fluorescein complex did not cross the cell membrane. The effects of divalent cations, the nonspecific nature of the ionic current suggested by its reversal potential, the entry of Lucifer yellow, and the ability of acetate, halothane, cobalt, and octanol to block the current lead us to hypothesize that the Q‐current results from the opening of hemi‐gap junctional channels that mediate electrical coupling between skate horizontal cells. © 1993 Wiley‐Liss, Inc.
Dye‐induced photolesion in the mammalian retina: Glial and neuronal reactions
Authors: Picaud S. , Peichl L. , Franceschini N.
Published: January 2005

Irradiation in the presence of a dye applied to the extracellular space is known to produce degenerative features in irradiated neurones and fibers. In the present study, we confirmed the potential use of this procedure as a lesion technique by showing the removal of degenerating elements as part of the glial reaction to the lesion. The dye Rose Bengal was applied to the vitreous body of a rat eye and a T‐shaped irradiation pattern was projected onto the retina within the absorption band of the dye. Degenerative features were restricted to the irradiated area, which could be readily identified from its shape. Retinae examined after various survival times showed that macrophages invaded the damaged area within 1 day, and that mitotic activity of reactive glial cells subsequently occurred in the vicinity of the wound. Both cell types were identified by their structural features. Macrophages were also revealed by a staining technique using the dye Nile Red, whereas reactive glial cells were immunolabeled with an antibody directed against the glial fibrillary acidic protein. Reactive glial cells helped the macrophages to gradually remove injured cells and damaged processes. Their main task, however, appeared to be in scar formation, since their processes seemed to suture the lips of the wound together and restore the limiting membrane at the inner retina. After 2 months' survival time, the parent ganglion cells of most disrupted axon bundles had retrogradely degenerated, but regenerating ganglion cell axons were also observed. These results provide some new data about healing processes in the retina. They demonstrate that the dyeinduced photolesion technique can be used to either remove or axotomize selected neurones in neural networks which have been made optically accessible. c 1993 Wiley‐Liss, Inc. © 1993 Wiley‐Liss, Inc.
Amphiphilic and hydrophilic forms of acetyl‐ and butyrylcholinesterase in human brain
Authors: Sáez‐Valero J. , Tornel P. L. , Muñoz‐Delgado E. , Vidal C. J.
Published: January 2005

Human brain acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were sequentially extracted, first with a Tris‐saline buffer (S1) and then with 1% (w/v) Triton X‐100 (S2). About 20 and 30% of the AChE and BuChE activities were recovered in S1 and most of the remaining enzymes in S2. Main molecular forms of about 10.5 S and 12.0 S, G4 forms of AChE and BuChE, and smaller amounts of 4.5 S and 5.5 S forms, G1 species of AChE and BuChE, were measured in S1. Application of Triton X‐114 phase partitioning and affinity chromatography on phenyl‐agarose to S1 revealed that 25% of the AChE and none of the BuChE molecules displayed amphiphilic properties. Analysis of the enzyme activity retained by the phenyl‐agarose showed that G1 AChE constituted the bulk of the amphiphilic molecules released without detergent. Main G4 forms of AChE and BuChE were found in the S2 extract. Eighty and 45% of the AChE and BuChE activities in S2 were measured in the detergent‐rich phase by Triton X‐114 phase partitioning. Thus, most of the AChE and about half of the BuChE molecules in S2 displayed amphiphilic properties. The main peak of BuChE, a 12.0 S form in gradients made with Triton X‐100, splits into two peaks of 9.5 S and 12.5 S in Brij 96‐containing gradients. This suggests that hydrophilic G4 BuChE forms are predominant in S1 and that hydrophilic and amphiphilic isoforms coexist in S2. © 1993 Wiley‐Liss, Inc.