bluuebunny/crossref_metadata_2025 huggingface dataset:
Cationic Lipids and Polymers Are Able to Enhance Adenoviral Infection of Cultured Mouse Myotubes
Authors: Dodds E. , Piper T. A. , Murphy S. J. , Dickson G.
Published: March 2003

Abstract: Adenovirus‐mediated gene transfer has been used to promote efficient expression of various reporter and therapeutic transgenes such as minidystrophin in skeletal muscle tissue. However, down‐regulation of the adenovirus internalisation receptors, αvβ3 and αvβ5, in adult myofibres and in mature cultured myotubes makes them less susceptible to infection than neonatal muscle or cultured myoblasts. It has been reported elsewhere that adenoviral transduction of cells that are normally refractory to infection can be enhanced by complexing virus particles with cationic lipids or cationic polymers. In this study we describe increased levels of adenovirusmediated transduction of cultured C2C12 myotubes, when the vector is complexed with either of the cationic lipids Lipofectamine or 1,3‐dioleoyloxy‐2‐(6‐carboxyspermyl)propylamide (DOSPER) or the cationic polymer polyethylenimine. The presence of polycations allowed a smaller dose of adenovirus vector to be used to attain the same level of infection seen with adenovirus alone, which has important relevance to future in vivo studies. Electron microscopic analysis of adenovirus/polycation complexes showed large aggregates as opposed to single adenovirus particles in the absence of polycations. Finally, by complexing adenovirus particles with polycations, partial protection against the neutralising effect of adenovirus antiserum was observed.
High‐Affinity Agonist Binding Correlates with Efficacy (Intrinsic Activity) at the Human Serotonin 5‐HT2A and 5‐HT2C Receptors: Evidence Favoring the Ternary Complex and Two‐State Models of Agonist Action
Authors: Fitzgerald Lawrence W. , Conklin Deborah S. , Krause Carol M. , Marshall Anne P. , Patterson John P. , Tran David P. , Iyer Geeta , Kostich Walter A. , Largent Brian L. , Hartig Paul R.
Published: March 2003

Abstract: Many modern models of receptor‐G protein function assume that there is a direct relationship between high‐affinity agonist binding and efficacy. The validity of this assumption has been recently questioned for the serotonin 5‐HT2A receptor. We examined the intrinsic activities of various ligands in activating phosphoinositide hydrolysis and measured their respective binding affinities to the high‐ and low‐affinity states of the 5‐HT2C (VNV isoform) and 5‐HT2A receptors. Ligand binding affinities for the high‐affinity state of the receptors were determined using 1‐(4‐[125I]iodo‐2,5‐dimethoxyphenyl)‐2‐aminopropane, whereas [3H]mesulergine and N‐[3H]methylspiperone were used, in the presence of excess guanine nucleotide [guanosine 5′‐O‐(3‐thiotriphosphate)], to define binding to the low‐affinity state of the 5‐HT2C and 5‐HT2A receptors, respectively. Antagonists labeled the high‐ and low‐affinity states of each receptor with comparable affinities. Previously identified inverse agonists of the 5‐HT2C receptor behaved as silent antagonists in our systems even when the receptor was over‐expressed at a relatively high density. In contrast, the ability of agonists to bind differentially to the high‐ and low‐affinity states of the 5‐HT2A and 5‐HT2C receptors was highly correlated (r2= 0.86 and 0.96, respectively) with their intrinsic activities. These data suggest that high‐affinity agonist states can account for agonist efficacy at human 5‐HT2A or 5‐HT2C receptors without the need for considering additional transition or active states of the receptor‐ligand complex. The procedure described herein may expedite drug discovery efforts by predicting intrinsic activities of ligands solely from ligand binding assays.
Murine Microglial Cells Produce and Respond to Interleukin‐18
Authors: Prinz Marco , Hanisch Uwe‐Karsten
Published: March 2003

Abstract: Interleukin (IL)‐18 (interferon‐δ‐inducing factor or IL‐1δ) belongs structurally to the IL‐1 cytokine family and shares biological properties with IL‐12. Expression, intracellular signaling, and functional relevance of IL‐18 within the CNS are mostly unknown. We show that IL‐18 protein is synthesized within mouse brain, preferentially during early postnatal stages, and that microglial cells but not astrocytes are a potential source. IL‐18 is produced by cultured microglia on exposure to lipopolysaccharide (LPS). Microglia also express major components of the IL‐1/IL‐18 receptor system. On IL‐18 stimulation, microglial IL‐1 receptor‐associated kinase (IRAK) can be coprecipitated with tumor necrosis factor (TNF) receptor‐associated factor 6 (TRAF6) but not with IL‐1 receptor type I, indicating that IRAK recruits TRAF6 during IL‐18 signaling. IL‐18 inhibits the LPS‐induced release of IL‐12 and attenuates that of TNF‐α, whereas the production of IL‐6 and macrophage inflammatory protein‐1 a is only marginally affected. IL‐18 may play a role during CNS development and can be produced by activated microglia, thus probably contributing to immune and inflammatory processes in the brain.
Differential Activation of Mitogen‐Activated Protein Kinase Pathways in PC12 Cells by Closely Related α1‐Adrenergic Receptor Subtypes
Authors: Zhong Hongying , Minneman Kenneth P.
Published: March 2003

Abstract: Coupling of the three known α1‐adrenergic
receptor (α1‐AR) subtypes to mitogen‐activated
protein kinase (MAPK) pathways were studied in stably transfected PC12 cells.
Subclones stably expressing α1A‐,
α1B‐, and α1D‐ARs
under control of an inducible promoter, or at high and low receptor density,
were isolated and characterized. Radioligand binding showed similar ranges of
expression of each subtype. Norepinephrine (NE) increased inositol phosphate
formation and intracellular Ca2+ level in these cells in a manner
dependent on receptor density. However, α1A‐ARs
activated these second messenger responses more effectively than
α1B‐ARs, whereas α1D‐ARs were
least effective. NE stimulated activation of extracellular signal‐regulated
kinases (ERKs) in cells expressing all three α1‐AR
subtypes, although α1A‐ and
α1B‐ARs caused larger ERK activation than did
α1D‐ARs. Nerve growth factor (NGF) caused similar
levels of ERK activation in all subclones. NE also activated p38 MAPK in
α1A‐ and α1B‐ but not
α1D‐transfected cells and activated c‐Jun
NH2‐terminal kinase (JNK) only in α1A‐transfected
cells. NE, but not NGF, strongly stimulated tyrosine phosphorylation of a
70‐kDa protein only in α1A‐transfected PC12 cells. NE caused
neutrite outgrowth only in α1A‐expressing PC12 cells, but not
in α1B‐ or α1D‐transfected cells,
whereas NGF caused neurite outgrowth in all cells. These studies show that
α1A‐ARs activate all three MAPK pathways,
α1B‐ARs activate ERKs and p38 but not JNKs, and
α1D‐ARs only activate ERKs. Only the
α1A‐AR‐expressing cells differentiated in response to NE. The relationship of these responses to second messenger pathways activated by these subtypes is discussed.
The Glutathione System of Peroxide Detoxification Is Less Efficient in Neurons than in Astroglial Cells
Authors: Dringen Ralf , Kussmaul Lothar , Gutterer Jan Mirko , Hirrlinger Johannes , Hamprecht Bernd
Published: March 2003

Abstract: The ability of neurons to detoxify exogenously applied
peroxides was analyzed using neuron‐rich primary cultures derived from
embryonic rat brain. Incubation of neurons with H2O2 at
an initial concentration of 100 μM (300 nmol/3 ml) led to a
decrease in the concentration of the peroxide, which depended strongly on the
seeding density of the neurons. When 3 × 106 viable cells
were seeded per dish, the half‐time for the clearance by neurons of
H2O2 from the incubation buffer was 15.1 min.
Immediately after application of 100 μM H2O2
to neurons, glutathione was quickly oxidized. After incubation for 2.5 min,
GSSG accounted for 48% of the total glutathione. Subsequent removal of
H2O2 caused an almost complete regeneration of the
original ratio of GSH to GSSG within 2.5 min. Compared with confluent
astroglial cultures, neuron‐rich cultures cleared H2O2
more slowly from the incubation buffer. However, if the differences in protein
content were taken into consideration, the ability of the cells to dispose of
H2O2 was identical in the two culture types. The
clearance rate by neurons for H2O2 was strongly reduced
in the presence of the catalase inhibitor 3‐aminotriazol, a situation
contrasting with that in astroglial cultures. This indicates that for the
rapid clearance of H2O2 by neurons, both glutathione peroxidase and catalase are essential and that the glutathione system cannot functionally compensate for the loss of the catalase reaction. In addition, the protein‐normalized ability of neuronal cultures to detoxify exogenous cumene hydroperoxide, an alkyl hydroperoxide that is reduced exclusively via the glutathione system, was lower than that of astroglial cells by a factor of 3. These results demonstrate that the glutathione system of peroxide detoxification in neurons is less efficient than that of astroglial cells.
Effects of Erythropoietin on Neuronal Activity
Authors: Koshimura Kunio , Murakami Yoshio , Sohmiya Motoi , Tanaka Junko , Kato Yuzuru
Published: March 2003

Abstract: Recently, erythropoietin (EPO) receptors and synthesis of
EPO have been identified in the brain. To clarify the effects of EPO on
neuronal cells, we investigated the effects of EPO on Ca2+ uptake,
intracellular Ca2+ concentration, membrane potential, cell
survival, release and biosynthesis of dopamine, and nitric oxide (NO)
production in differentiated PC12 cells, which possess EPO receptors. EPO
(10‐12‐10‐10M) increased
45Ca2+ uptake and intracellular Ca2+
concentration in PC12 cells in a dose‐related manner; these increases were
inhibited by nicardipine (1 μM) or anti‐EPO antibody (1:100
dilution). EPO induced membrane depolarization in PC12 cells. After a 5‐day
culture without serum and nerve growth factor (NGF), viable cell number
decreased to 50% of that of the control cells cultured with serum and NGF. EPO
(10‐13‐10‐10M) increased the number of viable
cells cultured without serum and NGF; this increase was blunted by nicardipine
or anti‐EPO antibody. Incubation with EPO (10‐13‐10‐10M) stimulated mitogen‐activated protein kinase activity in PC12
cells. EPO (10‐13‐10‐10M) increased dopamine
release from PC12 cells and tyrosine hydroxylase activity; these increases
were sensitive to nicardipine or anti‐EPO antibody. Following a 4‐h incubation
with EPO (10‐14‐10‐10M), NO production was
increased, which was blunted by nicardipine and anti‐EPO antibody. In
contrast, maximal NO synthase activity was not changed by EPO. These results
suggest that EPO stimulates neuronal function and viability via activation of
Ca2+ channels.
Differential Effects of Glial Cell Line‐Derived Neurotrophic Factor and Neurturin on Developing and Adult Substantia Nigra Dopaminergic Neurons
Authors: Åkerud Peter , Alberch Jordi , Eketjäll Susanna , Wagner Joseph , Arenas Ernest
Published: March 2003

Abstract: Neurturin (NTN) and glial cell line‐derived neurotrophic factor (GDNF), two members of the GDNF family of growth factors, exert very similar biological activities in different systems, including the substantia nigra. Our goal in the present work was to compare their function and define whether nonoverlapping biological activities on midbrain dopaminergic neurons exist. We first found that NTN and GDNF are differentially regulated during postnatal development. NTN mRNA progressively decreased in the ventral mesencephalon and progressively increased in the striatum, coincident with a decrease in GDNF mRNA expression. This finding suggested distinct physiological roles for each factor in the nigrostriatal system. We therefore examined their function in ventral mesencephalon cultures and found that NTN promoted survival comparable with GDNF, but only GDNF induced sprouting and hypertrophy of developing dopaminergic neurons. We subsequently examined the ability of NTN to prevent the 6‐hydroxydopamine‐induced degeneration of adult dopaminergic neurons in vivo. Fibroblasts genetically engineered to deliver high levels of GDNF or NTN were grafted supranigrally. NTN was found to be as potent as GDNF at preventing the death of nigral dopaminergic neurons, but only GDNF induced tyrosine hydroxylase staining, sprouting, or hypertrophy of dopaminergic neurons. In conclusion, our results show selective survival‐promoting effects of NTN over wider survival, neuritogenic, and hypertrophic effects of GDNF on dopaminergic neurons in vitro and in vivo. Such differences are likely to underlie unique roles for each factor in postnatal development and may ultimately be exploited in the treatment of Parkinson’s disease.
Effect of Dibutyryl Cyclic AMP on the Kinetics of myo‐Inositol Transport in Cultured Astrocytes
Authors: Isaacks Russell E. , Bender Alexander S. , Reuben Jayne S. , Kim Chang Y. , Shi You F. , Norenberg Michael D.
Published: March 2003

Abstract: Dibutyryl cyclic AMP (dBcAMP) is known to induce maturation and differentiation in astrocytes. As myo‐inositol is an important osmoregulator in astrocytes, we examined the effects of maturation and biochemical differentiation on the kinetic properties of myo‐inositol transport. Treatment of astrocytes with dBcAMP significantly decreased the Vmax of myo‐inositol uptake, but the effect on Km was not significant. The myo‐inositol content of astrocytes was significantly decreased in cells treated for 5 days with dBcAMP as compared with untreated controls. Maximum suppression of myo‐inositol uptake occurred 7 days after exposure of astrocytes to dBcAMP; this was gradually reversible when dBcAMP was removed from the medium. After exposure to hypertonic medium for 6 h, mRNA expression of the myo‐inositol co‐transporter was diminished by ∼36% in astrocytes treated with dBcAMP as compared with untreated cells. It appears that myo‐inositol transporters in astrocytes treated with dBcAMP are either decreased in number or inactivated during maturation and differentiation, suggesting that the stage of differentiation and biochemical maturation of astrocytes is an important factor in osmoregulation.
Neurotrophins Differentially Regulate the Survival and Morphological Complexity of Human CNS Model Neurons
Authors: Piontek Jörg , Chen Chi Chiung , Kempf Martina , Brandt Roland
Published: March 2003

Abstract: To determine the effect of neurotrophins on the survival and morphological differentiation of CNS neurons, we examined NT2‐N cells, which provide a unique culture model for terminally differentiated and polar human neurons. Here we report the development of conditions for the long‐term culture of NT2‐N cells in low density and in chemically defined medium. We show that NT2‐N cells express mRNAs for TrkA, TrkB, and TrkC tyrosine kinase receptors and the low‐affinity nerve growth factor receptor (p75NTR). All members of the nerve growth factor‐related family of neurotrophic factors promote neuronal survival in long‐term cultures with ∼1 ng/ml for half‐maximal survival. At high concentrations (>20 ng/ml), the neurotrophins reversed the survival‐promoting effect as judged by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] conversion. In contrast to the uniform effect of all neurotrophins on neuronal survival, brain‐derived neurotrophic factor selectively induced an increased dendritic complexity. These results demonstrate that NT2‐N cells provide a useful model to analyze the effect of neurotrophins on the survival and morphological differentiation of CNS neurons in vitro. In addition, the data indicate that neuronal survival and the development of morphological complexity are differentially regulated in a multireceptor context.
Intracellular Redox State Determines Whether Nitric Oxide Is Toxic or Protective to Rat Oligodendrocytes in Culture
Authors: Rosenberg Paul A. , Li Ya , Ali Sanjida , Altiok Nedret , Back Stephen A. , Volpe Joseph J.
Published: March 2003

Abstract : We found that several nitric oxide donors had similar

potency in killing mature and immature forms of oligodendrocytes (OLs).

Because of the possibility of interaction of nitric oxide with intracellular

thiols, we tested the effect of the nitrosonium ion donor

S‐nitrosylglutathione (SNOG) in OL cultures in the setting of cystine

deprivation, which has been shown to cause intracellular glutathione

depletion. Surprisingly, the presence of 200 μM SNOG completely

protected OLs against the toxicity of cystine depletion. This protection

appeared to be due to nitric oxide, because it could be blocked by hemoglobin

and potentiated by inclusion of superoxide dismutase. We tested the effect of

three additional NO• donors and found that protection was not

seen with diethylamine NONOate, a donor with a half‐life measured in minutes,

but was seen with dipropylenetriamine NONOate and diethylaminetriamine

NONOate, donors with half‐lives measured in hours. This need for donors with

longer half‐lives for the protective effect suggested that NO• was required when intracellular thiol concentrations were falling, a process evolving over hours in medium depleted of cystine. These studies suggest a novel protective role for nitric oxide in oxidative stress injury and raise the possibility that intracerebral nitric oxide production might be a mechanism of defense against oxidative stress injury in OLs.
Apoptotic Cell Death and Caspase‐3 Activation Induced by N‐Methyl‐d‐Aspartate Receptor Antagonists and Their Prevention by Insulin‐Like Growth Factor I
Authors: Takadera Tsuneo , Matsuda Ikumi , Ohyashiki Takao
Published: March 2003

Abstract : The effect of N‐methyl‐d‐aspartate

(NMDA) receptor antagonists on cell viability was studied in rat primary

cortical cells. NMDA antagonists [MK‐801 and 2‐amino‐5‐phosphonovalerate

(APV)] induced cell shrinkage, nuclear condensation or fragmentation, and

internucleosomal DNA fragmentation. Treatment of cells with MK‐801 (an NMDA

antagonist) for 1‐2 days induced apoptotic cell death in a dose‐dependent

manner (1 nM to 10 μM). NMDA (25 μM),

however, inhibited the MK‐801 (0.1 μM)‐induced apoptotic cell death. MK‐801 and APV decreased the concentration of intracellular calcium ion. Activation of caspase‐3 was accompanied by MK‐801‐induced cell death in a dose‐dependent manner, and an inhibitor of caspase‐3 reduced the cell death. Further, cycloheximide (0.2 μg/ml) completely protected the cells from MK‐801‐induced apoptotic cell death and caspase‐3 activation. Insulin‐like growth factor I completely attenuated MK‐801‐induced apoptotic cell death and caspase‐3 activation. These results demonstrated that the moderate NMDA receptor activation is probably involved in the survival signal of the neuron.
3′,5′‐Cyclic Guanosine Monophosphate Activates Mitogen‐Activated Protein Kinase in Rat Pinealocytes
Authors: Ho A. K. , Hashimoto K. , Chik C. L.
Published: March 2003

Abstract : The role of 3′,5′‐cyclic guanosine monophosphate (cGMP) in the activation of mitogen‐activated protein kinases (MAPKs) was investigated in rat pinealocytes. Treatment with dibutyryl cGMP (DBcGMP) dosedependently increased the phosphorylation of both p44 and p42 isoforms of MAPK. This effect of DBcGMP was abolished by PD98059 (a MAPK kinase inhibitor), H7 (a nonspecific protein kinase inhibitor), and KT5823 [a selective cGMP‐dependent protein kinase (PKG) inhibitor]. Elevation of cellular cGMP content by treatment with norepinephrine, zaprinast (a cGMP phosphodiesterase inhibitor), or nitroprusside was effective in activating MAPK. Natriuretic peptides that were effective in elevating cGMP levels in this tissue were also effective in activating MAPK. Our results indicate that, in this neuroendocrine tissue, the cGMP/PKG signaling pathway is an important mechanism used by hormones and neurotransmitters in activating MAPK.
Arachidonic Acid‐Induced Oxidative Injury to Cultured Spinal Cord Neurons
Authors: Toborek Michal , Malecki Andrzej , Garrido Rosario , Mattson Mark P. , Hennig Bernhard , Young Byron
Published: March 2003

Abstract : Spinal cord trauma can cause a marked release of free

fatty acids, in particular, arachidonic acid (AA), from cell membranes. Free

fatty acids, and AA by itself, may lead to secondary damage to spinal cord

neurons. To study this hypothesis, cultured spinal cord neurons were exposed

to increasing concentrations of AA (0.01‐10 μM). AA‐induced injury

to spinal cord neurons was assessed by measurements of cellular oxidative

stress, intracellular calcium levels, activation of nuclear factor‐κB

(NF‐κB), and cell viability. AA treatment increased cell intracellular

calcium concentrations and decreased cell viability. Oxidative stress

increased significantly in neurons exposed to 1 and 10 μM AA. In addition, AA treatment activated NF‐κB and decreased levels of the inhibitory subunit, IκB. It is interesting that manganese superoxide dismutase protein levels and levels of intracellular total glutathione increased in neurons exposed to this fatty acid for 24 h, consistent with a compensatory response to increased oxidative stress. These results strongly support the hypothesis that free fatty acids contribute to the tissue injury observed following spinal cord trauma.
Repeated Administration of Dexamethasone Increases Phosphoinositide‐Specific Phospholipase C Activity and mRNA and Protein Expression of the Phospholipase C β1 Isozyme in Rat Brain
Authors: Dwivedi Yogesh , Pandey Ghanshyam N.
Published: March 2003

Abstract : Altered hypothalamic‐pituitary‐adrenal (HPA) function has

been shown to be associated with changes in mood and behavior. The enzyme

phosphoinositide‐specific phospholipase C (PI‐PLC), an important component of

the PI signal transduction system, plays a major role in mediating various

physiological functions. In the present study, we investigated the effects of

a single dose and of repeated administration (0.5 or 1.0 mg/kg for 10 days) of

dexamethasone (DEX), a synthetic glucocorticoid, on PI‐PLC activity and on

expression of PLC isozymes (β1, δ1, and

γ1) in rat brain. Repeated administration of DEX (1.0 mg/kg)

caused a significant increase in PI‐PLC activity and in protein expression of

the PLC β1 isozyme in both membrane and cytosol fractions of

cortex and hippocampus ; however, the repeated administration of a smaller

dose of DEX (0.5 mg/kg) caused these changes only in hippocampus but not in

cortex. The increase in PLC β1 protein was associated with an

increase in its mRNA level, as measured by competitive RT‐PCR. A single

administration of DEX (0.5 or 1.0 mg/kg) to rats had no significant effects on

PI‐PLC activity or on the protein expression of PLC isozymes. These results

suggest that DEX up‐regulates PI‐PLC in rat brain, which presumably is due to

a selective increase in expression of the PLC β1 isozyme, and that these changes in PI‐PLC may be related to HPA axis‐mediated changes in mood and behavior.
Effects of Cannabinoids on Dopamine Release in the Corpus Striatum and the Nucleus Accumbens In Vitro
Authors: Szabo Bela , Müller Tobias , Koch Helga
Published: March 2003

Abstract : Cannabinoid receptors are widely distributed in the nuclei of the extrapyramidal motor and mesolimbic reward systems ; their exact functions are, however, not known. The aim of the present study was to characterize the effects of cannabinoids on the electrically evoked release of endogenous dopamine in the corpus striatum and the nucleus accumbens. In rat brain slices dopamine release elicited by single electrical pulses was determined by fast cyclic voltammetry. Dopamine release was markedly inhibited by the OP2 opioid receptor agonist U‐50488 and the D2/D3 dopamine receptor agonist quinpirole, indicating that our method is suitable for studying presynaptic modulation of dopamine release. In contrast, the CB1/CB2 cannabinoid receptor agonists WIN55212‐2 (10‐6M) and CP55940 (10‐6‐10‐5M) and the CB1 cannabinoid receptor antagonist SR141716A (10‐6M) had no effect on the electrically evoked dopamine release in the corpus striatum and the nucleus accumbens. The lack of a presynaptic effect on terminals of nigrostriatal and mesolimbic dopaminergic neurons is in accord with the anatomical distribution of cannabinoid receptors : The perikarya of these neurons in the substantia nigra and the ventral tegmental area do not synthesize mRNA, and hence protein, for CB1 and CB2 cannabinoid receptors. It is therefore unlikely that presynaptic modulation of dopamine release in the corpus striatum and the nucleus accumbens plays a role in the extrapyramidal motor and rewarding effects of cannabinoids.
Pharmacological Characterization of Morphine‐Induced In Vivo Release of Cholecystokinin in Rat Dorsal Horn
Authors: Gustafsson Henrik , Afrah Abdullahi , Brodin Ernst , Stiller Carl‐Olav
Published: March 2003

Abstract : Previous studies indicate that an increased release of cholecystokinin (CCK) in response to morphine administration may counteract opioid‐induced analgesia at the spinal level. In the present study we used in vivo microdialysis to demonstrate that systemic administration of antinociceptive doses of morphine (1‐5 mg/kg, s.c.) induces a dose‐dependent and naloxone‐reversible release of CCK‐like immunoreactivity (CCK‐LI) in the dorsal horn of the spinal cord. A similar response could also be observed following perfusion of the dialysis probe for 60 min with 100 μM but not with 1 μM morphine. The CCK‐LI release induced by morphine (5 mg/kg, s.c.) was found to be calcium‐dependent and tetrodotoxin‐sensitive (1 μM in the perfusion medium). Topical application of either the L‐type calcium channel blocker verapamil (50 μg) or the N‐type calcium channel blocker ω‐conotoxin GVIA (0.4 μg) onto the dorsal spinal cord completely prevented the CCK‐LI release induced by morphine (5 mg/kg, s.c.). Our data indicate that activation of L ‐ and N‐type calcium channels is of importance for morphineinduced CCK release, even though the precise site of action of morphine in the dorsal horn remains unclear. The present findings also suggest a mechanism for the potentiation of opioid analgesia by L‐and N‐type calcium channel blocking agents.
Internalization and Sorting of Plasma Membrane Sphingolipid Analogues in Differentiating Oligodendrocytes
Authors: Watanabe Rikio , Asakura Kunihiko , Rodriguez Moses , Pagano Richard
Published: March 2003

Abstract : We studied the formation of early endosomes in

differentiating oligodendrocytes and type‐2 astrocytes, which are derived from

common precursor cells in rat neonates, using fluorescent analogues of

lactosylceramide (LacCer) and sulfatide labeled with

4,4‐difluoro‐5,7‐dimethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene‐3‐pentanoic

acid (BODIPY FL C5). These sphingolipid analogues exhibit a

concentration‐dependent shift in their fluorescence emission maximum from

green to red wavelengths that can be used to estimate the relative

concentration of an analogue in the intracellular membranes of living

cells by quantitative fluorescence microscopy. When oligodendrocytes at

various stages of differentiation were incubated with 1 μM

BODIPY‐sphingolipid at 10°C and washed, yellow/green plasma membrane

fluorescence was observed. Quantitative studies confirmed that the amount of

BODIPY‐LacCer or‐sulfatide incorporated into the plasma membrane of a given

cell type was identical. When these cells were subsequently warmed to 37°C

for 2‐10 min to allow internalization to occur, the BODIPY‐sphingolipid

analogues were distributed in a punctate pattern throughout the cytoplasm.

Within individual cells labeled with BODIPY‐sulfatide, some endosomes

exhibited green fluorescence, whereas others emitted red/orange fluorescence.

In contrast, when BODIPY‐LacCer was used, only green endosomes were observed.

Although this phenomenon could be observed at earlier stages of

differentiation, it was most obvious in mature oligodendrocytes, where

quantitative measurements of the red/green ratio of individual endosomes

suggested about a threefold difference between the concentration of the LacCer

and sulfatide analogues in endosomes. These results suggest that “lipid

sorting” takes place during endocytosis in mature oligodendrocytes,

resulting in selective exclusion of certain lipid species during the internalization process. This sorting event may result in the net addition of lipids to the differentiated oligodendrocyte plasma membrane.
Decrease in Peptide Methionine Sulfoxide Reductase in Alzheimer's Disease Brain
Authors: Gabbita S. Prasad , Aksenov Michael Y. , Lovell Mark A. , Markesbery William R.
Published: March 2003

Abstract : Previous studies have shown that the pathophysiology of

Alzheimer's disease (AD) is linked to oxidative stress. Oxidative damage to

different biomolecular components of the brain is a characteristic feature of

AD. Recent evidence suggests that methionine may act as an antioxidant defense

molecule in proteins by its ability to scavenge oxidants and, in the process,

undergo oxidation to form methionine sulfoxide. The enzyme peptide, methionine

sulfoxide reductase (MsrA), reverses methionine sulfoxide back to methionine,

which once again is able to scavenge oxidants. The purpose of this study was

to measure the activity of MsrA in the brain of AD patients compared with

control subjects. Our results showed that there was a decline in MsrA activity

in all brain regions studied in AD and this decline reached statistical

significance in the superior and middle temporal gyri (p < 0.001),

inferior parietal lobule (p < 0.05), and the hippocampus

(p < 0.05) in AD. An elevation of protein carbonyl content was found in all brain regions except the cerebellum in AD and reached statistical significance in the superior and middle temporal gyri and hippocampus. Messenger RNA analysis suggests that the loss in enzyme activity may be the result of a posttranslational modification of MsrA or a defect of translation resulting in inferior processing of the MsrA mRNA. Our results suggest that a decline in MsrA activity could reduce the antioxidant defenses and increase the oxidation of critical proteins in neurons in the brain in AD.
{Different Signalling Pathways Mediate the Opposite Effects of Endogenous Versus Exogenous Nitric Oxide on Hydroperoxide Toxicity in CHP100 neuroblastoma Cells
Authors: Guidarelli Andrea , Clementi Emilio , Sciorati Clara , Cantoni Orazio
Published: March 2003

Abstract : The results presented in this study indicate that the

toxic response brought about by increasing concentrations of

tert‐butylhydroperoxide in CHP100 cells was mitigated significantly

by exogenously added nitric oxide donors via a cyclic GMP‐independent

mechanism. In contrast with these results, endogenous nitric oxide generated

by the Ca2+‐mobilizing agent caffeine was found to increase

hydroperoxide toxicity. Under these conditions, nitric oxide was not directly

toxic to the cells. Rather, nitric oxide was found to promote the

caffeine‐mediated release of Ca2+ from ryanodine‐sensitive

Ca2+ stores via a cyclic GMP‐independent mechanism. Release of the

cation from ryanodine‐sensitive Ca2+ stores was causally linked

with the caffeine/nitric oxide‐mediated enhancement of

tert‐butylhydroperoxide toxicity. It is concluded that endogenous and

exogenous nitric oxide activate diverging signalling pathways independent of

cyclic GMP formation and causing opposite effects on the toxic response evoked

by tert‐butylhydroperoxide in CHP100 cells.
Prolactin Induction of Nitric Oxide Synthase in Rat C6 Glioma Cells
Authors: Raso Giuseppina Mattace , Meli Rosaria , Gualillo Oreste , Pacilio Maria , Carlo Raffaele Di
Published: March 2003

Abstract : We have examined the neuroimmunoregulatory function of

prolactin (PRL) on astrocytic inducible nitric oxide synthase (iNOS)

expression in the C6 glioma cell line. After 24 h of PRL (5‐100 nM)

stimulation, a concentration‐dependent increase of NO release, evaluated as

nitrite, was observed in C6 culture medium. Moreover, both NO release and iNOS

expression induced by interferon‐γ (250 U/ml) were enhanced by PRL

(18‐100 nM). PRL‐induced NO release was inhibited by dexamethasone, an inhibitor of de novo iNOS synthesis. We used erbstatin (5 μg/ml), a potent inhibitor of protein tyrosine kinases, to test whether these proteins were required for signaling events evoked by PRL in these cells. This inhibitor was able to inhibit completely the PRL‐induced NO production and iNOS expression. In conclusion, we provide evidence that NO production in glial cells can be regulated not only by cytokines but also by neuroimmunoregulatory hormones such as PRL, which is present in normal brain but may be elevated in several pathological states.
Isoform Pattern of 14‐3‐3 Proteins in the Cerebrospinal Fluid of Patients with Creutzfeldt‐Jakob Disease
Authors: Wiltfang J. , Otto M. , Baxter H. C. , Bodemer M. , Steinacker P. , Bahn E. , Zerr I. , Kornhuber J. , Kretzschmar H. A. , Poser S. , Rüther E. , Aitken A.
Published: March 2003

Abstract : Two‐dimensional polyacrylamide gel electrophoresis of CSF has been used in the diagnosis of Creutzfeldt‐Jakob disease (CJD). One of the two diagnostic protein spots was identified as isoform(s) of the 14‐3‐3 family of abundant brain proteins. This has led to the development of one‐dimensional 14‐3‐3 sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot, which is currently used to support the diagnosis of CJD. In the present study employing western blot analysis, we have identified the panel of 14‐3‐3 isoforms that appear in the CSF of 10 patients with CJD compared with 10 patients with other dementias. The results clearly show that the 14‐3‐3 isoforms β, γ, ε, and η are present in the CSF of patients with CJD and can be used to differentiate other dementias. 14‐3‐3η also gave a baseline signal in all patients with other dementias, including six patients with Alzheimer's disease. The presence of 14‐3‐3η in the CSF of a patient with herpes simplex encephalitis was particularly noteworthy. This study has determined that isoform‐specific 14‐3‐3 antibodies against β, γ, and ε should be considered for the neurochemical differentiation of CJD from other neurodegenerative diseases.
Salmonid Olfactory System‐Specific Protein (N24) Exhibits Glutathione S‐Transferase Class Pi‐Like Structure
Authors: Kudo Hideaki , Ueda Hiroshi , Mochida Kazuhiko , Adachi Shinji , Hara Akihiko , Nagasawa Hiromichi , Doi Yoshiaki , Fujimoto Sunao , Yamauchi Kohei
Published: March 2003

Abstract: A salmonid olfactory system‐specific protein (N24) that has been identified in lacustrine sockeye salmon (Oncorhynchus nerka) was characterized by biochemical and molecular biological techniques. N24 is a homodimer, and the intact molecular mass is estimated as ∼43.3 kDa by gel filtration. Furthermore, N24 was located only in the cytosolic fraction of the olfactory tissues as determined by subcellular fractionation. cDNA encoding the lacustrine sockeye salmon N24 was isolated and sequenced. This cDNA contained a coding region encoding 216 amino acid residues and the molecular mass of this protein is calculated to be 242,224.77. The protein and nucleotide sequencing demonstrates the existence of a remarkable homology between N24 and glutathione S‐transferase (GST; EC 2.5.1.18) class pi enzymes. Northern analysis showed that N24 mRNA with a length of 950 bases is expressed in lacustrine sockeye salmon olfactory epithelium. Olfactory receptor cells showed strong hybridization signals for N24 mRNA in the olfactory epithelium. N24 demonstrated glutathione binding activity in affinity‐purified GST column experiments. The present study describes for the first time cDNA cloning of GST in fish olfactory epithelium.
Agonist Stimulation of the Serotonin1A Receptor Causes Suppression of Anoxia‐Induced Apoptosis via Mitogen‐Activated Protein Kinase in Neuronal HN2‐5 Cells
Authors: Adayev Tatyana , El‐Sherif Yasir , Barua Madhabi , Penington Nicholas J. , Banerjee Probal
Published: March 2003

Abstract: Previous studies have indicated that stimulation of neuronal inhibitory receptors, such as the serotonin1A receptor (5‐HT1A‐R), could cause attenuation of the activity of both N‐type Ca2+ channels and N‐methyl‐D‐aspartic acid receptors, thus resulting in protection of neurons against excitotoxicity. The purpose of this study was to investigate if the 5‐HT1A‐R is also coupled to an alternative pathway that culminates in suppression of apoptosis even in cells that are deficient in Ca2+ channels. Using a hippocampal neuron‐derived cell line (HN2‐5) that is Ca2+ channel‐deficient, we demonstrate here that an alternative pathway is responsible for 5‐HT1A‐R‐mediated protection of these cells from anoxia‐triggered apoptosis, assessed by deoxynucleotidyl‐transferase‐mediated dUTP nick end‐labeling (TUNEL). The 5‐HT1A‐R agonist‐evoked protection was eliminated in the presence of pertussis toxin and also required phosphorylation‐mediated activation of mitogen‐activated protein kinase (MAPK), as evidenced by the elimination of the agonist‐elicited rescue of neuronal cells by the MAPK kinase inhibitor PD98059 but not by the phosphatidylinositol 3‐kinase (PI‐3K) inhibitor wortmannin. Furthermore, agonist stimulation of the 5‐HT1A‐R caused a 60% inhibition of anoxia‐stimulated caspase 3‐like activity in the HN2‐5 cells, and this inhibition was abrogated by PD98059 but not by wortmannin. Although agonist stimulation of the 5‐HT1A‐R caused an activation of PI‐3Kγ in HN2‐5 cells, our results showed that this PI‐3Kγ activity was not linked to the 5‐HT1A‐R‐promoted regulation of caspase activity and suppression of apoptosis. Thus, in the neuronal HN2‐5 cells, agonist binding to the 5‐HT1A‐R results in MAPK‐mediated inhibition of a caspase 3‐like enzyme and a 60‐70% suppression of anoxia‐induced apoptosis through a Ca2+ channel‐independent pathway.
Changes in Serotonin2A and GABAA Receptors in Schizophrenia
Authors: Dean Brian , Hussain Tabasum , Hayes Wendy , Scarr Elizabeth , Kitsoulis Susie , Hill Christine , Opeskin Kenneth , Copolov David L.
Published: March 2003

Abstract: Having shown a decrease in serotonin2A receptors in the dorsolateral prefrontal cortex (DLPFC) from schizophrenic subjects, we have now determined if this change was reflective of widespread changes in neurochemical markers in DLPFC in schizophrenia. In Brodmann’s area (BA) 9 from 19 schizophrenic and 19 control subjects, we confirmed a decrease in the density of [3H]ketanserin binding to serotonin2A receptors in tissue from the schizophrenic subjects [39 ± 3.3 vs. 60 ± 3.6 fmol/mg estimated tissue equivalents (ETE); p < 0.005]. In addition, the density of [3H]muscimol binding to GABAA receptors was increased in the schizophrenic subjects (526 ± 19 vs. 444 ± 28 fmol/mg ETE; p < 0.02). [3H]YM‐09151‐2, N‐[1‐(2‐thienyl)cyclohexyl]‐3,4‐[3H]piperidine, [3H]SCH 23390, [3H]mazindol, and NG‐nitro‐L‐[3H]arginine binding to BA 9 did not differ between groups, and there was no specific binding of [3H]raclopride or 7‐hydroxy‐2‐(di‐n‐[3H]propylamino)tetralin to BA 9 from either cohort of subjects. This suggests the density of dopamine D1‐like and NMDA receptors, the dopamine transporter, and nitric oxide synthase activity are not altered in BA 9 from schizophrenic subjects. The selective nature of the changes in serotonin2A and GABAA receptors in DLPFC could indicate that these changes are involved in the pathology of schizophrenia.
Prominent 85‐kDa Oligomannosidic Glycoproteins of Rat Brain Are Signal Regulatory Proteins and Include the SHP Substrate‐1 for Tyrosine Phosphatases
Authors: Bartoszewicz Z. P. , Jaffe H. , Sasaki M. , Möller J. R. , Stebbins J. W. , Gebrekristos H. , Quarles R. H.
Published: March 2003

Abstract: The glycoprotein component in rat brain reacting most strongly with Galanthus nivalis agglutinin (GNA) on western blots migrates as an 85‐kDa band. GNA identifies mannose‐rich oligosaccharides because it is highly specific for terminal α‐mannose residues. After purification of this 85‐kDa glycoprotein band by chromatography on GNA‐agarose and preparative gel electrophoresis, binding of other lectins demonstrated the presence of fucose and a trace of galactose, but no sialic acid. Treatment with N‐Glycanase or endoglycosidase H produced a 65‐kDa band, indicating that it consisted of about one‐fourth N‐linked oligomannosidic carbohydrate moieties. High‐performance anion‐exchange chromatography and fluorescence‐assisted carbohydrate electrophoresis indicated that the major carbohydrate moiety is a heptasaccharide with the structure Manα1‐6(Manα1‐3)Manα1‐6(Manα1‐3)Manβ1‐4Glc‐NAcβ1‐4GlcNAc (Man5GlcNAc2). Determination of amino acid sequences of peptides produced by endoproteinase digestion demonstrated that this 85‐kDa mannose‐rich glycoprotein component contained the SHP substrate‐1 for phosphotyrosine phosphatases and at least one other member of the signal‐regulatory protein (SIRP) family. The unusually high content of oligomannosidic carbohydrate moieties on these receptor‐like members of the immunoglobulin superfamily in neural tissue could be of functional significance for intercellular adhesion or signaling.
Differential Subcellular Redistribution of Protein Kinase C Isozymes in the Rat Hippocampus Induced by Kainic Acid
Authors: McNamara Robert K. , Wees Elizabeth A. , Lenox Robert H.
Published: March 2003

Abstract: Protein kinase C (PKC) consists of a family of Ca2+/phospholipid‐dependent isozymes that has been implicated in the delayed neurotoxic effects of glutamate in vitro. In the present study, we assessed the effect of the glutamate analogue kainic acid (KA) on the subcellular expression of PKC isozymes in the hippocampus (HPC) in the period preceding (0.5, 1.5, 12, and 24 h) and during (120 h) hippocampal necrosis using western blot analysis and PKC isozyme‐specific antibodies. Before subcellular fractionation (cytosol + membrane), hippocampi were microdissected into “HPC” (fields CA1‐CA3) and “dentate gyrus” (DG; granule cells + hilus) regions. Four general patterns of alterations in PKC isozyme expression/distribution were observed following KA treatment. The first pattern was a relative stability in expression following KA treatment and was most apparent for cytosol PKCα (HPC + DG) and membrane (HPC) and cytosol (DG) PKCβII. The second pattern, observed with PKCγ and PKCε, was characterized by an initial increase in expression in both membrane and cytosolic fractions before seizure activity (0.5 h) followed by a gradual decrease until significant reductions are observed by 120 h. The third pattern, exhibited by PKCδ, involved an apparent translocation, increasing in the membrane and decreasing in the cytosol, followed by down‐regulation in both fractions and subsequent recovery. The fourth pattern was observed with PKCζ only and entailed a significant reduction in expression before and during limbic motor seizures followed by a dramatic fivefold increase in the membrane fraction during the period of hippocampal necrosis (120 h). Although these patterns did not segregate according to conventional PKC isozyme classifications, they do indicate dynamic isozyme‐specific regulation by KA. The subcellular redistribution of PKC isozymes may contribute to the histopathological sequelae produced by KA in the hippocampus and may model the pathogenesis associated with diseases involving glutamate‐induced neurotoxicity.
The Mood‐Stabilizing Agent Valproate Inhibits the Activity of Glycogen Synthase Kinase‐3
Authors: Chen Guang , Huang Li‐Dong , Jiang Yi‐Ming , Manji Husseini K
Published: March 2003

Abstract : Valproic acid (VPA) is a potent broad‐spectrum
anti‐epileptic with demonstrated efficacy in the treatment of bipolar
affective disorder. It has previously been demonstrated that both VPA and
lithium increase activator protein‐1 (AP‐1) DNA binding activity, but the
mechanisms underlying these effects have not been elucidated. However, it is
known that phosphorylation of c‐jun by glycogen synthase kinase
(GSK)‐3β inhibits AP‐1 DNA binding activity, and lithium has recently
been demonstrated to inhibit GSK‐3β. These results suggest that lithium
may increase AP‐1 DNA binding activity by inhibiting GSK‐3β. In the
present study, we sought to determine if VPA, like lithium, regulates GSK‐3.
We have found that VPA concentration‐dependently inhibits both GSK‐3α
and ‐3β, with significant effects observed at concentrations of VPA
similar to those attained clinically. Incubation of intact human neuroblastoma
SH‐SY5Y cells with VPA results in an increase in the subsequent in vitro
recombinant GSK‐3β‐mediated 32P incorporation into two
putative GSK‐3 substrates (~85 and 200 kDa), compatible with inhibition of
endogenous GSK‐3β by VPA. Consistent with GSK‐3β inhibition, incubation of SH‐SY5Y cells with VPA results in a significant time‐dependent increase in both cytosolic and nuclear β‐catenin levels. GSK‐3β plays a critical role in the CNS by regulating various cytoskeletal processes as well as long‐term nuclear events and is a common target for both lithium and VPA ; inhibition of GSK‐3β in the CNS may thus underlie some of the long‐term therapeutic effects of mood‐stabilizing agents.
S100β Induction of the Proinflammatory Cytokine Interleukin‐6 in Neurons
Authors: Yuekui Li , Barger Steven W. , Liu Ling , Mrak Robert E. , Griffin W. Sue T.
Published: March 2003

Abstract: Levels of the neurotrophic cytokine S100β and the proinflammatory cytokine interleukin‐6 (IL‐6) are both elevated in Alzheimer’s brain, and both have been implicated in β‐amyloid plaque formation and progression. We used RT‐PCR and electrophoretic mobility shift assay to assess S100β induction of IL‐6 expression and the role of κB‐dependent transcription in this induction in neuron‐enriched cultures and in neuron‐glia mixed cultures from fetal rat cortex. S100β (10 or 100 ng/ml × 24 h) increased IL‐6 mRNA levels two‐ and fivefold, respectively (p < 0.05 in each case), and S100β (100‐1,000 ng/ml) induced increases in medium levels of biologically active IL‐6 (30‐80%). Combined in situ hybridization and immunohistochemistry preparations localized IL‐6 mRNA to neurons in these cultures. S100β induction of IL‐6 expression correlated with an increase in DNA binding activity specific for a κB element and was inhibited (75%) by suppression of κB binding with double‐stranded “decoy” oligonucleotides. The low levels of S100β required to induce IL‐6 overexpression in neurons, shown here, suggest that overexpression of S100β induces neuronal expression of IL‐6 and of IL‐6‐induced neurodegenerative cascades in Alzheimer’s disease.
Reserpine Pretreatment Prevents Increases in Extracellular Striatal Dopamine Following L‐DOPA Administration in Rats with Nigrostriatal Denervation
Authors: Kannari Kazuya , Tanaka Hiroyasu , Maeda Tetsuya , Tomiyama Masahiko , Suda Toshihiro , Matsunaga Muneo
Published: March 2003

Abstract: The influence of L‐DOPA and reserpine on extracellular dopamine (DA) levels in the striatum of intact and dopaminergic denervated rats was studied using the brain microdialysis technique. In intact rats, reserpine (5 mg/kg s.c.) reduced extracellular DA levels to 4% of basal values. L‐DOPA (50 mg/kg i.p.) had no effect on extracellular DA levels in reserpine‐pretreated rats. In rats with 6‐hydroxydopamine‐induced lesion of the nigrostriatal dopaminergic system, basal levels of extracellular DA were low but markedly increased by L‐DOPA (50 mg/kg i.p.). In 6‐hydroxydopamine‐lesioned rats, pretreatment with reserpine (5 mg/kg s.c.) diminished L‐DOPA (50 mg/kg i.p.)‐induced increases in extracellular DA levels to 16% of those obtained in denervated animals not pretreated with reserpine (p < 0.01). These results suggest that in the intact striatum, extracellular DA stems mainly from vesicular storage sites and that in the striatum with dopaminergic denervation, a large part of the L‐DOPA‐derived extracellular DA is also derived from a vesicular pool that is released by an exocytosis mechanism.