bluuebunny/crossref_metadata_2025 huggingface dataset:
MT1 Melatonin Receptor mRNA Expressing Cells in the Pars Tuberalis of the European Hamster: Effect of Photoperiod
Authors: Dardente H. , Klosen P. , Pévet P. , Masson‐Pévet M.
Published: July 2010

Melatonin, secreted only during the night by the pineal gland, transduces the photoperiodic message to the organism. One important target for the hormone is the pars tuberalis (PT) of the adenohypophysis which displays a very high number of melatonin binding sites in mammals and is implicated in the seasonal regulation of prolactin secretion. To gain insight into the mechanism by which the melatonin signal is decoded in the PT, we studied the effect of photoperiod on the PT cells expressing the MT1 melatonin receptor in a highly photoperiodic species, the European hamster. Recently, we showed that, in the rat, the MT1 receptor mRNA is expressed in PT‐specific cells characterized by their expression of β‐thyroid stimulating hormone (β‐TSH) along with the α‐glycoprotein subunit (α‐GSU). As the cellular composition of the PT shows variability among species, we first identified the cell type expressing the MT1 receptor in the European hamster by combining immunocytochemistry and nonradioactive in situ hybridization for the MT1 receptor mRNA. Our results show that, in the European hamster, as in the rat, the MT1 receptor is only expressed by the PT‐specific‐cells, β‐TSH and α‐GSU positive. In a second step, we analysed the effects of photoperiod on the MT1 mRNA, and on β‐TSH and α‐GSU both at the mRNA and protein levels. Our data show that, compared to long photoperiod, short photoperiod induces a dramatic decrease of MT1, β‐TSH and α‐GSU expression. Protein levels of β‐TSH and α‐GSU were also dramatically reduced in short photoperiod. Together, our data suggest that melatonin exerts its seasonal effects in the PT by signalling to PT specific‐cells through the MT1 receptor subtype.
Elevated Levels of Glucose and L‐Fucose Reduce 22Na+ Uptake and Whole Cell Na+ Current in Cultured Neuroblastoma Cells
Authors: Yorek Mark A. , Stefani Mark R. , Wachtel Ruth E.
Published: July 2010

Abstract: Na+ flux was studied in cultured neuroblastoma cells grown in medium containing increased glucose or L‐fucose concentrations. Chronic exposure of neuroblastoma cells to 30 mM glucose or 30 mM L‐fucose caused a decrease in ouabain‐sensitive and veratridine‐stimulated 22Na+ uptake compared with cells cultured in unsupplemented medium. The Na+ current, determined by using whole‐cell configuration of the patch clamp, was also decreased in these cells. Tetrodotoxin (3 μM), which blocked whole cell Na+ currents, also blocked veratridine‐stimulated 22Na+ accumulation. Culturing cells in medium containing 30 mM fructose as an osmotic control had no effect on Na+ flux. Specific [3H] saxitoxin binding was not affected by 30 mM glucose or 30 mM L‐fucose compared with cells grown in unsupplemented medium, suggesting that the number of Na+ channels was not decreased. These studies suggest that exposing cultured neuronal cells to conditions that occur in the diabetic milieu alters Na+ transport and Na+‐channel activity.
Glycolipids Isolated from Aplysia kurodaiCan Activate Cyclic Adenosine 3′, 5′‐Monophosphate‐Dependent Protein Kinase from Rat Brain
Authors: Arakane Futoshi , Fukunaga Kohji , Araki Shigeko , Abe Sachiko , Satake Mei , Miyazaki Kohji , Okamura Hitoshi , Miyamoto Eishichi
Published: July 2010

Abstract: Cyclic AMP (cAMP)‐dependent protein kinase (cAMP‐kinase) partially purified from the membrane fractions of rat brains was stimulated by novel phosphonogly‐cosphingolipids (glycolipids) derived from the skin and nerve fibers of Aplysia kurodai. Among various glycolipids tested, a major glycolipid from the skin, 3‐O‐MeGalβ 1→3GalNAcα 1→3 [6′‐O‐(2‐aminoethylphosphonyl) Galα1→2] (2‐aminoethylphosphonyl→6) Glcβ 1→4GICβ1→1ceramide (SGL‐II), was most potent, giving half‐maximal activation at 32.2 μM. Activation of cAMP‐kinase was maximal with 250 μM SGL‐II using kemptide as substrate. The effect of SGL‐II was additive on kinase activity at submaximal concentrations of cAMP. The kinase activity activated with SGL‐II was inhibited by the addition of protein kinase inhibitor peptide, a specific peptide inhibitor for cAMP‐kinase. Its inhibitory pattern was similar to that for the catalytic subunit. Of the various substrates tested, the glycolipid‐stimulated cAMP‐kinase could phosphorylate microtubule‐associated protein 2, synapsin I, and myelin basic protein but not histone H1 and casein. The regulatory subunit strongly inhibited the activity of purified catalytic subunit of cAMP‐kinase. This inhibition was reversed by addition of SGL‐II, as observed for cAMP. SGL‐II was capable of partially dissociating cAMP‐kinase, which was observed by gel filtration column chromatography. However, the binding activity of cAMP to the holoenzyme was not inhibited with SGL‐II. These results demonstrate that the glycolipids can directly activate cAMP‐kinase in a manner similar, but not identical, to that of cAMP.
In Vivo, Ex Vivo, and In Vitro One‐ and Two‐Dimensional Nuclear Magnetic Resonance Spectroscopy of an Intracerebral Glioma in Rat Brain: Assignment of Resonances
Authors: Rémy C. , Arús C. , Ziegler A. , Lai E. Sam , Moreno A. , Fur Y. Le , Décorps M.
Published: July 2010

Abstract: An in vivo study of intracerebral rat glioma using proton‐localized NMR spectroscopy showed important modifications of the spectra in the tumor as compared with the contralateral brain. To carry out the assignment of the resonances of the glioma spectra, tumoral and normal rat brain tissues were studied in vivo, ex vivo, and in vitro by one‐dimensional and two‐dimensional proton spectroscopy. N‐Acetylaspartate was found at an extremely low level in the glioma. The change of peak ratio total creatine/3.2 ppm peak was found to be due to a simultaneous decrease of the total creatine content and an increase of the 3.2 ppm peak. The 3.2 ppm resonance in the glioma spectra has been shown to originate from choline, phosphocholine, glycerophosphocholine, taurine, inositol, and phosphoethanolamine. The increase of the 3.2 ppm peak in the glioma was found to result from the increase of taurine and phosphoethanolamine contents. The peak in the 1.3 ppm region of the glioma spectra was due to both lactate and mobile fatty acids. Moreover, two‐dimensional spectroscopy of excised tissues and extracts showed the presence of hypotaurine only in the tumor.
Changes in Extracellular and Rat Brain Tissue Concentrations of D‐β‐Hydroxybutyrate After 1, 3‐Butanediol Treatment
Authors: Gueldry S. , Marie C. , Christofi G. , Sarna G. S. , Obrenovitch T. P.
Published: July 2010

Abstract: 1, 3‐Butanediol (BD) treatment was previously shown to produce a dose‐related increase of the plasma levels of D‐β‐hydroxybutyrate (BHB) and to protect brain tissue against hypoxia and ischemia. The purpose of this study was to test whether BD‐induced hyperketonemia was associated with changes in brain extracellular and tissue concentrations of BHB. Changes in extracellular levels of BHB were continuously monitored in anesthetized rats before and after intraperitoneal injection of BD (25 mmol/kg), using intracerebral microdialysis coupled to online analysis of BHB in the dialysate. Cortical tissue concentrations of BHB were determined in control and BD‐ treated rats (25 and 50 mmol/kg, i.p.) after freezing of the brain in situ. Butanediol produced a rapid increase in dialysate levels of BHB, with a linear relationship between dialysate and plasma BHB concentrations (r=0.81, p < 0.001). In contrast, and although brain tissue levels of BHB were markedly increased after BD treatment, they were not related to the plasma concentration of BHB. Our results suggest that BHB produced from BD did not accumulate in brain and that BD protects against hypoxia or ischemia by increasing brain BHB availability.
Sialosylcholesterol Effects on Reconstitution of Microfilament and Glia Filament
Authors: Ito Jin‐ichi , Masuda Mari , Tanaka Ryo
Published: July 2010

Abstract: The effects of α‐sialosylcholesterol (α‐SC) on formation of either microfilament or glia filament of rat astrocytes were investigated using a reconstitution system. Polymerization of the depolymerized microfilament preparation that had been extracted from a crude cytoskeletal fraction of rat astrocytes, in the presence of 100 mM KCI and 10 mM MgCI2, was suppressed in a dose‐dependent manner by α‐SC. α‐SC inhibited polymerization of G‐actin in a similar manner. The intensity of a‐SC inhibition of G‐ actin polymerization was as great as that of microfilament polymerization, suggesting that the inhibition of microfilament polymerization by α‐SC was due to the direct action of α‐SC on actin, the main component of microfilament. α‐SC depolymerized partly the polymerized microfilament preparation, which resembled F‐actin (microfilament‐like filaments). α‐SC suppressed, in a dose‐dependent manner, polymerization of a glia filament preparation that had been extracted from astrocyte cytoskeletons in the presence of phalloidin. An increase in the amount of added α‐SC (up to 15 nM) decreased the amount of the larger glia filament‐like filaments, which were 10 nm thick and centrifuged down at 16,000 g for 30 min, and increased that of smaller ones precipitated only after centrifugation at 100,000 g for 1 h. The lower the concentration of the depolymerized glia filament extract, the greater was the inhibition by α‐SC of the polymerization. α‐SC repressed polymerization of vimentin, the dominant component of glia filament. Vimentin polymerization was more strongly inhibited by α‐SC than polymerization of glia filament was. The findings suggested that α‐SC suppressed polymerization of glia filament through a direct action on vimentin and that the glia filament‐associated proteins increased its structural stability in the presence of α‐SC.
Characterization and Developmental Expression of Lactotriosylceramider: Galactosyltransferase for the Synthesis of Neolactotetraosylceramide in the Nervous System
Authors: Chou Denise K. H. , Jungalwala Firoze B.
Published: July 2010

Abstract: Neolactoglycolipids are derived from neolactotetraosylceramide (nLcOse4Cer). They are found during the embryonic and neonatal developmental periods in the rat cerebral cortex and disappear shortly after birth. These glycolipids are, however, abundant in the adult cerebellum. Lactotriosylceramide (LcOse3Cer):galactosyltrans‐ ferase (GT), which catalyzes the terminal step in the biosynthesis of nLcOse4Cer, was characterized in mammalian brain. The enzyme was highly specific for LcOse3Cer, with a terminal GlcNAcβ1 ‐3Gal‐residue, and it did not catalyze the transfer of galactose to other glycolipids studied with alternate carbohydrate residues. The microsomal membrane enzyme required Mn2+ and a detergent for in vitro activity. The optimal pH was 7.4, and the Km value for LcOse3Cer was 34 μM (Vmax=∼2 nmol/mg/h). The LcOse3Cer:GT was shown to be different from the GM2:GT and the soluble enzyme lactose synthase A. The specific activity of LcOse3Cer:GT was enriched fivefold higher in the white matter than in the gray matter of young adult rat brain, whereas GM2:GT was enriched only about 1.5‐fold higher in the white matter. The developmental expression of LcOse3Cer:GT in the cerebral cortex and cerebellum was not correlative with the levels of nLcOse4Cer in these neural areas. Despite the complete absence of nLcOse4Cer in the cerebral cortex of animals older than 5 days, significant activity of the LcOse3Cer:GT was found even in the adult cortex. In cerebellum, the levels of nLcOse4Cer increased with development, but the specific activity of the enzyme was reduced by 50% soon after birth and then remained practically the same with development. The results indicate that LcOse3Cer:GT is not a regulatory enzyme that controls the expression of nLcOse4Cer and its derived neolactoglycolipids in the brain.
Induction of Nitric Oxide Synthase in Rat C6 Glioma Cells
Authors: Feinstein Douglas L. , Galea Elena , Roberts Steven , Berquist Henrik , Wang Hong , Reis Donald J.
Published: July 2010

Abstract: We have examined the induction of nitric oxide syhthase (NOS) activity in the rat astrocyte‐derived C6 glioma cell line. In contrast to the previous results with primary astrocyte cultures, incubation of C6 cells with bacterial endotoxin lipopolysaccharide (LPS; 1 μg/ml for 24 h) did not stimulate NO2 production. However, addition of either tumor necrosis factor‐a (TNF‐α) or interferon‐γ (IFN‐γ), cytokines that by themselves had no effect on NOS activity, imparted LPS responsiveness onto these cells in a dose‐dependent manner (EC50 values of 39 ng/ml of TNF‐α and 9.4 U/ml of IFN‐γ), and the effect of TNF‐α could be further potentiated (twofold) by the presence of interleukin‐1β. The simultaneous presence of TNF‐α and IFN‐γ yielded a greater response than either cytokine alone; however, the respective EC50 values were not affected. A cytoplasmic extract from induced C6 cells catalyzed the Ca2+‐independent conversion of l‐arginine to l‐ citrulline, with an apparent Km of 51.2 nM, and this activity could be blocked by l‐arginine analogues in the potency order amino > methyl > nitroarginine. Immunoblot analysis revealed an apparent molecular mass of 125 kDa for the NOS protein induced in C6 cells. These results indicate that the combination of LPS plus cytokines can induce NOS activity in C6 glioma cells with properties similar to those of the enzyme expressed in primary astrocyte cultures.
Tissue‐Specific Expression of Isoaspartyl Protein Carboxyl Methyltransferase Gene in Rat Brain and Testis
Authors: Mizobuchi Mutsuhiko , Murao Koji , Takeda Ryohei , Kakimoto Yasuo
Published: July 2010

Abstract: Isoaspartyl protein carboxyl methyltransferase (PIMT) is widely distributed in mammalian tissues. Using a polymerase chain reaction‐generated 124‐bp DNA fragment from brain cDNA as a probe, four different sizes (∼4.0, 2.5, 1.7, and 1.1 kb) of transcripts were detected with northern blot analysis. They were expressed predominantly in rat brain and testis. The major transcripts were 2.5 and 1.7 kb in the brain and 2.5 and 1.1 kb in the testis. One of the major transcripts specific to the testis (1.1 kb) was determined to study the structural difference of major transcripts in the two tissues. This testicular cDNA had neither the 5’(94 nucleotides) nor the 3’(594 nucleotides) end of previously reported brain cDNA corresponding to 1.7 kb. The mRNA levels and enzyme activities of different regions and developmental changes were examined in the brain. The mRNA levels and enzyme activities were concomitantly high in cerebral cortex and hippocampus. Although they increased rapidly ~30 days after birth in the testis and decreased in aged rats, they increased gradually after birth and remained high during the aging of the brain. Both structural and developmental studies show that the expression of the PIMT gene in brain and testis is regulated in a tissue‐specific manner.
Rapid Communication: Cu/Zn Superoxide Dismutase Activity in Familial and Sporadic Amyotrophic Lateral Sclerosis
Authors: Robberecht Wim , Sapp Peter , Viaene Maria Kristina , Rosen Daniel , McKenna‐Yasek Dianne , Haines Jonathan , Horvitz Robert , Theys Paul , Brown Robert
Published: July 2010

Abstract: Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease that is inherited as an autosomal dominant trait in ~ 10% of cases. Recently we and others identified several single‐base mutations in the Cu/Zn superoxide dismutase (SOD1) gene in patients with familial ALS (FALS). Using single‐strand conformational polymorphism, we studied the C to G mutation in exon 2 of the SOD1 gene (resulting in a leucine to valine substitution in position 38) in affected and unaffected members of a large Belgian family with FALS. We measured the SOD1 activity in red blood cell lysates in 14 members of this family, including the only surviving clinically affected patient. SOD1 activity of the family members carrying the mutation was less than half that of members without the mutation. In addition, in 11 patients with sporadic ALS and 11 age‐ and sex‐matched controls, red blood cell SOD1 activity was normal. These studies indicate that SOD1 activity is reduced in these FALS patients but not in sporadic ALS patients. Moreover, this SOD1 enzyme abnormality is detectable years before onset of clinical ALS in carriers of this FALS mutation.
Lesions of Mouse Striatum Induced by 6‐Hydroxydopamine Differentially Alter the Density, Rate of Synthesis, and Level of Gene Expression of D1 and D2 Dopamine Receptors
Authors: Qin Zheng‐Hong , Chen Jiang Fan , Weiss Benjamin
Published: July 2010

Abstract: To study the effects of altering dopaminergic input on the levels and rate of synthesis of dopamine receptors, corpora striata of mice were lesioned unilaterally with 6‐hydroxydopamine (6‐OHDA), and the densities and levels of the mRNAs for D1 and D2 dopamine receptors were determined. The results showed that 6‐OHDA caused significant reductions in D1 dopamine receptors and in D1 dopamine receptor mRNA in dorsolateral and dorsomedial regions of the lesioned striatum. By contrast, 6‐OHDA lesions caused significant increases in D2 dopamine receptors and in D2 dopamine receptor mRNA in dorsolateral and ventrolateral regions of the lesioned striatum. To assess the effects of 6‐OHDA lesions on the rate of synthesis of D1 and D2 dopamine receptors, the irreversibly acting dopamine receptor antagonist 2‐ethoxy‐1‐ethoxycarbonyl‐1,2‐dihydroquinoline (EEDQ) was administered, and the rate of recovery of these receptors determined. The lesions decreased the rate of synthesis of D1 dopamine receptors in dorsolateral striatum but increased the rate of synthesis of D2 dopamine receptors in dorsolateral striatum. Correlation of these molecular events with dopaminergic behaviors showed that the rate of recovery from EEDQ‐induced cataleptic activity and the recovery from inhibition of quinpirole‐induced rotational behavior was more rapid than the recovery of either the D1 or D2 dopamine receptor. These results suggest that dopaminergic denervation differentially affects the rate of synthesis of D1 and D2 dopamine receptors in mouse striatum, and that these alterations in the rates of synthesis of the receptors may be explained by corresponding alterations in the levels of the respective transcripts for these receptors.
Expression of Tyrosine Hydroxylase Gene in Cultured Hypothalamic Cells: Roles of Protein Kinase A and C
Authors: Kedzierski Wojciech , Aguila‐Mansilla Nelson , Kozlowski Gerald P. , Porter John C.
Published: July 2010

Abstract: In hypothalamic cells cultured in serum‐free medium, the quantity of tyrosine hydroxylase mRNA increases after treatment with an activator of the protein kinase A pathway (8‐bromoadenosine cyclic AMP, 3‐isobutyl‐1‐methylxanthine, or forskolin) or an activator of protein kinase C (12‐O‐tetradecanoylphorbol 13‐acetate or sn‐1,2‐diacylglycerol). The tyrosine hydroxylase mRNA level decreases in the cells after inhibition of protein kinase C with calphostin C or after depletion of protein kinase C by extended phorbol ester treatment. These data suggest that both protein kinase pathways regulate tyrosine hydroxylase gene expression in hypothalamic cells. As simultaneous activation of both pathways has less than an additive effect on the tyrosine hydroxylase mRNA level, they appear to be interrelated. Compared with the rapid and dramatic increase of the tyrosine hydroxylase mRNA level in pheochromocytoma cells, activation of the protein kinase A or protein kinase C pathway in the cultured hypothalamic cells induces slow changes of a small magnitude in the amount of tyrosine hydroxylase mRNA. The slow regulation of tyrosine hydroxylase gene expression in hypothalamic dopaminergic neurons corresponds to the relatively high stability of tyrosine hydroxylase mRNA (half‐life = 14 ± 1 h) in these cells.
Protein Kinase Inhibitor H‐7 Differentially Affects Early and Delayed Nerve Growth Factor Responses in PC12 Cells
Authors: Kahle Philipp , Mangold Martine , Kuwahara Toshikazu , Schubenel Robert , Hertel Cornelia
Published: July 2010

Abstract: The effects of the protein kinase inhibitor H‐7 on early and delayed responses to nerve growth factor (NGF) were investigated in PC12 cells. H‐7 reduced the NGF‐induced expression of c‐Fos in a dose‐dependent manner without affecting the time course of c‐Fos appearance. Conversely, H‐7 potentiated delayed NGF effects, i.e., neurite outgrowth and Ca2+/phospholipid‐dependent protein kinase (PKC) induction, but not choline acetyltransferase induction. Long‐term treatment with NGF resulted in an increase of at least four tyrosine‐phosphorylated protein bands with molecular masses between 39 and 48 kDa, which was also potentiated by H‐7. In the absence of NGF, H‐7 had no significant effect on c‐Fos expression, tyrosine phosphorylation of the 45 kDa protein, or choline acetyltransferase activity. However, 4 days of exposure to H‐7 alone induced PKC activity and tyrosine phosphorylation of the 39‐kDa protein. The action of H‐7 derivatives on neurite outgrowth did not correlate with their inhibition profile of cyclic nucleotide‐dependent protein kinases. Down‐regulation of PKC activity by prolonged exposure to phorbol ester did not completely abolish the effects of NGF and H‐7 on induction of c‐Fos, choline acetyltransferase activity, and neurite outgrowth, indicating that PKC‐independent pathways contribute to these actions. These results suggest that additional pathway(s) sensitive to H‐7 may exist, which induce immediate early gene expression and suppress neuronal differentiation of PC12 cells.
Age‐Associated Changes of Rat Brain Neuronal and Astroglial Poly(ADP‐Ribose) Polymerase Activity
Authors: Messripour Manoucher , Weltin Denis , Rastegar Ali , Ciesielski Lucien , Kopp Patrick , Chabert Magali , Mandel Paul
Published: July 2010

Abstract: Nuclear poly(ADP‐ribose) polymerase levels as well as the DNA strand break levels of whole‐brain neuronal and astroglial cells were investigated. Three‐ and 30‐month‐old rats were used. Low‐molecular‐weight neurofilaments and glutamine synthetase served as neuronal and astroglial markers, respectively. A large increase in the poly(ADP‐ribose) polymerase activity was observed in the neurons (threefold) and astrocytes (3.7‐fold) derived from 30‐month‐old rats. Similarly, the amount of poly(ADP‐ribose) polymerase, evaluated per milligram of DNA, increased ∼3.5‐fold in neurons and 3.9‐fold in astrocytes prepared from 30‐month‐old rats. Whether the increase in the poly(ADP‐ribose) polymerase activity was due to an enhanced rate of DNA strand break was investigated by determining the rate of DNA unwinding. A significant increase in DNA unwinding rate was detected in the neurons (2.7‐fold), although a lower increase was observed in the astroglia (1.3‐fold) of aged animals.
Age‐Dependent Alterations in Dopamine Content, Tyrosine Hydroxylase Activity, and Dopamine Uptake in the Striatum of the Weaver Mutant Mouse
Authors: Simon J. R. , Richter J. A. , Ghetti B.
Published: July 2010

Abstract: Mice of different ages and homozygous or heterozygous for the weaver gene (wv) were used to study the time course for the effect of the weaver gene on several striatal dopaminergic parameters. Dopamine uptake was decreased in the homozygous weaver at all ages examined. The deficit in uptake at the earliest age studied, postnatal day 3, was approximately 50% and increased to greater than 70% at older ages. In control mice, dopamine uptake reached a maximum by postnatal day 22, but in homozygous weaver mice, development of uptake activity was curtailed by postnatal day 7. Dopamine content and tyrosine hydroxylase activity were significantly decreased in the homozygous weaver at all ages studied except postnatal days 7 and 10. The magnitude of the deficit in dopamine content ranged from approximately 40% at postnatal days 3 and 5 to about 70% in adults (6 months to 1 year of age). The magnitude of the deficit in tyrosine hydroxylase activity ranged from 40 to 70%. In general, no major differences between heterozygotes and controls were observed for any of the dopaminergic parameters investigated. The results of the present investigation indicate that neurochemical alterations can be observed in the striata of weaver mice as early as postnatal day 3 and raise the possibility that the striatal dopamine transporter may be an early target of the weaver mutation.
Endothelin‐1 Receptor Binding and Cellular Signal Transduction in Cultured Human Brain Endothelial Cells
Authors: Stanimirovic Danica B. , Yamamoto Toshifumi , Uematsu Sumio , Spatz Maria
Published: July 2010

Abstract: The kinetic properties of endothelin‐1 (ET‐1) binding sites and the production of inositol phosphates (IPs; IP1, IP2, IP3), cyclic AMP, thromboxane B2, and prostaglandin F2α induced by various endothelins (ET‐1, ET‐2, ET‐3, and sarafotoxin S6b) were examined in endothelial cells derived from human brain microvessels (HBECs). The presence of both high‐ and low‐affinity binding sites for ET‐1 with KD1 = 122 pM and KD2 = 31 nM, and Bmax1 = 124 fmol/mg of protein and Bmax2 = 909 fmol/mg of protein, respectively, was demonstrated on intact HBECs. ET‐1 dose‐dependently stimulated IP accumulation with EC50 (IP3) = 0.79 nM, whereas ET‐3 was ineffective. The order of potency for displacing ET‐1 from high‐affinity binding sites (ET‐1 > ET‐2 > sarafotoxin S6b > ET‐3) correlated exponentially with the ability of respective ligands to induce IP3 formation. ET‐1‐induced IP3 formation by HBEC was inhibited by the ETA receptor antagonist, BQ123. The protein kinase C activator phorbol myristate ester dose‐dependently inhibited the ET‐1‐stimulated production of IPs, whereas pertussis toxin was ineffective. Cyclic AMP production by HBECs was enhanced by both phorbol myristate ester and ET‐1, and potentiated by combined treatment with ET‐1 and phorbol myristate ester. Data indicate that protein kinase C plays a role in regulating the ET‐1‐induced activation of phospholipase C, whereas interaction of different messenger systems may regulate ET‐1‐induced accumulation of cyclic AMP. ET‐1 also stimulated endothelial prostaglandin F2α production, suggesting that activation of phospholipase A2 is most likely secondary to IP3‐mediated intracellular calcium mobilization because both ET‐1‐induced IP3 and prostaglandin F2α were inhibited by BQ123. These findings are the first demonstration of ET‐1 (ETA‐type) receptors linked to phospholipase C and phospholipase A2 activation in HBECs.
Regulation of Intracellular pH in Guinea Pig Cerebral Cortex Ex Vivo Studied by 31P and 1H Nuclear Magnetic Resonance Spectroscopy: Role of Extracellular Bicarbonate and Chloride
Authors: Pirttilä Tiina‐R. M. , Kauppinen Risto A.
Published: July 2010

Abstract: The role of transmembrane processes that are dependent on external anions in the regulation of cerebral intracellular pH (pHi), high‐energy metabolites, and lactate was investigated using 31P and 1H NMR spectroscopy in an ex vivo brain slice preparation. During oxygenated superfusion, removal of external HCO3−/CO2 in the presence of Na+ led to a sustained split of the inorganic phosphate (Pi) peak so that the pHi indicated by one part of the peak was 0.38 pH units more alkaline and by the other part 0.10 pH units more acidic at 5 min than in the presence of HCO3−. The pH in the compartment with a higher pHi value returned to 7.29 ± 0.04 by 10.5 min of superfusion in a HCO3−‐free medium, whereas the pHi in an acidic compartment was reduced to 7.02. In the presence of 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid or the absence of external Cl−, removal of HCO3− caused alkalinization without split of the Pi peak. Both treatments reduced the rate of pHi normalization following alkalinization. Simultaneous omission of external HCO3− and Na+ did not inhibit alkalinization of the pHi following CO2 exit. All these data show that the acid loading mechanism at neutral pHi is mediated by an Na+‐independent anion transport. During severe hypoxia, pHi dropped from 7.29 ± 0.05 to 6.13 ± 0.16 and from 7.33 ± 0.03 to 6.67 ± 0.05 in the absence and presence of HCO3−, respectively, in Na+‐containing medium. Lactate accumulated to 18.7 ± 2.8 and 19.6 ± 1.5 mmol/kg under the respective conditions. In the HCO3−‐free medium supplemented with 1 mM amiloride, the pHi fell only to 6.94 ± 0.08 despite the lactate concentration of 18.9 ± 2.4 mmol/kg. Acidification caused by hypoxia was also small in the slice preparations superfused in the absence of both HCO3− and Cl−, as the pHi was 7.01 ± 0.12 at a lactate concentration of 24.5 ± 2.4 mmol/kg. These data indicate that apart from anaerobic glucose metabolism, separate acidifying mechanisms are functioning during hypoxia under these conditions. Recovery of phosphocreatine levels following reoxygenation was >75% relative to the prehypoxic level in the slice preparations superfused in the absence of HCO3− but <47% in those preparations superfused without HCO3− and Cl−. This indicates that either neutral pHi or absence of Cl− during hypoxia was deleterious to the energy metabolism. The present data indicate that Cl−/HCO3− exchange mechanisms have distinct roles in cerebral H+ homeostasis depending on the level of pHi and energy state.
Neurochemical and Neuroprotective Effects of Some Aliphatic Propargylamines: New Selective Nonamphetamine‐Like Monoamine Oxidase B Inhibitors
Authors: Yu P. H. , Davis B. A. , Durden D. A. , Barber A. , Terleckyj I. , Nicklas W. G. , Boulton A. A.
Published: July 2010

Abstract: Aliphatic N‐propargylamines have recently been discovered to be highly potent, selective, and irreversible monoamine oxidase B (MAO‐B) inhibitors. N‐Methyl‐N‐(2‐pentyl)propargylamine (M‐2‐PP) and N‐methyl‐N‐(2‐hexyl) propargylamine (2‐HxMP), for example, are approximately fivefold more potent than I‐deprenyl at inhibiting mouse brain MAO‐B activity following oral administration. These inhibitors are nonaromatic compounds and are chemically quite different from other known MAO‐B inhibitors. Some of their neurochemical and neuroprotective properties have been evaluated and compared with those of I‐deprenyl. We have confirmed that these new inhibitors selectively inhibit MAO‐B activity both in vitro and in vivo. 2‐Phenylethylamine levels were substantially increased following administration of M‐2‐PP, but the levels of dopamine, 3,4‐dihydroxyphenylacetic acid, homovanillic acid, 5‐hydroxytryptamine, and 5‐hydroxyindoleacetic acid were not affected except at high, nonselective doses. Chronic oral administration of I‐deprenyl and M‐2‐PP causes selective inhibition of MAO‐B activity and increases dopamine levels in mouse caudate. M‐2‐PP, like I‐deprenyl, has been shown to be potent in protecting against MPTP‐induced damage in the mouse. N‐(2‐Chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP‐4), a noradrenaline neurotoxin, is not an MAO substrate. Its noradrenaline‐depleting effects were substantially mitigated by I‐deprenyl as well as by M‐2‐PP and 2‐HxMP in the mouse hippocampus. Administration of 2‐phenylethylamine, however, failed to reverse the effect of DSP‐4. The neuroprotective effect of M‐2‐PP and 2‐HxMP is apparently unrelated to the uptake of DSP‐4.
Purification and Characterization of Kynurenine Aminotransferase I from Human Brain
Authors: Baran Halina , Okuno Etsuo , Kido Ryo , Schwarcz Robert
Published: July 2010

Abstract: Two kynurenine aminotransferases (KATs), arbitrarily termed KAT I and KAT II, are capable of producing the neuroinhibitory brain metabolite kynurenic acid from l‐kynurenine in human brain tissue. Here we describe the purification of KAT I to homogeneity and the subsequent characterization of the enzyme using physicochemical, biochemical, and immunological methods. KAT I was purified from human brain ∼2,000‐fold with a yield of 2%. Assessed by polyacrylamide gel electrophoresis, KAT I migrated toward the anode as a single protein with a mobility of 0.5. The pure enzyme was found to be a dimer consisting of two identical subunits of ∼60 kDa. Among several oxo acids tested, KAT I showed highest activity with 2‐oxoisocaproate. Kinetic analyses of the pure enzyme revealed an absolute Km of 2.0 mM and 10.0 mM for l‐kynurenine and pyruvate, respectively. KAT I activity was substantially inhibited by l‐glutamine, l‐phenylalanine, and l‐tryptophan, using either pyruvate (1 mM) or 2‐oxoisocaproate (1 mM) as a cosubstrate. l‐Tryptophan inhibited enzyme activity noncompetitively with regard to pyruvate (Ki = 480 µM) and competitively with regard to l‐kynurenine (Ki = 200 µM). Anti‐KAT I antibodies were produced against pure KAT I and were partially purified by conventional techniques. Immunotitration and immunoblotting analyses confirmed that KAT I is clearly distinct from both human KAT II and rat kynurenine‐pyruvate aminotransferase. Pure human KAT I and its antibody will serve as valuable tools in future studies of kynurenic acid production in the human brain under physiological and pathological conditions.
Distinctly Phosphorylated Neurofilaments in Different Classes of Neurons
Authors: Soussan Lior , Barzilai Ari , Michaelson Daniel M.
Published: July 2010

Abstract: Recent immunohistochemical experiments revealed that specific anti‐neurofilament monoclonal antibodies yield distinct patterns in different types of neurons. This led to the suggestion that neurofilaments are a family of heterogeneous molecules whose occurrence and distribution are a function of cell type. In the present study we examined the hypothesis that this heterogeneity is due to differences in the extent of phosphorylation of neurofilament proteins in distinct types of neurons. In view of the large number of potential phosphorylation sites on the heavy neurofilament protein (NF‐H), we focused on this protein and examined its extent of phosphorylation in different types of neurons. This was performed using neurofilaments isolated from axons of the cholinergic bovine ventral root motor neurons and of the chemically heterogeneous bovine dorsal root neurons. Two‐dimensional gel electrophoresis revealed that the isoelectric point of ventral root NF‐H (pl 5.10) was ∼0.2 pl units more acidic than that of dorsal root NH‐F. This difference was abolished by treating the neurofilaments with alkaline phosphatase, suggesting that the excess negative charge of ventral root NF‐H is due to increased levels of phosphorylation. Amino acid analysis confirmed that the phosphoserine content of ventral root NF‐H (27.2 ± 2.5% of the serines) is markedly higher than that of dorsal root NF‐H (15.5 ± 6.2% of the serines). These findings provide a novel system for studying the biochemistry and function of distinctly phosphorylated neurofilaments in different types of neurons.
Amino Acid Residues 226–240 of τ Which Encompass the First Lys‐Ser‐Pro Site of τ, Are Partially Phosphorylated in Alzheimer Paired Helical Filament‐τ
Authors: Liu Wan‐Kyng , Dickson Dennis W. , Yen Shu‐Hui C.
Published: July 2010

Abstract: A synthetic peptide corresponding to residues 226–240 (E9 peptide) of human τ, which contains an Lys‐Ser‐Pro motif, was used to raise a polyclonal antibody. The antibody, E9, was 10‐fold less reactive with phospho‐E9 peptide than with native E9 peptide. E9 antibody was used to study the extent of phosphorylation in a modified form of τ (PHF‐τ) that is found in Alzheimer's disease brain and is incorporated into paired helical filaments (PHFs). E9 immunolabeled Alzheimer's disease neurofibrillary tangles and abnormal neurites in brain sections intensely, with increased immunoreactivity detected after pretreatment of sections with phosphatase. On immunoblots and ELISA, E9 reacted with PHF‐τ and recombinant human τ but not with the high and middle molecular weight neurofilament proteins. Phosphatase treatment of PHF‐τ improved the E9 immunoreactivity by 30–50%. Dephosphorylated high but not middle molecular weight neurofilament protein became reactive with E9. These results indicate that <50% of the PHF‐T is phosphorylated in the subregion corresponding to residues 226–240 of τ and suggest that the phosphorylation of this region may not be essential for PHF formation.
Phosphorylation‐Dependent Immunoreactivity of Neurofilaments and the Rate of Slow Axonal Transport in the Central and Peripheral Axons of the Rat Dorsal Root Ganglion
Authors: Archer D. R. , Watson D. F. , Griffin J. W.
Published: July 2010

Abstract: The rate of axonal transport of tubulin, actin, and the neurofilament proteins was measured in the peripheral and central projections of the rat L5 dorsal root ganglion (DRG). [35S]Methionine was injected into the DRG, and the “front” of the radiolabeled protein was located 7, 14, and 20 days postinjection. Transport rates calculated for the neurofilament triplet proteins, tubulin, and actin in the peripheral nerve were ∼ 1.5‐fold faster than those in the dorsal root. A progressive decrease in the rate of transport was observed from 7 to 20 days after radiolabeling in both the central and peripheral directions (neurofilaments, ∼ 1.7‐fold; tubulin/actin, 2.1‐fold). A surgical preparation, leaving the peripheral sciatic nerve with predominantly sensory fibers, was the basis for ELISAs for phosphorylation‐dependent immunoreactivity of the high‐molecular‐weight neurofilament protein. In both dorsal roots and peripheral sensory axons the degree of phosphorylation was greater in nerve segments further away from the cell bodies. The degree of phosphorylation‐related immunoreactivity correlates with the slowing of transport of radiolabeled cytoskeletal protein.
Identification of the Palmitoylation Site in Rat Myelin P0 Glycoprotein
Authors: Bizzozero Oscar A. , Fridal Kirsa , Pastuszyn Andrzej
Published: July 2010

Abstract: P0 glycoprotein, the major protein of PNS myelin, contains approximately 1 mol of covalently bound long‐chain fatty acids. To determine the chemical nature of the fatty acid‐protein linkage, P0 was labeled in rat sciatic nerve slices with [3H]palmitic acid and subsequently treated with various reagents. The protein‐bound palmi‐tate was released by incubation with the reducing agents dithiothreitol and 2‐mercaptoethanol, and with 1M hydrox‐ylamine at pH 7.5. In addition, P0 was deacylated by treatment with 10 mM NaBH4 with the concomitant production of [3H]hexadecanol, indicating that the fatty acid is bound in a thioester linkage. This conclusion was supported further by the fact that deacylation with hydroxylamine generated free thiol groups, which were titrated with [14C]‐iodoacetamide. To identify the cysteine residue involved in the thioester linkage, [14C]carboxyamidomethylated P0was digested with trypsin and the resulting peptides analyzed by reversed‐phase HPLC. Identification of the radioactive protein fragments by amino acid analysis and amino‐terminal peptide sequencing revealed that Cys153 in rat P0 glycoprotein is the acylation site. The acylated cysteine is located at the junction of the putative transmem‐brane and cytoplasmic domains. This residue is also present in the P0 glycoprotein of other species, including human, bovine, mice, and chicken.
Interrelationships of Leucine and Glutamate Metabolism in Cultured Astrocytes
Authors: Yudkoff Marc , Daikhin Yevgeny , Lin Zhi‐Ping , Nissim Liana , Stern Janet , Pleasure David , Nissim Itzhak
Published: July 2010

Abstract: The aim was to study the extent to which leu‐cine furnishes α‐NH2 groups for glutamate synthesis via branched‐chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [15N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography‐mass spectrometry. The ratio of labeling in both [15N]glutamate/[15N]leucine and [2‐15N]glutamine/[15N]leucine suggested that at least one‐fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [15N]leucine declined, reflecting dilution of the 16N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [16N]‐isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of 15N in valine was more gradual. Measurement of branched‐chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α‐ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ∼50% in only 5 min. Extracellular concentrations of α‐ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched‐chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [15N]glutamate. After 45 min, the most highly labeled amino acid was [15N]alanine, which was closely followed by [15N]leucine and [15N]isoleucine. Relatively little 15N was detected in any other amino acids, except for [15N]serine. The transamination of leucine was ∼17 times greater than the rate of [1‐14C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a‐ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.
Structural Features of Myelin Basic Protein mRNAs Influence Their Translational Efficiencies
Authors: Ueno Shu‐ichi , Handley Vance W. , Byravan Sujatha , Campagnoni Anthony T.
Published: July 2010

Abstract: The myelin basic protein (MBP) gene expresses several alternatively spliced products with the same 5’and 3’untranslated regions (UTRs). It has been reported that its expression may be regulated not only at the transcriptional level but also at the translational level during development. We engineered several MBP mRNA deletion mutants with 5’(‐48, ‐37, ‐27, ‐22, and ‐10) and 3’UTRs of differing lengths and examined the translational efficiencies of these constructs in cell‐free systems. The translational efficiencies of the constructs differed significantly over a range of almost 10‐fold. A deletion of 11 nucleotides from the 5’end of the natural (i.e., ‐48) MBP mRNA resulted in an approximate fourfold reduction in translational efficiency. Further truncation of the 5’UTR increased the translational efficiencies of the constructs as has been observed with many RNAs. These results suggest that there may be a positive control element between ‐48 and ‐37 nucleotides in the 5’UTR of MBP mRNA. The effects of modifying the lengths of the 5’UTR on the translational efficiency of mRNAs encoding the 21.5‐kDa and 14‐kDa MBPs were the same, suggesting that the effect observed was not unique to the 21.5‐kDa MBP mRNA. Truncating the 3’UTR of four different alternatively spliced MBP mRNAs also altered their translational efficiencies. Thus, the 8 and 3’UTRs of MBP mRNAs appear to influence the translation of these mRNAs, and such factors may be involved in the translational regulation of MBP gene expression.
Evidence for the Colocalization of Parvalbumin and Glutamate, but Not GABA, in the Perforant Path of the Gerbil Hippocampal Formation: A Combined Immunocytochemical and Microquantitative Analysis
Authors: Nitsch Cordula , Maly I. Piotr , Mori David , Scotti Alessandra L.
Published: July 2010

Abstract:Gerbils (Meriones unguiculatus) are known for their seizure sensitivity, which is dependent on an intact perforant path from the entorhinal cortex to the hippocampus. In contrast with other species, the perforant path in gerbils contains parvalbumin, a cytosolic high‐affinity calcium‐binding protein. Parvalbumin is known to be present in a subpopulation of GABA‐containing neurons and is thought to be responsible for their physiological characteristics of fast spiking activity and lack of spike adaptation. Therefore, the question arose of whether this projection in gerbils is GABAergic or glutamatergic as in other species. In a first approach to this question, the effect of lesioning the origin of the perforant path, the entorhinal cortex, on levels of GABA and glutamate was determined by enzymatic‐luminometric assay in single layers of the dentate gyrus of lyophilized brain sections. Parallel sections were cryofixed using an acidified acetone‐formaldehyde mixture at ‐20°C for 48 h, and subsequently stained for parvalbumin immunocytochemistry. Seven days after ablation of the entorhinal cortex, parvalbumin staining was undetectable in the termination zone of the perforant path, the outer two‐thirds of the stratum moleculare. In parallel, glutamate content was reduced to 80% of controls (and of the unoperated contralateral side) but unchanged in the inner third of the stratum moleculare and in stratum granulare. GABA content was not significantly altered by the lesion. From these results, we conclude that in the gerbil as in other species, the perforant path contains glutamate. The association of glutamate with parvalbumin suggests, however, that in the gerbil the calcium‐dependent release of the transmitter is carefully controlled in the entorhinal perforant path.
Subcellular Fractionation on Percoll Gradient of Mossy Fiber Synaptosomes: Morphological and Biochemical Characterization in Control and Degranulated Rat Hippocampus
Authors: Taupin Philippe , Zini Sylvie , Cesselin François , Ben‐Ari Yezekiel , Roisin Marie‐Paule
Published: July 2010

Abstract: A method for preparation of hippocampal mossy fiber synaptosomes directly from the postnuclear pellet is presented. This method represents an adaption of that previously described for the isolation of synaptosomes by centrifugation through Percoll gradients directly from the supernatant fraction. We have characterized by electron microscopy two fractions, PII and PIII, enriched in mossy fiber synaptosomes; fraction PIII had 75% mossy fiber synaptosomes with well‐preserved morphology (large size 3 μm, complex morphology, high synaptic vesicle density, multisynapses), whereas fraction PII contained 12%. These fractions were enriched in lactate dehydrogenase activity indicating that the integrity of synaptosomes was preserved. Compared with the other synaptosomal fractions, these fractions showed greater levels of dynorphin A (1–8) immunoreactivity and endogenous zinc, which are particularly concentrated in hippocampal mossy fiber terminals. Furthermore, we prepared synaptosomes from adult hippocampus after neonatal irradiation, which destroys the majority of granule cells and associated mossy fibers. The levels of dynorphin and zinc decreased by 88 and 70% in fraction PII and by 95 and 90%, respectively, in PIII. These results suggest that the rapid Percoll procedure is convenient for the purification of mossy fiber synaptosomes.
The Pyruvate Dehydrogenase Complex: Cloning of the Rat Somatic El α Subunit and Its Coordinate Expression with the mRNAs for the E1β E2, and E3 Catalytic Subunits in Developing Rat Brain
Authors: Cullingford Tim E. , Clark John B. , Phillips Ian R.
Published: July 2010

Abstract: We report the isolation of cDNA clones encoding the somatic form of the E1α subunit of the pyruvate dehydrogenase complex of rat. The deduced amino acid sequence has 99.5, 98, and 97% identity, respectively, with the orthologous proteins of mouse, human, and pig and 98.5% identity with a rat E1α sequence reported previously. The cDNAs isolated in this and earlier studies predict different E1α subunit mRNA sizes and amino acid sequences. These differences have been investigated by PCR, northern blot hybridization, and RNase protection. We have used our E1α cDNA, in conjunction with cDNA probes to the E1β, E2, and E3 catalytic subunits of rat pyruvate dehydrogenase complex and also to rat citrate synthase, to perform RNase protection assays of developing rat whole brain RNA. The results show a 2.5‐fold increase in the concentration of each of the subunit mRNAs and a 1.2‐fold increase in citrate synthase mRNA from late foetal stage to 5 days post partum. Thereafter, the mRNA levels remained constant. These data indicate that the respective six‐and threefold increases in the amounts of pyruvate dehydrogenase complex and citrate synthase found to occur in rat brain between birth and adulthood are mediated principally by translational and/or posttranslational mechanisms.