bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Candida species by PCR in self‐collected vaginal swabs of women after taking antibiotics
Authors: Tabrizi Sepehr N. , Pirotta Marie V. , Rudland Elice , Garland Suzanne M.
Published: October 2006

Women undergoing antibiotic treatment can develop vulvovaginal candidiasis. This study evaluated polymerase chain reaction (PCR) for the detection of Candida species in comparison with the conventional laboratory culture methods in samples from women with and without symptoms of postantibiotic candidiasis. The self‐collected vaginal swabs from 90 women, with no recent symptoms of vulvovaginal candidiasis (VVC), who were prescribed antibiotics for non‐genital infection were evaluated 8 days after completion of antibiotics and/or at the time of developing symptoms of VVC. Broad‐spectrum fungal PCR was performed on extracted DNA from each sample. Overall PCR detected four additional Canidida albicans, three Candida parapsilosis and one Candida tropicalis when compared with culture. All but one case additionally detected by PCR were found in patients with no VVC symptoms. PCR, although more sensitive than conventional culture methods, in this small number of cases, has not been able to detect Candida species in significantly more patients with symptoms suggestive of candidiasis. The results of this study may indicate that other agents including other yeast species may be responsible for symptoms of postantibiotic vulvovaginitis.
Mise en Evidence de la Carcinine Synthétase, une Nouvelle Enzyme Catalysant le Métabolisme de l'Histamine dans le Systeme Nerveux Central de Carcinus maenas
Authors: Arnould Jean‐Marie
Published: October 2006

Résumé: La biosynthèse de la carcinine est réalisée in vitro à partir de ses deux constituants: la β‐alanine et l'histamine. La réaction est catalysée par des extraits de muscles, de coeur, et de CNS de Carcinus maenas. L'activité spécifique de l'enzyme, la carcinine synthétase, est 15 fois plus élevée dans le CNS que dans les autres organes. Seul le CNS réalise la biosynthèse de la carcinine à partir de l'histidine et uniquement en présence de phosphate‐5’ de pyridoxal. Le siège de la biosynthèse de la carcinine serait done le CNS. Dans cet organe, l'histidine serait transformée en histamine qui serait ensuite catabolisée sous forme de carcinine. Celle‐ci serait alors transportée jusque dans le tissu cardiaque où elle s'accumulerait. L'histamine dont le métabolisme s'effectue en totalité dans le CNS serait done impliquée dans l'activité neuronale du Crustacé. La carcinine synthétase est une enzyme soluble qui requiert la présence d'ATP, de β‐alanine, et d'histamine. Mg2+ et le dithiothréitol sont aussi essentiels pour son activité. Son pH optimum se situe à∼7,6. La carcinine synthétase se distingue de la carnosine synthétase et de la γ‐glutamylhistamine synthétase car elle ne catalyse ni la synthèse de la β‐alanylhistidine, ni celie de la γ‐glutamylhistamine. Mots Clés: Métabolisme de l'histamine—β‐Alanylhistamine—Carcinine—Carcinus maenas. Arnould J.‐M. Mise en evidence de la carcinine synthétase, une nouvelle enzyme catalysant le métabolisme de l'histamine dans le systeme nerveux central de Carcinus maenas. J. Neurochem. 48, 1316–1324 (1987).
Influence of Hydrocortisone on Chick Embryo Retina Development
Authors: Vento Renza , D'Ancona Giuseppina , Giuliano Michela , Taibi Gennaro , Tesoriere Giovanni
Published: October 2006

Abstract: Treatment of chick embryos in ovo with hydro‐cortisone‐21‐phosphate (a single dose of 150 μg) caused a marked reduction of retinal thymidine kinase activity 24 h later. The inhibitory effect was highest (65–70%) in 8–10‐day‐old embryos and declined with age, disappearing after day 15. It was accompanied by a reduction in thickness of the retinal layers. Adrenocorticotropic hormone (ACTH) treatment (10 μg daily for 2 days) also produced an age‐dependent inhibitory effect on retinal thymidine kinase, whereas treatment with a single dose of 200 μg of metopir‐one, a compound that prevents the 11β‐hydroxylation of steroid molecules in the adrenal glands, impeded the decrease in thymidine kinase activity that normally occurs in chick embryo retina after day 9 of development. In addition, metopirone prevented the inhibition exerted by ACTH on thymidine kinase activity but had no effect on the action of hydrocortisone.
Inhibition of Muscarinic‐Coupled Phosphoinositide Hydrolysis by N‐Methyl‐d‐Aspartate Is Dependent on Depolarization via Channel Activation
Authors: Morrisett Richard A. , Chow Carolyn C. , Sakaguchi Takuya , Shin Cheolsu , McNamara James O.
Published: October 2006

Abstract: The intent of this work was to elucidate the mechanism by which N‐methyl‐d‐aspartate (NMDA) receptor agonists inhibit a second messenger system, namely, the stimulation of phosphoinositide (PI) hydrolysis activated by muscarinic cholinergic receptor agonists. NMDA inhibited cholinergic stimulation of PI hydrolysis in a dose‐ and time‐dependent manner. NMDA exerts this effect indirectly through channel activation, because both MK‐801 and N‐[1‐(2‐thienyl)cyclohexyl]piperidine (TCP) prevented this action. Prevention of the NMDA effect by removal of sodium, but not calcium, from the incubation buffer suggested that depolarization may be the responsible mechanism. Depolarization alone proved sufficient to inhibit cholinergic activation of PI hydrolysis, because both veratridine and an elevated extracellular potassium level inhibited cholinergic stimulation of PI hydrolysis. The effect of NMDA appeared to require sodium flux through NMDA channels rather than through voltage‐dependent sodium channels, because tetrodotoxin failed to inhibit the effect of NMDA. In correlative electrophysiologic experiments, NMDA profoundly inhibited evoked excitatory postsynaptic potentials and population action potentials of CA1 neurons, an effect almost certainly due to depolarization. The dose and time course of the electrophysiologic effects correlated well with the biochemical effects. Taken together, the data support the assertion that NMDA receptor activation inhibits PI hydrolysis by depolarization mediated by sodium flux through NMDA channels.
Depolarization‐Induced Phosphorylation of the Protein Kinase C Substrate B‐50 (GAP‐43) in Rat Cortical Synaptosomes
Authors: Dekker L. V. , De Graan P. N. E. , De Wit M. , Hens J. J. H. , Gispen W. H.
Published: October 2006

Abstract: We studied the molecular events underlying K+‐induced phosphorylation of the neuron‐specific protein kinase C substrate B‐50. Rat cortical synaptosomes were prelabelled with 32P‐labelled orthophosphate. B‐50 phosphorylation was measured by an immunoprecipitation assay. In this system, various phorbol esters, as well as a synthetic diacylglycerol derivative, enhance B‐50 phosphorylation. K+ depolarization induces a transient enhancement of B‐50 phosphorylation, which is totally dependent on extracellular Ca2+. Also, the application of the Ca2+ ionophore A23187 induces B‐50 phosphorylation, but the magnitude and kinetics of A23187‐induced B‐50 phosphorylation differ from those induced by depolarization. The protein kinase inhibitors 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H‐7), N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7), and staurosporine antagonize K+‐ as well as PDB‐induced B‐50 phosphorylation, whereas trifluoperazine and calmidazolium are ineffective under both conditions. We suggest that elevation of the intracellular Ca2+ level after depolarization is a trigger for activation of protein kinase C, which subsequently phosphorylates its substrate B‐50. This sequence of events could be of importance for the mechanism of depolarization‐induced transmitter release.
Molecular Species of Choline and Ethanolamine Phospholipids in Rat Cerebellum During Development
Authors: Leray C. , Pelletier A. , Massarelli R. , Dreyfus H. , Freysz L.
Published: October 2006

Abstract: The molecular species composition of rat cerebellar phospholipid subclasses has been studied by HPLC after phospholipase C treatment and dinitrophenyl derivatization. During rat cerebellum development (3–90 days postpartum), cholinephosphoglycerides and ethanolamine phosphoglycerides represented ∼80% of all phospholipids, with their relative amount changing after 1 month. Among ethanolamine phosphoglycerides, the molar ratio of diacylglycerophosphoethanolamine (diacylGPE) to alkenylacylGPE decreased from ∼1.4 at 3 days to ∼0.5 after 10 days. The phospholipids investigated contained up to 12 different molecular species. The rate of accumulation of the various molecular species of diacylglycerophosphocholine (diacylGPC), diacylGPE, and alkenylacylGPE during cerebellar development allowed a classification into three main groups. The overall increase of the molecular species of the first group (6‐diacylGPC, 5‐diacylGPE, and 4‐alkenylacylGPE) was ∼ 18‐fold between 3 and 90 days, with a faster rate of accumulation between 3 and 30 days. Those of the second group (3‐diacylGPC, 5‐diacyl‐GPE, and 5‐akenylacylGPE) increased by ∼45‐fold during the same developmental period, at a slow rate before day 15 and at a faster one thereafter. The molecular species of the third group (3‐alkenylacylGPE) increased by >250‐fold between 3 and 90 days, at a very slow rate before day 21 and more quickly thereafter. The different rates of accumulation of the components of the three groups during cerebellar development suggest a preferential location of the first group in membranes of neuronal perikaryons, glial cells, and synaptosomal structures. Those of the second group appear to be located in both synaptosomal membranes and myelin sheets, and those of the third group can be considered as myelin markers.
N‐Methyl‐D‐Aspartate Recognition Site Ligands Modulate Activity at the Coupled Glycine Recognition Site
Authors: Hood William F. , Compton Robert P. , Monahan Joseph B.
Published: October 2006

Abstract: In synaptic plasma membranes from rat forebrain, the potencies of glycine recognition site agonists and antagonists for modulating [3H]1‐[1‐(2‐thienyl)cyclohexyl]piperidine ([3H]TCP) binding and for displacing strychnine‐insensitive [3H]glycine binding are altered in the presence of N‐methyl‐D‐aspartate (NMDA) recognition site ligands. The NMDA competitive antagonist, cis‐4‐phosphonomethyl‐2‐piperidine carboxylate (CGS 19755), reduces [3H]glycine binding, and the reduction can be fully reversed by the NMDA recognition site agonist, L‐glutamate. Scatchard analysis of [3H]glycine binding shows that in the presence of CGS 19755 there is no change in Bmax (8.81 vs. 8.79 pmol/mg of protein), but rather a decrease in the affinity of glycine (KD of 0.202 γM vs. 0.129 γM). Similar decreases in affinity are observed for the glycine site agonists, D‐serine and 1‐aminocyclopropane‐1‐carboxylate, in the presence of CGS 19755. In contrast, the affinity of glycine antagonists, 1‐hydroxy‐3‐amino‐2‐pyrrolidone and 1‐aminocyclobutane‐1‐carboxylate, at this [3H]glycine recognition site increases in the presence of CGS 19755. The functional consequence of this change in affinity was addressed using the modulation of [3H]TCP binding. In the presence of L‐glutamate, the potency of glycine agonists for the stimulation of [3H]TCP binding increases, whereas the potency of glycine antagonists decreases. These data are consistent with NMDA recognition site ligands, through their interactions at the NMDA recognition site, modulating activity at the associated glycine recognition site.
Evidence for Functionally Distinct Subclasses of γ‐Aminobutyric Acid Receptors in Rabbit Retina
Authors: Lee Friedman David , Redburn Dianna A.
Published: October 2006

Abstract: γ‐Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the mammalian retina, where it serves many roles in establishing complex response characteristics of ganglion cells. We now provide biochemical and physiological evidence that at least three subclasses of GABA receptors (A1, A2, and B) contribute to different types of synaptic integration. Receptor binding studies indicate that approximately three‐fourths of the total number of [3H]GABA binding sites in retina are displaced by the GABAA receptor antagonist, bicuculline, whereas one‐fourth are displaced by the GABA‐B receptor agonist, baclofen. GABAA receptors can be described by a three‐site binding model with KD values of 19 nM, 122 nM, and 5.7 μM. Benzodiazepines and barbiturates potentiate binding to the GABAA site, which suggests that significant numbers of GABAA receptors are coupled to regulatory sites for these compounds and thus are classified as GABAA1 receptors. The response to pentobarbital appears to involve a conversion of low‐affinity sites to higher‐affinity sites, and is reflected in changes in the densities of sites at different affinities. Functional studies were used to establish which of the different receptor subclasses regulate release from cholinergic amacrine cells. Our results show that GABA suppresses light‐evoked [3H]acetylcholine release via GABAA2 receptors not coupled to a benzodiazepine or barbiturate regulatory site, and enhances release via GABAB receptors. GABAA1 sites do not appear to control acetylcholine release in rabbit retina.
Mutation of Serine 41 in the Neuron‐Specific Protein B‐50 (GAP‐43) Prohibits Phosphorylation by Protein Kinase C
Authors: Nielander H. B. , Schrama L. H. , van Rozen A. J. , Kasperaitis M. , Oestreicher A. B. , Gispen W. H. , Schotman P.
Published: October 2006

Abstract: The neuron‐specific, calmodulin‐binding protein B‐50 (also known as GAP‐43, F1, or neuromodulin) is an endogenous substrate of protein kinase C (PKC). PKC exclusively phosphorylates Ser residues in B‐50. As potential phosphorylation sites for PKC, Ser41, Ser110, and Ser122 were indicated, of which Ser41 is contained in the sequence ASF, which matches with the sequence of a synthetic PKC substrate. N‐terminally 35S‐labeled B‐50, produced from cDNA, was subjected to digestion with Staphylococcus aureus V8 protease (SAP). Consecutively, 35S‐labeled 28‐ and 15‐kDa fragments were formed, similar to those after digestion of 32P‐labeled B‐50. In a previous study, we showed that the 32P‐labeled 15‐kDa SAP fragment contains all 32P radioactivity. The present data indicate that it contains the N‐terminus of B‐50 as well. The 15‐kDa fragment, with a calculated length ranging from amino acid residue 1 to 65, contains only one potential PKC phosphorylation site, at Ser41. Mutagenesis of Ser41 into Thr or Ala resulted in recombinant B‐50 products with mobilities on two‐dimensional electrophoresis similar to those of the nonmutated recombinant B‐50 and the rat brain B‐50. Only [Ser41]B‐50 was phosphorylated by PKC, whereas [Thr41]‐ or [Ala41]B‐50 did not show any phosphorylation at the positions indicated on the immunoblots. This leads us to the conclusion that Ser41 is the sole phosphorylation site for PKC in vitro.
Role of the Blood‐Brain Barrier in the Formation of Long‐Chain ω‐3 and ω‐6 Fatty Acids from Essential Fatty Acid Precursors
Authors: Moore Steven A. , Yoder Elizabeth , Spector Arthur A.
Published: October 2006

Elongated, more highly polyunsaturated derivatives of linoleic acid (18:2ω‐6) and linolenic acid (18:3ω‐3) accumulate in brain, but their sites of synthesis and mechanism of entry are not well characterized. To investigate the role of the blood‐brain barrier in this process, cultured murine cere‐bromicrovascular endothelia were incubated with [1‐14C]18: 2ω‐6 or [1‐14C]18:3ω‐3 and their elongation/desaturation products determined. The major metabolite of 18:2ω‐6 was 20:4ω‐6, whereas the primary product from 18:3ω‐3 was 20: 5ω‐3. Although these products were found primarily in cell lipids, they were also released from the cells and gradually accumulated in the extracellular fluid. Eicosanoid production was observed from the 20:4ω‐6 and 20:5ω‐3 that were formed. No 22:5ω‐6 or 22:6ω‐3 fatty acids were detected, suggesting that these endothelial cells are not the site of the final desaturation step. Although the uptake of 18:3ω‐3 and 18:2ω‐6 was nearly identical, 18:3ω‐3 was more extensively elongated and desaturated. Competition experiments demonstrated a preference for 18:3ω‐3 by the elongation/desaturation pathway. These findings suggest that the blood‐brain barrier can play an important role in the elongation and desaturation of ω‐3 and ω‐6 essential fatty acids during their transfer from the circulation into the brain.
Demyelination Induced in Aggregating Brain Cell Cultures by a Monoclonal Antibody Against Myelin/Oligodendrocyte Glycoprotein
Authors: de Rosbo Nicole Kerlero , Honegger Paul , Lassmann Hans , Matthieu Jean‐Marie
Published: October 2006

A monoclonal antibody (8–18C5) directed against myelin/oligodendrocyte glycoprotein (MOG) induced demyelination in aggregating brain cell cultures. With increasing doses of anti‐MOG antibody in the presence of complement, myelin basic protein (MBP) concentrations decreased in a dose‐related manner. A similar, albeit less pronounced, effect was observed on specific activity of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase. In the absence of complement, anti‐MOG antibody did not induce detectable demyelination. In contrast to the effect of anti‐MOG antibody and as expected, anti‐MBP antibody did not demyelinate aggregating brain cell cultures in the presence of complement. These results provide additional support to the suggestion that MOG, a quantitatively minor myelin component located on the external side of the myelin membrane, is a good target antigen for antibody‐induced demyelination. Indeed, they show that a purified anti‐MOG antibody directed against a single epitope on the glycoprotein can produce demyelination, not only in vivo as previously shown, but also in cultures. Such an observation has not been made with polyclonal antisera raised against purified myelin proteins like MBP and proteolipid protein, the major protein components of the myelin membrane, or myelin‐associated glycoprotein. These observations may have important implications regarding the possible role of anti‐MOG antibodies in demyelinating diseases.
Maitotoxin Induces Phosphoinositide Turnover and Modulates Glutamatergic and Muscarinic Cholinergic Receptor Function in Cultured Cerebellar Neurons
Authors: Lin Wan‐Wan , Lee Chen Yuan , Yasumoto Takeshi , Chuang De‐Maw
Published: October 2006

Abstract: Maitotoxin (MTX) stimulated inositol phosphate (IP) formation in primary cultures of rat cerebellar granule cells. MTX‐induced IP production was dependent on extracellular Ca2+ but independent of extracellular Na+. The stimulation of IP formation elicited by MTX was unaffected by pretreatment of cells with phorbol dibutyrate. pertussis toxin, and a variety of Ca2+ entry blockers, such as nimodipine, nisoldipine, Co2+, and Mn2+. The presence of MTX markedly attenuated IP production induced by carbachol and glutamate, with no apparent effect on the responses to norepinephrine (NE), histamine, 5‐hydroxytryptamine (5‐HT), and endothelin‐1. The inhibition of the carbachol‐ and glutamate‐induced responses by MTX was dose dependent with IC50 values of 1.2 and 0.5 ng/ml, respectively. Pretreatment of cells with a lower concentration of MTX (0.3 ng/ml) also attenuated carbachol‐ and glutamate‐induced IP formation, in a time‐dependent manner, with a decrease observed after 30 min prestimulation, but failed to affect NE‐, histamine‐, 5‐HT‐, endothelin‐1, and sarafotoxin S6b‐induced responses. Thus, MTX elicited a marked Ca2+‐dependent phosphoinositide (PI) turnover in cerebellar granule cells and selectively inhibited carbachol‐ and glutamate‐induced PI hydrolysis. Possible mechanisms underlying these selective modulations are discussed.
Synthesis of Urokinase‐Type Plasminogen Activator and of Type‐ 1 Plasminogen Activator Inhibitor in Neuronal Cultures of Human Fetal Brain: Stimulation by Phorbol Ester
Authors: Presta M. , Ennas M. G. , Torelli S. , Ragnotti G. , Gremo F.
Published: October 2006

Abstract: Human neuronal brain cultures established from 12‐ and 14‐week‐old fetuses synthesize and secrete urokinase‐type plasminogen activator (uPA) and limited amounts of tissue‐type plasminogen activator (tPA). These cells also produce and secrete the endothelial cell‐type PA inhibitor (PAII), which forms sodium dodecyl sulfate‐stable tPA/PAI‐I complexes in the culture medium. Immunocytochemistry shows a predominant localization of uPA, tPA, and PAI‐1 in neuronal cells, with only a very weak positivity detectable in the few glial cells present in these cultures. The protein kinase C (PKC) activator 12‐0‐tetradecanoylphorbol 13‐ac‐etate (TPA) stimulates the synthesis of both uPA and PAI‐I, resulting in a final increase in the plasmin‐generating capacity of neuronal cell cultures. No significant effect is observed, however, when cells are treated with the TPA analogue 4a‐phorbol 12,13‐didecanoate, which is inactive as a PKC inducer, or with the neurotrophic polypeptide basic fibroblast growth factor. These data represent the first characterization of the plasmin‐generating system in human fetal brain neurons and suggest a role for PKC in the modulation of uPA and PAI‐I synthesis.
6,7‐Dinitroquinoxaline‐2,3‐Dione Blocks the Cytotoxicity of N‐Methyl‐D‐Aspartate and Kainate, but Not Quisqualate, in Cortical Cultures
Authors: Patel Jitendra , Zinkand William C. , Klika Andrea B. , Mangano Thomas J. , Keith Richard A. , Salama Andre I.
Published: October 2006

Abstract: Based on radioligand binding and electrophysiological studies, quinoxalinediones such as 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX) have been shown to be potent competitive antagonists at the quisqualate and kainate subtypes of the glutamate receptor. In this report we have examined the effects of DNQX on excitatory amino acid neurotoxicity and evoked neurotransmitter release. DNQX was found to be a potent neuroprotective agent against glutamate and N‐methyl‐D‐aspartate (NMDA) neurotoxicity. The data suggest that this neuroprotective activity of DNQX is due to its antagonism of the coagonist activity of glycine at the NMDA receptor–channel complex. The specificity of DNQX for the glycine site associated with the NMDA receptor–channel complex was confirmed in radioligand binding and neurotransmitter release studies. DNQX also prevented kainate neurotoxicity and kainate‐evoked neurotransmitter release, presumably by direct competition for the kainate receptor. DNQX, however, did not prevent quisqualate neurotoxicity, suggesting that a novel quisqualate‐preferring receptor insensitive to DNQX may mediate quisqualate toxicity.
Neuropeptide Y Receptor in Vascular Smooth Muscle
Authors: Shigeri Yasushi , Mihara Shin‐ichi , Fujimoto Masafumi
Published: October 2006

Abstract: 125I‐Bolton‐Hunter (125I‐BH) neuropeptide Y (NPY) was used to identify specific high‐affinity NPY binding sites in porcine aortic smooth muscle membrane fractions and to characterize the binding sites in comparison with those in porcine hippocampal membrane fractions. Ca2+, but not Mg2+ or Mn2+, enhanced specific 125I‐BH‐NPY binding in aortic smooth muscle, while all of the cations did in hippocampus. The fast, saturable, and selective binding was to a single population of sites, with a KD of 0.99 ± 0.11 nM and a Bmax of 0.35 ± 0.06 fmol/mg protein. GTP and its non‐hydrolyzable analogues reduced 125I‐BH‐NPY binding dose dependently. Neither calcium channel blockers nor nor‐adrenaline had any effect on the binding. Among structurally related peptides, peptide YY displaced 125I‐BH‐NPY binding as potently as NPY, while human, avian, and rat pancreatic polypeptides displaced 125I‐BH‐NPY binding 100 times less potently than NPY. The C‐terminal fragment NPY(13–36) for inhibiting 125I‐BH‐NPY binding in aortic smooth muscle was ∼40 times less potent than in the hippocampus. When we examined the effect of these peptides on cytosolic free Ca2+ ([Ca2+]i) in cultured porcine aortic smooth muscle cells, only the peptides showing high affinity for 125I‐BH‐NPY binding sites increased [Ca2+]i. These results indicate that the NPY binding sites labeled by 125I‐BH‐NPY in aortic smooth muscle are functional receptors and have different properties from those in the hippocampus with respect to dependency on divalent cations, sensitivity to GTP analogues, and affinity for NPY(13–36) and pancreatic polypeptides.
L‐3,4‐Dihydroxyphenylalanine Synthesis by Genetically Modified Schwann Cells
Authors: Owens Geoffrey C. , Johnson Randall , Bunge Richard P. , O'Malley Karen L.
Published: October 2006

Abstract: We have investigated whether Schwann cells can be modified by gene transfer to synthesize L‐3,4‐dihydroxy‐phenylalanine (L‐DOPA), the immediate precursor in the formation of dopamine. By using a retrovirus containing a rat tyrosine hydroxylase (TH) cDNA, we established an immortalized rodent Schwann cell line that stably expressed high levels of TH and secreted L‐DOPA in vitro when supplied with tyrosine and the essential cofactor biopterin. We also infected primary Schwann cells and demonstrated that cells expressing TH secreted L‐DOPA while maintaining their capacity to myelinate neurons in vitro. This study indicates that it may be feasible to utilize autotransplantation of genetically modified Schwann cells to alleviate the movement disorders in Parkinson's disease.
Multicatalytic, High‐Mr Endopeptidase from Postmortem Human Brain
Authors: McDermott J. R. , Gibson A. M. , Oakley A. E. , Biggins J. A.
Published: October 2006

Abstract: The main high molecular weight (650K) multica‐talytic endopeptidase has been purified from postmortem human cerebral cortex. As in other tissues and species, this enzyme is composed of several subunits of 24‐3 IK and has three distinct catalytic activities, as shown by the hydrolysis of the fluorogenic tripeptide substrates glutaryl‐Gly‐GIy‐Phe‐7‐amido‐4‐methylcoumarin, benzyloxycarboxyl ‐ Gly ‐ Gly ‐Arg‐7‐amido‐4‐methylcoumarin, and benzyloxycarboxyl‐Leu‐Leu‐Glu‐2‐naphthylamide with hydrophobic (Phe), basic (Arg), and acidic (Glu) residues in the P, position, respectively. These activities are distinguishable by their differential sensitivity to peptidase inhibitors. The enzyme hydrolysed neu‐ropeptides at pH 7.4 at multiple sites with widely differing rates, ranging from 113 nmol/min/mg for substance‐P, down to 2 nmol/min/mg for bradykinin. The enzyme also had pro‐teinase activity as shown by the hydrolysis of casein. For the hydrolysis of the Tyr5‐Gly6 bond in luteinizing hormone‐releasing hormone, the Km was 0.95 mM and the specificity constant (kcat/Km) was 4.7 ± 103 M‐l s‐1. The bond specificity of the enzyme at neutral pH was determined by identifying the degradation products of 15 naturally occurring peptide sequences. The bonds most susceptible to hydrolysis had a hydrophobic residue at P1, and either a small (e.g., ‐Gly or ‐NH2) or hydrophobic residue at P1. Hydrolysis of ‐Glu‐X bonds (most notably in neuropeptide Y) and the Arg6‐Arg7 bond in dynorphin peptides was also seen. Thus the three activities identified with fluorogenic substrates appear to be expressed against oligopeptides.
Inhibition of the Chick Pineal Rhythm in Rate of Thymidine Incorporation by Cyclic AMP
Authors: Wainwright S. D. , Wainwright Lillian K.
Published: October 2006

Abstract: Chick pineal glands in organ culture showed a circadian rhythm in the rate of thymidine incorporation. Thymidine incorporation was very markedly inhibited when 3‐isobutyl‐1‐methylxanthine (IBMX) was continuously present. When IBMX was added to cultures in control medium during the photoperiod of the second day in culture, the extent of inhibition of incorporation during that photoperiod increased with the increase in length of the photoperiod remaining. Incorporation did not resume at the start of a second photoperiod if IBMX was added within the first 10 h of the first photoperiod. Corresponding results were obtained with glands continuously cultured in constant darkness. Similar results were also obtained using glands treated with 4‐(3‐butoxy‐4‐methoxybenzyl)‐2‐imidazolidinone (Ro 20‐1724), 7β‐acetoxy‐8,13‐epoxy‐1 α,6β,9α‐trihydroxy‐1abd‐14‐ene‐11‐one (forskolin), or 8‐bromo‐cyclic AMP, but not with 8‐bromo‐cyclic GMP. When glands cultured with IBMX were transferred to control medium, incorporation remained inhibited until the start of the next photoperiod. We conclude that the increase in the rate of thymidine incorporation at the start of each new photoperiod is dependent on a “switch’ process that is inhibited by elevated concentrations of cyclic AMP.
Binding of Brain and Atrial Natriuretic Peptides to Cultured Mouse Astrocytes and Effect on Cyclic GMP
Authors: Yeung V. T. F. , Lai C. K. , Cockra C. S. , Young J. D. , Nicholls M. G.
Published: October 2006

Abstract: 125I‐Porcine brain natriuretic peptide (125I‐pBNP) bound to mouse astrocytes in primary culture in a time‐dependent manner (t1/2= 4.5 min), similar to 125I‐human atrial natriuretic peptide (125I‐hANP) (t1/2= 5 min). Binding was saturable and reached equilibrium after 90 min at 22°C for both radioligands. Scatchard analysis suggested a single class of binding sites for pBNP with a binding affinity and capacity (KD= 0.08 nM; Bmax= 78.3 fmol/mg of protein) similar to those of hANP1–28 (KD= 0.1 nM; Bmax= 90.3 fmol/mg of protein). In competition binding studies, pBNP or human/rat atrial natriuretic peptide (ANP) analogues [hANP1–28, rat ANP1–28 (rANP1–28), and rANP5–28] displaced 125I‐hANP, 125I‐pBNP, and 125I‐rANP1–28 completely, all with IC50 values of <1 nM (0.14–0.83 nM). All four peptides maximally stimulated cyclic GMP (cGMP) production by 10 min at 22°C at concentrations of 1 μM with EC50 values ranging from 50 to 100 nM. However, maximal cGMP induction by brain natriuretic peptide (BNP) (25.9 ± 2.1 pmol/mg of protein) was significantly greater than that by hANP1–28 (11.5 ± 2.2 pmol/mg of protein), rANP1–28 (16.5 ± 2.0 pmol/mg of protein), and rANP5–28 (15.8 ± 2.2 pmol/mg of protein). These studies indicate that BNP and ANPs act on the same binding sites and with similar affinities in cultured mouse astrocytes. BNP, however, exerts a greater effect on cGMP production. The difference in both affinity and selectivity between binding and cGMP production may indicate the existence of receptor subtypes that respond differentially to natriuretic peptides despite similar binding characteristics.
Sulfhydryl Groups Modulate the Allosteric Interaction Between Glycine Binding Sites at the Inhibitory Glycine Receptor
Authors: Ruiz‐Gómez Ana , Fernández‐Shaw Clara , Morato Esperanza , Marvizón Juan Carlos G. , Vázquez Jesús , Valdivieso Fernando , Mayor Federico
Published: October 2006

Abstract: We have investigated the effect of chemical reagents that modify sulfhydryl groups on the ligand binding properties of the glycine receptor (GlyR). The Hill coefficient (nH) for the displacement of [3H]strychnine binding by glycine was increased from ∼0.8 to values significantly above 1 (∼1.2–1.4) in membranes pretreated with the disulfide‐reducing agent dithiothreitol or glutathione. However, the affinity of strychnine or glycine for the GlyR was not affected by these treatments. This indicates that several glycine binding sites interact cooperatively for displacing bound strychnine under such experimental circumstances. A similar increase in the nH for glycine has been observed when the temperature of the binding assay was increased to 37°C. Combination of dithiothreitol pretreatment and increased binding temperature led to nH variations similar to those observed with either of these treatments alone, a finding suggesting that their mechanisms of action are not independent. Conversely, modification of rat spinal cord membranes or of purified and reconstituted GlyR preparations with the sulfhydryl‐alkylating agent N‐ethylmaleimide or fluorescein‐maleimide decreased nH values to ∼0.5, without affecting glycine or strychnine affinities. This effect may be caused by an increased heterogeneity of GlyR populations. It is interesting that occupancy of the receptor by glycine or β‐alanine (but not by antagonists) specifically protects from the effects of the different sulfhydryl reagents. Moreover, the presence of some of the Eccles' anions, i.e., anions that permeate through the channels associated with GlyRs and γ‐aminobutyric acidA receptors, seems to be required for the action of both dithiothreitol and N‐ethylmaleimide. Our results suggest the existence of thiol groups at the GlyR that are involved in the interaction between several glycine binding sites for displacing bound strychnine and that may also be functionally related to ionic and agonist sites. Furthermore, they raise the possibility that GlyR function could be regulated in vivo by reduction and oxidation.
Regional Variations in [3H]MK801 Binding to Rat Brain N‐Methyl‐D‐Aspartate Receptors
Authors: Reynolds Ian J. , Palmer Alan M.
Published: October 2006

Abstract: This study examined (+)‐[3H]5‐methyl‐10,11‐di‐hydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate ([3H]MK801) binding to the N‐methyl‐D‐aspartate (NMDA) receptor in membranes prepared from six regions of rat brain. Highest levels of binding were found in hippocampus and cortex, whereas much lower densities were found in brainstem and cerebellum. NMDA receptors in cerebellum exhibited a significantly lower affinity for [3H]MK801 than cortical NMDA receptors. To determine whether forebrain and hindbrain NMDA receptors were distinct, the actions of glutamate, NMDA, ibotenate, quinolinate, glycine, and spermine were investigated. These agents increased [3H]MK801 binding in all brain regions examined. However, agonists were uniformly less efficacious in hindbrain compared to forebrain regions. NMDA mimetics and spermine were less potent in cerebellum compared to cortex whereas glycine was equipotent. Antagonists that act at the various modulatory sites on the NMDA receptor were also examined. DL‐Amino‐phosphonopentanoic acid and 7‐chlorokynurenate were approximately equipotent in cortex and cerebellum. However, antagonists that are believed to act inside the NMDA‐operated ion channel, including Mg2+ and phencyclidine, were approximately threefold less potent in cerebellum. The diminished regulation of [3H]MK801 binding by glutamate and glycine in the cerebellum was associated with a smaller effect of these agonists on the dissociation of [3H]MK801 from its binding site. The levels of glutamate, aspartate, glycine, serine, and glutamine in the membrane preparations were determined. However, variations in the levels of endogenous amino acids were not sufficient to account for the regional differences in [3H]MK801 binding. These results do not support the hypothesis that a distinct NMDA receptor exists in hindbrain regions of the rat CNS. However, the extent to which NMDA receptors in the cerebellum can be activated by agonists and positive allosteric modulators may be significantly reduced compared to NMDA receptors in cortex.
Inositol 1,4,5‐Trisphosphate 3‐Kinase mRNA: High Levels in the Rat Hippocampal CA1 Pyramidal and Dentate Gyrus Granule Cells and in Cerebellar Purkinje Cells
Authors: Mailleux P. , Takazawa K. , Erneux C. , Vanderhaeghen J.‐J.
Published: October 2006

Abstract: The distribution of inositol 1,4,5‐trisphosphate (InsP3) 3‐kinase mRNA in the rat brain is reported using oligonucleotides based on a cDNA clone sequence that encodes rat brain InsP3 3‐kinase and the in situ hybridization technique. Moderate levels were found in CA2‐4 pyramidal neurons, in the cortex, and in the striatum. The cerebellar granule cells, thalamus, hypothalamus, brainstem, spinal cord, and white matter tracts were almost negative. The levels of InsP3 3‐kinase mRNA were highest in the hippocampal CA1 pyramidal neurons, granule cells of the dentate gyrus, and cerebellar Purkinje cells. These results contrast with the lower concentration of the InsP3 receptor already reported in the hippocampus versus the Purkinje cells and suggest a special role for inositol 1,3,4,5‐tetrakisphosphate in Ammon's horn.
A Monoclonal Antibody Recognizing K‐ but Not γ‐ and δ‐Opioid Receptors
Authors: Maderspach Katalin , Németh Klára , Simon József , Benyhe Sándor , Szücs Mária , Wollemann Mária
Published: October 2006

Abstract: A monoclonal antibody (mAb), KA8, that interacts with the k‐opioid receptor binding site was generated. BALB/ c female mice were immunized with a partially purified k‐opioid receptor preparation from frog brain. Spleen cells were hybridized with SP2/0AG8 myeloma cells. The antibodyproducing hybridomas were screened for competition with opioid ligands in a modified enzyme‐linked immunosorbent assay. The cell line KA8 secretes an IgG1 (k‐light chain) immunoglobulin. The mAb KA8 purified by affinity chromatography on protein A‐Sepharose CL4B was able to precipitate the antigen from a solubilized and affinity‐purified frog brain k‐opioid receptor preparation. In competition studies, the mAb KA8 decreased specific [3H]ethylketocyclazocine ([3H]EKC) binding to the frog brain membrane fraction in a concentration‐dependent manner to a maximum to 72%. The degree of the inhibition was increased to 86% when γ‐and δ‐opioid binding was suppressed by 100 nM [D‐Ala2,NMe‐Phe4,Gly‐ol]‐enkephalin (DAGO) and 100 nM [D‐Ala2,L‐Leu5]‐enkephalin (DADLE), respectively, and to 100% when γ‐, δ‐, and k2‐sites were blocked by 5 γM DADLE. However, the γ‐specific [3H]DAGO and the δ‐preferring [3H]DADLE binding to frog brain membranes cannot be inhibited by mAb KA8. These data suggest that this mAb is recognizing the k‐ but not the γ‐ and δ‐subtype of opioid receptors. The mAb KA8 also inhibits specific [3H]naloxone and [3H]EKC binding to chick brain cultured neurons and rat brain membranes, whereas it has only a slight effect on [3H]EKC binding to guinea pig cerebellar membranes. These findings suggest homologies in the k‐opioid binding site of frog brain and rat brain as well as chick neurons, but the k‐opioid receptor subtype in the guinea pig cerebellum may be different.
Genomic Effects of Cold and Isolation Stress on Magnocellular Vasopressin mRNA‐Containing Cells in the Hypothalamus of the Rat
Authors: Angulo Jesus A. , Ledoux Marie , McEwen Bruce S.
Published: October 2006

Abstract: We assessed the effects of cold and isolation stress on arginine vasopressin (AVP) mRNA in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Vasopressin mRNA levels were determined by in situ hybridization histochemistry at the cellular level. In posterior magnocellular neurons of the PVN isolation stress for 7 or 14 days increased vasopressin mRNA levels 28 and 29%, respectively, compared to group‐housed controls. No significant alterations in vasopressin gene expression were observed in the SON after 7 or 14 days of isolation stress. Scattered magnocellular AVP mRNA‐expressing cells of the medial parvocellular PVN showed increases of 19 and 34% after 7 and 14 days of isolation, respectively. We also studied the effect of cold or combined cold and isolation stress on vasopressin gene expression in the PVN and SON. Cold stress for 3 h daily for 4 consecutive days increased AVP mRNA levels in the posterior magnocellular PVN by 15%. Cold‐isolated animals showed an increase of 21%. No significant effect on AVP mRNA levels in the SON was observed. In contrast to the posterior magnocellular PVN, cold or cold‐isolation stress increased AVP mRNA in magnocellular neurons of the medial parvocellular region of the PVN by 25 and 43%, respectively, relative to control rats. These results suggest that psychological and metabolic stress may be added to the list of stressors that activate the hypothalamo–neurohypophysial system.
Characterization of [3H]Dopamine Uptake Sites and [3H]Cocaine Recognition Sites in Primary Cultures of Mesencephalic Neurons During In Vitro Development
Authors: Grilli Mariagrazia , Wright A. Gilbert , Hanbauer Ingeborg
Published: October 2006

Abstract: [3H]Dopamine uptake and [3H]cocaine binding sites were studied in primary cultures of ventral mesencephalon from 14‐day‐old rat embryos. Specific binding sites for [3H]cocaine and [3H]mazindol were detected only in intact cell cultures of ventral mesencephalon, and were absent in sonicated, washed membranes prepared from these cell cultures. [3H]Cocaine was not taken up by the cells through an active transport process because [3H]cocaine binding occurred also at 4°C. Moreover, the possibility of [3H]cocaine entering the cells by passive diffusion and ion trapping was also excluded because extensive washing failed to remove [3H]cocaine from the cells. [3H]Cocaine binding was reduced to 6% of control when cells were permeabilized with streptolysin O (0.2 U/ml, 5 min). Taken together, these results suggest that in cultured mesencephalic neurons, [3H]cocaine may enter the cell by passive diffusion and then be sequestered by a cytosolic compartment that is lost in the process of permeabilization or sonication and washing of membrane preparations. Permeabilization of cultured neurons failed to alter the storage of [3H]dopamine. When cells were permeabilized with streptolysin O (0.2 U/ml; 5 min) after [3H]dopamine was taken up, [3H]dopamine was retained by the cells and did not leak into the incubation medium, indicating that [3H]dopamine was stored in sites that could not pass through the perforated membranes. In contrast, [3H]dopamine uptake into already permeabilized cells was reduced by 33%, suggesting that a cytosolic protein that had leaked out may play a functional role in the uptake process. In contrast to striatal membrane preparations of adult rats, [3H]cocaine binding in intact mesencephalic cell cultures was Na+ independent. The expression of [3H]dopamine uptake and [3H]cocaine binding sites appeared to be developmentally linked to neuritic outgrowth, supporting the view that cocaine binding sites may be closely associated with the dopamine transporter.
Hexokinase Levels in Discrete Regions of Rat Brain: Evaluation by Quantitative Immuno fluoresence Using Interactive Laser Cytometry and Comparison with Basal Rates of Glucose Utilization
Authors: Burgess Robert W. , Wilson John E.
Published: October 2006

Relative levels of hexokinase (ATP:D‐hexose 6‐phosphotransferase, EC 2.7.1.1) have been determined in 16 discrete regions of adult rat brain by a quantitative immunofluorescence method. The distribution of immunofluorescence in brain sections was determined by interactive laser cytometry and related to hexokinase content by comparison with standard sections containing known amounts of the enzyme. In many of these regions, referred to here as group I regions, hexokinase content was correlated with previously reported basal rates of glucose utilization. However, several regions (group II regions) in which hexokinase content exceeded that expected from basal rates of glucose utilization were also detected. Compared with the corresponding regions from albino rat brain, higher hexokinase levels were found in the dorsal and ventral lateral geniculate (group I regions) of pigmented Norway rate, a result reflecting previously reported increased glucose utilization by these regions in pigmented rats. There was no difference in hexokinase levels in the superior colliculus, a group II structure, from albino and pigmented rats, a finding implying that a reported increase in rate of glucose utilization in the superior colliculus of pigmented rats is effected without an increase in hexokinase content. It is suggested that group II regions may be adapted to sustain increases in rates of glucose utilization that are, relative to basal rates, considerably greater than those experienced by group I regions.
Phosphatidylcholine Asymmetry in Electroplax from the Electric Eel: Use of a Phosphatidylcholine Exchange Protein
Authors: Supernovich Carrie , Grain Richard , Rosenberg Philip
Published: October 2006

Phosphatidylcholine asymmetry in the inner and outer leaflets of the plasma membrane bilayer of the innervated and noninnervated surfaces of the electroplax cell was determined, using a Phosphatidylcholine exchange protein. The exchange protein from bovine liver catalyzed the exchange of Phosphatidylcholine from small unilamellar vesicles to the outer monolayer of the plasma membrane bilayer. The exchange protein did not penetrate to the inner monolayer of the plasma membrane, did not modify the permeability of the electroplax, and did not alter the phospholipid or cholesterol content of the electroplax. In the innervated plasma membrane, 42% of the Phosphatidylcholine is in the outer leaflet, 33% is in the inner leaflet, and 25% is inaccessible to the exchange protein. Corresponding values for the noninnervated plasma membrane are 56, 26, and 18%, respectively. These results are similar to Phosphatidylcholine asymmetry in other biological membranes. This unique cell can be used as a model to test the effects on phospholipid asymmetry of compounds that act on the membrane.
Monoclonal Antibodies Against Myelin Proteolipid Protein: Identification and Characterization of Two Major Determinants
Authors: Yamamura Takashi , Konola Jukka T. , Wekerle Hartmut , Lees Marjorie B.
Published: October 2006

: This report describes the preparation and characterization of a panel of monoclonal antibodies (mAbs) against the myelin proteolipid protein (PLP). A Lewis rat was immunized with bovine proteolipid apoprotein and 27 mAbs were selected based on their reactivity against bovine PLP on enzyme‐linked immunosorbent assays. Eleven mAbs recognized the PLP carboxyl‐terminal sequence when tested against a panel of synthetic peptides in a solid‐phase assay. A carboxyl‐terminal pentapeptide (residues 272–276) was sufficient for antibody binding and the terminal phenylalanine residue was found particularly important. Deletion, modification, or replacement of this residue markedly reduced or obliterated antigen‐antibody interaction. Nine mAbs reacted with a second antigenic determinant, residues 209–217, but these could be identified only by competitive immunoassays. This peptide was a more effective inhibitor than the longer peptides 202–217 and 205–221, suggesting that flanking residues may interfere with peptide‐antibody interaction. Seven antibodies did not react with any of the synthetic peptides tested and their determinants remain unidentified. Immunoblot analysis showed that the mAbs reacted with both the PLP and the DM‐20 isoforms. Twenty‐three of the mAbs were of the immunoglobulin G2a or b isotype; the remaining antibodies were immunoglobulin M and all of these were specific for residues 209–217. Cultured murine oligodendrocytes were stained by most of the mAbs tested, but the most intense reactivity was observed with the carboxyl‐terminus‐specific mAbs. The immunocytochemical analyses demonstrate that the mAbs react with the native PLP in situ and show their potential usefulness for studies of the cell biology of myelin and oligodendrocytes.
Tellurium‐Induced Alterations in 3‐Hydroxy‐3‐Methylglutaryl‐CoA Reductase Gene Expression and Enzyme Activity: Differential Effects in Sciatic Nerve and Liver Suggest Tissue‐Specific Regulation of Cholesterol Synthesis
Authors: Toews A. D. , Goodrum J. F. , Lee S. Y. , Eckermann C. , Morell P.
Published: October 2006

The demyelination of peripheral nerves that results from exposure of developing rats to tellurium is due to inhibition of squalene epoxidase, a step in cholesterol biosynthesis. In sciatic nerve, cholesterol synthesis is greatly depressed, whereas in liver, some compensatory mechanism maintains normal levels of cholesterol synthesis. This tissue specificity was further explored by examining, in various tissues, gene expression and enzyme activity of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase, the rate‐limiting enzyme in cholesterol biosynthesis. Exposure to tellurium resulted in pronounced increases in both message levels and enzyme activity in liver, the expected result consequent to up‐regulation of this enzyme in response to decreasing levels of intracellular sterols. In contrast to liver, levels of mRNA and enzyme activity in sciatic nerve were both decreased during the tellurium‐induced demyelinating period. The temporal pattern of changes in 3‐hydroxy‐3‐methylglutaryl‐CoA reductase message levels in sciatic nerve seen following exposure to tellurium was similar to the down‐regulation seen for mRNA specific for PNS myelin proteins. Possible mechanisms for differential control of cholesterol biosynthesis in sciatic nerve and liver are discussed.