bluuebunny/crossref_metadata_2025 huggingface dataset:
2‐Oxoglutarate: Oxidation and Role as a Potential Precursor of Cytosolic Acetyl‐CoA for the Synthesis of Acetylcholine in Rat Brain Synaptosomes
Authors: Willoughby Jane , Craig Fiona E. , Harvey Stephen A. K. , Clark John B.
Published: October 2006

Abstract: The possibility that 2‐oxoglutarate may supply acetyl units for the cytosolic synthesis of acetylcholine in rat brain synaptosomes was investigated. The contribution of [14C]2‐oxoglutarate to the synaptosomal synthesis of [14C]acetylcholine was found to be negligible despite evidence for its uptake and oxidation. The activity of the enzymes NADP‐isocitrate dehydrogenase (EC 1.1.1.42), aconitate hydratase (EC 4.2.1.3), and ATP citrate‐lyase (EC 4.1.3.8) were measured in the synaptosol. NADP‐isocitrate dehydrogenase and aconitate hydratase are present at three‐ to 1.5‐fold higher activities than ATP citrate‐lyase. It seems likely that these enzymes contribute to the metabolism of citrate and prevent detectable formation of cytosolic acetyl‐CoA from exogenously added 2‐oxoglutarate (or citrate). The data further suggest that ATP citrate‐lyase may in part be associated with the mitochondrial fraction.
High‐Affinity Cannabinoid Binding Site: Regulation by Ions, Ascorbic Acid, and Nucleotides
Authors: Nye Jeffrey S. , Snowman Adele M. , Voglmaier Susan , Snyder Solomon H.
Published: October 2006

Abstract: The high‐affinity cannabinoid site in rat brain is an integral component of brain membranes that recognizes cannabinoids with inhibitory constants (Ki) in the nanomolar range. To clarify its physiological role, we studied the regulation of [3H]5′‐trimethylammonium Δ8‐tetrahydrocannabinol ([3H]TMA) binding. The site is inhibited by heavy metal ions, such as La3+, at low micromolar concentrations; divalent cations, such as Ca2+ and Mg2+, inhibit [3H]TMA binding, though at somewhat higher concentrations. In contrast, [3H]TMA binding is stimulated by Fe2+, Cu2+, and Hg2+ ions. Ascorbic acid and its analogs are also stimulators of cannabinoid binding at low micromolar concentrations. Stimulation of [3H]TMA binding by ascorbate or ions is dependent upon molecular oxygen, but is not inhibited by metabolic poisons. Metabolically stable nucleoside triphosphate analogs enhance [3H]TMA binding by different mechanisms, with hydrolysis of a high‐energy phosphate bond apparently requisite for these influences. These results suggest that the cannabinoid binding site is associated with a nucleotide‐utilizing protein possessing multiple regulatory subsites.
Equilibrium Binding Analysis of Neural Cell Adhesion Molecule Binding to Heparin
Authors: Nybroe Ole , Moran Niamh , Bock Elisabeth
Published: October 2006

Abstract: The kinetics of neural cell adhesion molecule (NCAM) binding to heparin were studied in a heparin‐Sepharose‐based solid‐phase binding assay. The observed binding is time dependent and saturable. A binding constant of 5.2 ± 1.4 × 10−8M is observed for binding of newborn rat NCAM to heparin. This is ∼25 times lower than the binding constant determined for newborn rat NCAM homophilic binding. Both Scatchard and Hill plot analyses suggest the presence of only one binding site. Fab' fragments of antibodies to rat NCAM significantly inhibit binding, a result indicating that a specific site on NCAM is involved in binding to heparin. The binding is inhibited by heparin (IC50, ∼5 μg/ml), whereas chondroitin sulfate is a less potent inhibitor (IC50, ∼15 μg/ml).
Periodate‐Modified Gangliosides Enhance Surface Binding of Tetanus Toxin to PC 12 Pheochromocytoma Cells
Authors: Nathan Annetta , Yavin Ephraim
Published: October 2006

Abstract: The interaction of 125I‐labeled tetanus toxin with PC 12 pheochromocytoma cells in monolayer cultures has been examined. Under regular growth conditions, the PC 12 cells bind 125I‐tetanus toxin to a limited degree compared with dissociated cerebral neuron cultures. After exposure to nerve growth factor for 2 days in low serum‐containing media with growth factor supplements, binding of toxin increases over twofold compared with untreated PC 12 cells. Binding can also be enhanced (>2.5‐fold) after treatment of cells with 2mM sodium metaperiodate for 20 min. Dissociated cerebral neurons but not fibroblasts in cell culture bind more toxin after periodate treatment. The effect of periodate can be abolished by 5 mM sodium borohydride. A ganglioside isolated from periodate‐treated PC 12 cells and tentatively identified as GT1b [(N‐acetylneuraminyl)galactosyl‐N‐acetyl‐galactosaminyl(N‐acetylneuraminyl‐N‐acetylneuraminyl)‐galactosyl‐glucosylceramide] binds 125I‐tetanus toxin on silica gel chromatoplates and on nitrocellulose paper. There are no indications to suggest binding to a polypeptide from treated cells after polyacrylamide gel electrophoresis. Cells artificially supplemented with GTlb and subsequently treated with periodate effectively bind the toxin. The data suggest that modified sialyl groups linked to gangliosides, and not to proteins, are preferential targets for tetanus toxin.
In Vitro Synthesis of Dopamine and Noradrenaline in the Isolated Rat Pineal Gland: Day‐Night Variations and Effects of Electrical Stimulation
Authors: Racké Kurt , Krupa Heike , Schröder Hannsjörg , Vollrath Lutz
Published: October 2006

Abstract: Isolated rat pineal glands were incubated in vitro in a medium containing [I4C]dopamine or [14C]tyrosine, and the tissue contents of l4C‐labelled and total dopamine and noradrenaline were determined by HPLC followed by electrochemical detection and scintillation spectrometry. During incubation with [l4C]dopamine, the labelled amine accumulated in pineal glands and was partially converted into [l4C]noradrenaline. Nomifensine, a neuronal amine uptake blocker, largely inhibited the accumulation of [l4C]dopamine and the formation of [14C]noradrenaline. These experiments demonstrated dopamine β‐hydroxylase activity in the sympathetic nerves of the pineal gland. During incubation with [14C]tyrosine, formation of [l4C]dopamine and [14C]noradrenaline was observed in the pineal tissue, indicating that noradrenaline can also be synthesized from dopamine, endogenously formed in the gland. Electrical stimulation of the stalk region of the pineal gland during incubation with [l4C]dopamine enhanced the accumulation of [l4C]dopamine and synthesis of [14C]noradrenaline. Electrical stimulation also enhanced the formation of [l4C]dopamine during incubation with [l4C]tyrosine. Compared to that at midday, the tissue content of endogenous noradrenaline at midnight was enhanced by 50% and that of dopamine by 450%. The in vitro accumulation of [14C]dopamine, as well as the synthesis of [14C]dopamine and [14C]noradrenaline, was also increased at midnight. In conclusion, sympathetic nerves in the rat pineal gland contain tyrosine hydroxylase and dopamine β‐hydroxylase, the two enzymes required for the synthesis of noradrenaline. The capacity of the pineal gland to synthesize dopamine and noradrenaline is enhanced during the night.
Spontaneous Glutamate Release by a “Fibrous”‐Like Cerebellar Astroglial Cell Clone
Authors: Cambier Danièle , Pessac Bernard
Published: October 2006

Abstract: To investigate the role of astrocytes in the metabolism of glutamate, the neurotransmitter of the granule cells of the cerebellar cortex, we have analyzed various parameters related to the synthesis of glutamate in astroglial cell clones that may be the in vitro counterparts of the cerebellar astrocytes. The “fibrous”‐like clone spontaneously released large quantities of glutamate, even in the absence of glutamine in the culture medium, but did not release alanine. In contrast, the “Golgi‐Bergmann”‐like cells released alanine but not glutamate, whereas the “velate‐protoplasmic”‐like astrocytes released little glutamate and alanine. However, the glutamate oxaloacetate transaminase and glutamate pyruvate transaminase activities of the three astroglial cell lines, measured in the direction of glutamate synthesis, were comparable. In addition, the “velate protoplasmic” and “Golgi‐Bergmann” clones did not consume glutamine present at 2 mM in the culture medium. These data suggest that the different types of in vivo cerebellar astrocytes may have distinct roles regarding glutamate‐glutamine metabolism.
Age‐Related Loss of t‐[35S]Butylbicyclophosphorothionate Binding to the γ‐Aminobutyric AcidA Receptor‐Coupled Chloride Ionophore in Rat Cerebral Cortex
Authors: Erdö Sandor L. , Wolff Joachim R.
Published: October 2006

Abstract: Muscimol and t‐butylbicyclophosphorothionate (TBPS) are known to label two distinct sites within the γ‐aminobutyric acidA(GABAA) receptor complex, i.e., the GABA recognition site and the chloride ionophore, respectively. Age‐dependent changes in the specific binding of [3H]muscimol and [3SS]TBPS were compared in membranes prepared from the cerebral cortex of rats, 2‐800 days old. Perinatal (day 2) binding of muscimol and TBPS represented 8 and 20% of the respective values for adults (day 180). After the first week, muscimol binding increased more rapidly than TBPS binding. Levels near those of adults were reached at day 20 and remained practically unchanged in adulthood (day 180). In aged (780‐day‐old) rats, the binding of TBPS was significantly reduced, whereas muscimol binding did not change compared with adult values This decrease of TBPS binding derived from a reduced density of binding sites, rather than from affinity changes. The allosteric responsiveness of TBPS binding to exogenous GABA was also reduced in aged animals. These findings indicate an age‐related change in the molecular (structural) organization of the GABAA receptor‐chloride ionophore complex in rat cerebral cortex.
α2‐Adrenergic, k‐Opiaie, and PrPurinergic Autoreceptors Have Mutually Antagonistic Effects: A New Regulatory Mechanism?
Authors: Adamson Peter , Mantzofiridis Timoleon , Xiang Jian‐Zhong , Hajimohammadreza Iradj , Bfammer Michael J. , Campbell Iain C.
Published: October 2006

Abstract: Rat cortical synaptosomes prepared on four‐step discontinuous Percoll density gradients were loaded with the fluorescent Ca2+‐indicator fura‐2 to allow measurement of the intrasynaptosomal free calcium concentration ([Ca2+]i). When P1‐purinergic, α2‐adrenergic, or k‐opiate agonists were incubated with these synaptosomes for 1 min, there was a highly significant, dose‐dependent reduction in [Ca2+]i. The effects of these agonists were blocked by inclusion of appropriate specific antagonists. When α2‐adrenergic and P1‐puri‐nergic agonists were coincubated, a mutual antagonism of their effects was observed, and, in fact, an increase rather than a decrease in [Ca2+]i was apparent. This mutual antagonism was reversed by addition of either a P1‐purinergic or a α2‐adrenergic antagonist. Parallel studies in which k‐opiate and P1‐purinergic agonists were coincubated also demon‐strated a mutual antagonism between the individual effects that was reversed by prior inclusion of either a k‐opiate or P1‐purinergic antagonist. As these mutually antagonistic effects have been observed between α2‐adrenergic, k‐opiate, and P1‐purinergic receptor‐mediated events, we suggest that this may be a general phenomenon and may be a regulatory mechanism at nerve endings.
Oligosaccharide Composition, Localization, and Developmental Changes of a CNS‐Specific (F3‐87‐8) Glycoprotein
Authors: Ripellino J. A. , Margolis R. U. , Margolis R. K.
Published: October 2006

Abstract: The F3‐87‐8 glycoprotein was isolated from rat brain by immunoaffinity chromatography after biosynthetic labeling by intracerebral administration of [3H]glucosamine, and the oligosaccharide composition of pronase‐derived gly‐copeptides was determined by sequential lectin affinity chro‐matography and alkali treatment. Triantennary complex oli‐gosaccharides (65%) and O‐glycosidic oligosaccharides (18%) were the predominant types present, accompanied by 7‐10% each of biantennary and high‐mannose oligosaccharides. Twenty‐two percent of the complex oligosaccharides had a fucose residue linked to the proximal (N‐acetylgluco|samine of the chitobiose units. No poly(N‐acetyllactosaminyl) or hybrid oligosaccharides were detected. Immunocytochemical studies on the localization of this glycoprotein in developing rat brain demonstrated that in 1‐week postnatal cerebellum, there is light staining of the internal granule cell layer and surrounding the Purkinje cells. By 2 weeks, an intense Staining of myelinating fiber tracts appears, accompanied by much lighter staining in the granule cell layer and at the base of the molecular layer. Staining of the white matter remains strong at 3 weeks postnatal, together with significant staining throughout the molecular layer, and then decreases in both areas by 1 month. In adult brain there is relatively uniform staining of approximately equal intensity in the white matter. granule cell layer, and molecular layer, whereas the Purkinje cell bodies appear unstained throughout development. In agreement with a previously reported immunochemical analysis, no staining was seen in other tissues, confirming the CNS‐specific localization of this glycoprotein.
Allosteric Activation off Brain Mitochondrial Ca2+ Uptake by Spermine and by Ca2+: Developmental Changes
Authors: Jensen John R. , Lynch Gary , Baudry Michel
Published: October 2006

Abstract: Kinetic analysis of 45Ca2+ uptake by raj brain mitochondria in Ca2+‐l,2‐bis(o‐aminophenoxy)ethane‐N, N,N′.N′‐tetraacetic acid buffers indicated that spermine both increased the apparent affinity for Ca2+ and decreased the cooperativity of uptake. Both effects are consistent with an allosteric activation of uptake by spermine. The stimulating effect of spermine on 45Ca2+ uptake was maximal with mitochondria from postnatal day 10 animals and then steadily decreased with increasing age to reach adult values by ∼30 postnatal days; this was observed independently of the substrates used to fuel mitochondria. Mitochondrial Ca2+ buffering was also analyzed by use of a Ca2+‐selective electrode. Addition of a large bolus of Ca2+ produced a decrease in the subsequent equilibrium extramitochondrial Ca2+ concentration (or a “rebound overshoot”) under some conditions. It is proposed that this effect is the result of an allosteric activation of Ca2+ uptake by Ca2+. This effect was slowly reversible, or hysteretic, and was blocked by spermine. The overshoot was increased in the presence of higher concentrations of Mg2+ and was absent when mitochondria were incubated with 0.3 mM Mg2+. It was maximal in mitochondria prepared from early postnatal brain, and changes in the magnitude of the effect during development paralleled those obtained with spermine stimulation of 45Ca2+ uptake. The data suggest that spermine produces an allosteric activation of Ca2+ uptake by binding to the same regulatory sites that are involved in the Ca2+‐induced activation. The results as a whole suggest that spermine could modulate mitochondrial buffering of the intracellular Ca2+ concentration in brain, particularly during the early postnatal period.
Effects of Diltiazem on Bioenergetics, K+ Gradients, and Free Cytosolic Ca2+ Levels in Rat Brain Synaptosomes Submitted to Energy Metabolism Inhibition and Depolarization
Authors: Dagani F. , Feletti F. , Canevari L.
Published: October 2006

Abstract: Diltiazem was able to decrease the oxygen consumption rate and lactate production in synaptosomes isolated from rat forebrains, both under control and depolarized (40 μM veratridine) conditions, starting from a concentration of 250 μM. This effect was particularly evident when synaptosomes were depolarized by veratridine. This depolarization‐counteracting action was evident also when transplasma membrane K+ diffusion potentials were measured after depolarization induced by veratridine and by rotenone with a glucose shortage. The concentrations of ATP, phosphocre‐atine, and creatine were less sensitive to diltiazem action. The concentration/response relationships were the same as those found for the oxygen consumption rate, lactate production, and K+ diffusion potentials. The effects of 0.5 mM diltiazem in counteracting inhibition of energy metabolism induced by rotenone without glucose were no longer detectable when either Ca2+ or Na+ was absent from the incubation medium of synaptosomes. Diltiazem at the same concentrations (starting from 250 μM) was able to inhibit both the veratridine‐induced and the rotenonc‐without‐glucose‐in‐duced increase in intrasynaptosomal free Ca2+ levels evaluated with the fluorescent probe quin2. The results are discussed in view of a possible effect of diltiazem on voltage‐dependent Na+ channels and the possibility of utilizing this approach for counteracting neuronal failure due to derangement of energy metabolism or hyperexcitation
Identification and Characterization of Multiple Tachykinin Immunoreactivities in Bovine Retina: Evidence for the Presence of a Putative Oxidative Inactivation System for Substance P
Authors: Shaw C. , Foy W. L. , Johnston C. F. , Buchanan K. D.
Published: October 2006

Abstract: Tachykinin immunoreactivity has been quantified and characterized in extracts of bovine retinae by combining radioimmunoassay, gel permeation chromatography, and reverse‐phase HPLC. Using an antiserum specific for the C‐terminal hexapeptide amide of substance P, levels of 3.43 ± 0.33 ng g−1 and 12.45 ± 0.76 ng g−1 (mean ± SD, n = 5) were measured in extracts prepared by acidified ethanol and boiling 0.5 M acetic acid, respectively. Levels of neurokinin A immunoreactivity, assayed using an antiserum cross‐reacting with neurokinin A (100%), neurokinin B (50%), neuropeptide K (85%), and substance P (<0.1%) were 12.46 ± 0.47 ng g−1 and 7.20 ± 0.37 ng g−1 in the same extracts. Gel permeation chromatography identified a single substance P immunoreactant eluting with substance P standard, whereas two neurokinin A immunoreactants were resolved eluting with neuropeptide K and neurokinin A standards. Reverse‐phase HPLC analysis resolved immunoreactivity eluting with substance P, neurokinin A, neuropeptide K, and neurokinin B and their respective methionine sulphoxides. The amount of immunoreactive material co‐eluting with the respective sulphoxides was higher in acidified ethanol extracts, and sub stance P was most susceptible to oxidative modification. Subsequent incubation of synthetic substance P with dispersed bovine retinal cells resulted in rapid conversion to three metabolites identified and isolated by reverse‐phase HPLC. Each had an amino acid composition identical to that of substance P, and the major product had the same retention time as substance P sulphoxide. Deamidation was ruled out on the basis of the unequivocal demonstration of both glutamine residues and a methionine amide residue on each metabolite. These data demonstrate the presence of the mammalian tachykinins substance P, neurokinin A, neurokinin B, and neuropeptide K in the bovine retina. The susceptibility of substance P to methionine oxidation has been confirmed by incubation with dispersed retinal cells, and this putative inactivation system may be of physiological relevance within the retina.
Chromatographic Analyses of the Serotonin 5‐HT1A Receptor Solubilized from the Rat Hippocampus
Authors: Mestikawy S. El , Taussig D. , Gozlan H. , Emerit M. B. , Ponchant M. , Hamon M.
Published: October 2006

Abstract: Serotonin 5‐HT1A receptors in rat hippocampal membranes were solubilized by 10 mM 3‐[3‐(cholamidopro‐pyl)dimethylammonio]‐l‐propane sulfonate (CHAPS) and chromatographed on various gels in an attempt to design a relevant protocol for their (partial) purification. In particular, an affinity gel made of the 8‐hydroxy‐2‐(di‐n‐propylami‐no)tetralin (8‐OH‐DPAT) derivative 8‐methoxy‐2‐[(N‐propyl, N‐butylamino)amino]tetralin (8‐MeO‐N‐PBAT) coupled to Affigel 202 was specially developed for this purpose. First, studies of the effects of various compounds (detergents, lipids, reducing agents, sugars, etc.) on the specific binding of [3H]8‐OH‐DPAT and on the rate of heat‐induced inactivation of solubilized 5‐HT1A sites led to a buffer composed of 50 mM Tris‐HCl, 50 μM dithiothreitol, 1 mM CHAPS, 10% glycerol, 0.1 mA/ MnCl2, and 50 μg/ml of cholesleryl hemisuccinate, pH 7.4, ensuring a high degree of stability of solubilized 5‐HT1A sites, compatible with chromatographic analyses for 2–4 days at 4°C. Adsorption and subsequent elution of [3H]8‐OH‐DPAT specific binding sites were found with several chromatographic gels, including wheat germ agglutinin‐agarose, phenyl‐Sepharose, hydroxylapatite‐Ultrogel, diethyl‐aminoethyl (DEAE)‐Sepharose, and DEAE‐Sephacel. Similarly, 8‐MeO‐N‐PBAT‐Affigel 202 allowed the adsorption and subsequent elution (by 1 mM 5‐HT) of active 5‐HT1Abinding sites solubilized from rat hippocampal membranes. The two‐step chromatography using 8‐MeO‐N‐PBAT‐Affigel 202 followed by wheat germ agglutinin‐agarose gave a fraction enriched (by at least 400‐fold) in 5‐HT1A sites. Sodium do‐decyl sulfate‐polyacrylamide gel electrophoresis of this partially purified fraction revealed a major protein band with Mr close to 60,000.
Differential Modulation of D1 and D2 Dopamine‐Sensitive Adenylate Cyclases by 17β‐Estradiol in Cultured Striatal Neurons and Anterior Pituitary Cells
Authors: Maus M. , Bertrand P. , Drouva S. , Rasolonjanahary R. , Kordon C. , Glowinski J. , Premont J. , Enjalbert A.
Published: October 2006

Abstract: Primary cultures of anterior pituitary cells from female rats and of mouse embryonic striatal neurons were used to study the effects of 17β‐estradiol on D1‐ and D2‐dopamine (DA)‐sensitive adenylate cyclase. 17β‐Estradiol pretreatment (10−9M, 72 h) suppressed the D2‐DA‐induced inhibition of adenylate cyclase activity in anterior pituitary cells. The steroid (10−9M, 24 h) also blocked the D2‐DA‐evoked response in striatal neurons whereas it enhanced by twofold the D1‐DA‐induced stimulation of the enzyme activity in these neurons. All these effects of the steroid were dose dependent and specific, as neither 17α‐estradiol, dexamethasone, nor progesterone used at the same concentration (10−9M) was effective. Furthermore, the modulation of DA‐sensitive adenylate cyclases by the steroid required long‐term exposure of living cells to 17β‐estradiol since neither 17β‐estradiol pretreatment for 4 h nor its addition to broken cells directly into the adenylate cyclase assay induced any alteration in the DA‐sensitive adenylate cyclase activity. These results are in agreement with a genomic effect of the steroid. Using both anterior pituitary cells and striatal neurons in culture, 17β‐estradiol affected neither the total number of DA (D1 and D2) receptors nor the estimated number of adenylate cyclase catalytic units. Therefore, it is suggested that the steroid modifies the coupling process by a mechanism that still has to be elucidated. These results demonstrate an effect of 17β‐estradiol on DA target cells in both systems. The steroid‐induced changes in DA receptivity could account, at least in part, for the modulation of hormonal (in the anterior pituitary) and behavioral (in the striatum) responses mediated by DA observed under various physiological or pathological conditions.
Oxidation of N‐Methyl‐1,2,3,4‐Tetrahydroisoquinoline into the N‐Methyl‐Isoquinolinium Ion by Monoamine Oxidase
Authors: Naoi Makoto , Matsuura Sadao , Parvez Hasan , Takahashi Tsutomu , Hirata Yoko , Minami Masayasu , Nagatsu Toshiharu
Published: October 2006

Abstract: N‐Methyl‐1,2,3,4‐tetrahydroisoquinoline (NMTIQ) was found to be oxidized by monoamine oxidase (MAO) into N‐methylisoquinolinium ion, which was proved to inhibit enzymes related to the metabolism of catecholamines, such as tyrosine hydroxylase, aromatic‐L‐amino acid decarboxylase, and MAO. NMTIQ was oxidized by both types A and B MAO in human brain synaptosomal mitochondria. Oxidation was dependent on the amount of MAO sample and the reaction time. Enzyme activity with respect to NMTIQ reached optimum at a pH of ∼7.25, as was the case with other substrates. Type A MAO had higher activity for this substrate than type B. The Km and Vmax values of the oxidation by types A and B MAO were 571 ± 25 μM and 0.29 ± 0.06 pmol/min/mg protein, and 463 ± 43 μM and 0.16 ± 0.03 pmol/min/mg protein, respectively. The Vmax values of types A and B MAO for NMTIQ were much smaller than those for other substrates such as kynuramine. NMTIQ was the first tetrahydroisoquinoline shown to be oxidized into the isoquinolinium ion by MAO in the brain.
Characterization of Binding Sites for the Angiotensin II Antagonist 125I‐[Sarc1,Ile8]‐Angiotensin II on Murine Neuroblastoma N1E‐115 Cells
Authors: Fluharty Steven J. , Reagan Lawrence P.
Published: October 2006

Abstract: The murine neuroblastoma N1E‐115 cell line contains binding sites for the angiotensin II (Ang II) receptor antagonist 125I‐[Sarc1,Ile8]‐Ang II (125I‐SARILE). Binding of 125I‐SARILE to N1E‐115 membranes was rapid, reversible, and specific for Ang II‐related peptides. The rank order potency of 125I‐SARILE binding was the following: [Sar1]‐Ang II = [Sar1Ile8]‐Ang II > Ang II > Ang III = [Sarc1, Thr8]‐Ang II > Ang I. Scatchard analysis of membranes prepared from confluent monolayers revealed a homogenous population of high affinity (KD= 383 ± 60 pM) binding sites with aBmax of 25.4 ± 1.6 fmol/mg of protein. Moreover, the density, but not the affinity, of the binding sites increased as the cells progressed from logarithmic to stationary growth in culture. Finally, agonist, but not antagonist, binding to N1E‐115 cells was regulated by guanine nucleotides. Collectively, these results suggest that the murine neuroblastoma N1E‐115 cell line may provide a useful model in which to investigate the signal transduction mechanisms utilized by neuronal Ang II receptors.
Formation of Ganglioside GD 1b‐Lactone in Rat Brain from Intracisternally Administered GD 1b
Authors: Riboni Laura , Ghidoni Riccardo , Tettamanti Guido
Published: October 2006

Abstract: The presence of ganglioside GD1b, in lactone form GD1b‐L, was ascertained in rat brain. The possible formation of GD1b‐L from GD1b in brain was explored by the intracisternal injection of GD1b, 3H‐labelled at the level of the terminal galactose. This was followed by recognition of the radioactive gangliosides formed at different times (1,3, and 7 days) after injection. Whereas at 0 time after injection the only radioactive ganglioside was GD1b, after 1, 3, and 7 days other radioactive gangliosides were also found, thus indicating GD1b penetration into the brain tissue, followed by metabolic processing. Besides GD1b, the following radioactive gangliosides were recognized: GM1 and GM2, derived from GD1b degradation; GT1b, formed by the direct sialylation of GD1b; and GD1b‐L, produced by metabolic lactonization. The radioactivity carried by GD1b‐L was maximal 3 days after injection; its time course was different from that of the other gangliosides, suggesting that the process of lactonization is separate from that of both degradation and glycosylation. Under the same experimental conditions, some radioactive gangliosides also appeared in the liver, although in much smaller amounts than in brain. Radioactive GD1b‐L could not be detected in liver, thus indicating that metabolic lactonization is a tissue‐ or organ‐specific process.
Accumulation of Inositol Phosphates Induced by Chlorpromazine in C6 Glioma Cells
Authors: Leli Ubaldo , Ananth Uma , Hauser George
Published: October 2006

Abstract: Chlorpromazine, a cationic amphiphilic drug known to affect phospholipid metabolism, greatly increases the generation of inositol phosphates in C6 glioma cells. When a pulse‐chase protocol with myo‐[2‐3H]inositol as the radioactive precursor was used, the peak increase in radioactivity of inositol phosphates was observed at 20 min. The drug decreased inositol tetrakisphosphate labeling as a percentage of inositol trisphosphate in a dose‐dependent manner. It also increased the labeling of the inositol‐containing phospholipids, the precursors of the inositol phosphates. The increase in radioactivity of both phospholipids and inositol phosphates was dose‐dependent, but appeared also to be a function of the time of exposure of the cultures to the drug, suggesting that the concentration of chlorpromazine in the cell, and not that in the medium, is the critical factor. The optimum concentration for maximum phospholipid labeling was lower than that eliciting maximum generation of inositol phosphates. The data suggest that the mechanism probably does not involve cell‐surface receptors, but rather may consist of a direct effect of chlorpromazine on phosphoinositidase C and possibly other enzymatic reactions concerned with the metabolism of inositol phosphates.
Purification of Neuronal Cell Surface Proteins and Generation of Epitope‐Specific Monoclonal Antibodies Against Cell Adhesion Molecules
Authors: Schlosshauer Burkhard
Published: October 2006

Abstract: To establish a procedure for the purification of a broad spectrum of cell surface proteins, three separate methods based on different principles were compared with the aid of four marker proteins. Membrane preparation by sedimentation–flotation centrifugation, temperature‐induced phase separation with Triton X‐114, and lectin affinity chromatography were used separately as well as in combination. The two‐step procedure of membrane preparation and lectin affinity chromatography provided by far the best enrichment of cell surface marker proteins. This result was further substantiated by screening >6,600 hybridoma cultures that originated from mice that had been immunized with protein fractions obtained by different purification protocols. In addition, it was found that solubilized glycoproteins used as immunogens led to many more cell surface‐specific monoclonal antibodies than glycoproteins immobilized on lectinagarose beads. Three monoclonal antibodies that recognize distinct epitopes of cell adhesion molecules (CAMs) were isolated. Monoclonal antibody C4 bound to a detergent‐labile epitope of G4 (neuron–glia CAM). Monoclonal antibody D1 recognized specifically nonreduced neural CAM (N‐CAM) with intact disulfide bridges, and monoclonal antibody D3 recognized only the 180‐kilodalton isoform of N‐CAM. Because of these specificities, these monoclonal antibodies promise to be useful tools for the elucidation of the structural organization of adhesion molecules.
Sulfation of Rat Apolipoprotein E
Authors: Gebicke‐Haerter Peter J. , Shooter Eric M.
Published: October 2006

The synthesis of a 37‐kilodalton (kDa) protein which has been shown recently to be identical with apolipoprotein E (apo‐E) was increased after sciatic nerve injury of the rat. When regeneration of the nerve was allowed, its synthesis returned to control levels at about 8 weeks post injury. In this report it is shown that similar time‐course studies of the protein in the rat optic nerve revealed a delayed increase of the protein but a comparably high level of synthesis at 3 weeks post injury. This level was maintained up to at least 18 weeks after crush. Furthermore, two‐dimensional electrophoresis revealed that the characteristic “trailing” of the protein is due to its sialylation, because it was reduced after neuraminidase treatment. This treatment, however, detected a neuraminidase‐resistant heterogeneous form in CNS tissue and a homogeneous form in peripheral nervous tissue. The trailing persisted up to 18 days of culture of optic nerve explants, of CNS glial cells, and of peritoneal macrophages, but disappeared during the first culture days of sciatic nerve explants and was not observed in Schwann cell culture media. Incorporation studies with 35SO4 revealed that apo‐E was the major sulfated protein in culture media conditioned by CNS glial cells, whereas sulfation of the protein was undetectable in Schwann cell cultures. Because macrophages are likely to be the major source of apo‐E in both peripheral and central glial cell cultures as well as in injured optic and sciatic nerves, it is hypothesized that resident cells of sciatic nerves secrete potent sulfatases. As a result, sialic acid residues may be more susceptible to degradation. Furthermore, the affinity of apo‐E toward heparan sulfate proteoglycans of the extracellular matrix may be increased, which results in its preferential accumulation in the peripheral nerve.
Fructose 2,6‐Bisphosphate Changes in Rat Brain During Ischemia
Authors: Yamamoto Masaaki , Hamasaki Naotaka , Maruta Yoshiro , Tomonaga Masamichi
Published: October 2006

Brain ischemia was produced by bilateral ligation of the common carotid arteries of spontaneously hypertensive rats. The concentrations of fructose 2,6‐bisphosphate and other glycolytic intermediates as well as of pyridine and adenine nucleotides were measured in frozen brain samples. In contrast to the decrease reported in hepatocytes under anoxic conditions, the fructose 2,6‐bisphosphate content was increased by 20–30% during the early stages of ischemia. Elevation in fructose 1,6‐bisphosphate level and lactate formation followed the rise in fructose 2,6‐bisphosphate content, a finding suggesting that this compound plays a key role in the compensatory acceleration of glycolysis under ischemic conditions in vivo.
Toxicity of 1‐Methyl‐4‐Phenylpyridinium for Rat Dopaminergic Neurons in Culture: Selectivity and Irreversibility
Authors: Michel P. P. , Dandapani B. K. , Knusel B. , Sanchez‐Ramos J. , Hefti F.
Published: October 2006

Cultures of dissociated embryonic rat mesencephalic cells were exposed to 10 μM 1‐methyl‐4‐phenylpyridinium (MPP+), a concentration shown earlier to result in loss of >85% of tyrosine hydroxylase (TH)‐positive neurons without affecting the total number of cells observed by phase‐contrast microscopy. To characterize better the selectivity of the toxic action of MPP+, other parameters were measured reflecting survival and function of dopaminergic or nondopaminergic neurons. Exposure of cultures to 10 μM MPP+ for 48 h reduced TH activity to 11% of control values without reducing protein levels. [3H]Dopamine uptake was reduced to < 4% of control values, whereas the uptake of γ‐[3H]aminobutyric acid ([3H]GABA) was not affected in these cultures. This same treatment failed to reduce the number of cholinergic cells visualized in septal cultures and did not affect either choline acetyltransferase activity or high‐affinity choline uptake. To assess for possible recovery of dopaminergic neurons, cultures were exposed to 10, 1.0, or 0.1 μM MPP+ for 48 h and then kept for up to 6 days in MPP+‐free medium. After exposure to 10 μM MPP+, the number of TH‐positive neurons, their neurite density, TH activity, and [3H]dopamine uptake remained at constant, reduced levels throughout the period of observation after termination of exposure, whereas GABA uptake remained normal. Treatment with lower concentrations of MPP+, i.e., 1.0 and 0.1 μM, induced less pronounced dopaminergic toxic effects. However, no recovery was seen after posttreatment incubation in toxin‐free medium. These findings provide evidence that MPP+ treatment results in highly selective and irreversible toxicity for cultured dopaminergic neurons.
Precursors for Cholesterol Synthesis (7‐Dehydrocholesterol, 7‐Dehydrodesmosterol, and Desmosterol): Cholesterol/7‐Dehydrocholesterol Ratio as an Index of Development and Aging in PNS but Not in CNS
Authors: Bourre J. M. , Clément M. , Gérard D. , Legrand R. , Chaudière J.
Published: October 2006

In rat sciatic nerve, the 7‐dehydrocholesterol content decreased dramatically during the postnatal period and slowly during adulthood and aging. In contrast, the 7‐dehydrodesmosterol content peaked at 14 days and was nearly undetectable after 60 days. The desmosterol content peaked at 21 days and was nearly undetectable after 1 year. The cholesterol content increased up to 21 days and remained nearly constant thereafter. In brain (in contrast to sciatic nerve), 7‐dehydrodesmosterol and desmosterol contents decreased dramatically during development and slightly during adulthood and aging; the 7‐dehydrocholesterol content peaked at 21 days and remained constant during aging. Only 7‐dehydrocholesterol was dramatically more concentrated in PNS than in CNS. In brain, the cholesterol/7‐dehydrocholesterol ratio increased during development and remained stable after 6 months. In contrast, in sciatic nerve, this ratio continuously increased during development and aging (950‐fold between 5 days and 18 months). Thus, the cholesterol/7‐dehydrocholesterol ratio is a useful biochemical index of development and aging in the PNS.
Expression of Functional D2 Dopamine Receptors Following Differentiation of Y‐79 Human Retinoblastoma Cells
Authors: Monsma Frederick J. , Barton Anne C. , Sibley David R.
Published: October 2006

Y‐79 human retinoblastoma cells grown in serumfree medium in monolayer culture have previously been shown to undergo differentiation in response to dibutyryl cyclic AMP (Bt2cAMP). We report here that Y‐79 cells treated in this manner also express very high levels of functional D2 dopamine receptors. In control Y‐79 cells, cultured in suspension, D2 dopamine receptors, quantified via saturation analysis with the D2 antagonist [3H]methylspiperone, are expressed at a level of ∼3 fmol/106 cells (∼1,800 receptor sites/cell). Differentiation is initiated by attachment of the cells to the culture dish with poly‐D‐lysine and fibronectin and continued culture in serum‐free medium. After 8 days in serumfree culture, differentiation is further induced with continuous Bt2cAMP treatment. Using this differentiation protocol, D2 receptor levels increase up to a maximum of 30 fmol/106 cells (18,000 receptors/cell) on day 20, the limit of culture viability. Cultures of 15–17 days (7–9 days of Bt2cAMP treatment) expressing receptor levels of 15–20 fmol/106 cells are used for pharmacological and functional characterization of D2 dopamine receptors. The pharmacology of competition for [3H]methylspiperone binding to differentiated Y‐79 (dY‐79) cell membranes by a series of dopaminergic antagonists verifies the D2 receptor nature of this site, exhibiting appropriate affinities and the following rank order of potency: YM‐09151‐2 ∼ spiperone > domperidone ∼ (+)‐butaclamol ∼ fluphenazine > chlorpromazine > (–)‐sulpiride > (+)‐sulpiride > promethazine > (+)‐SCH 23390 ≫ (–)‐butaclamol. Inhibition of [3H]methylspiperone binding by dopaminergic agonists in dY‐79 cell membranes indicates the presence of multiple agonist affinity states, which can be converted to a homogeneous low‐affinity state by addition of guanine nucleotides. In addition, incubation with dopamine or other D2‐selective agonists results in an ∼50% reduction in forskolin‐stimulated cyclic AMP production in lysed dY‐79 cells, which can be blocked by D2‐selective antagonists. These data indicate that the D2 receptors expressed in these cells are indeed functional. The ability to induce high levels of expression of functional D2 dopamine receptors in a cultured human cell line should provide an excellent model system with which to examine the molecular events involved in the expression and regulation of this receptor system.
Modulation of the Ca2+,Mg2+‐ATPase Activity of Synaptosomal Plasma Membrane by the Local Anesthetics Dibucaine and Lidocaine
Authors: Garcia‐Martin E. , Gutiérrez‐Merino C.
Published: October 2006

It has been previously shown that local anesthetics inhibit the total Ca2+,Mg2+‐ATPase activity of synaptosomal plasma membranes. We have carried out kinetic studies to quantify the effects of these drugs on the different Ca2+‐dependent and Mg2+‐dependent ATPase activities of these membranes. As a result we have found that this inhibition is not altered by washing the membranes with EDTA or EGTA. We have also found that the Ca2+‐dependent ATPase activity is not significantly inhibited in the concentration range of these local anesthetics and under the experimental conditions used in this study. The inhibition of the Mg2+‐dependent ATPase activities of these membranes was found to be of a noncompetitive type with respect to the substrate ATP‐Mg2+, did not significantly shift the Ca2+ dependence of the Ca2+,Mg2+‐ATPase activity, and occurred in a concentration range of local anesthetics that does not significantly alter the order parameter (fluidity) of these membranes. Modulation of this activity by the changes of the membrane potential that are associated with the adsorption of local anesthetics on the synaptosomal plasma membrane is unlikely, on the basis of the weak effect of membrane potential changes on the Ca2+,Mg2+‐ATPase activity. It is suggested that the local anesthetics lidocaine and dibucaine inhibit the Ca2+,Mg2+‐ATPase of the synaptosomal plasma membrane by disruption of the lipid annulus.
Sites of Tubulin Polymerization in PC 12 Cells
Authors: Keith Charles H. , Blane Kathleen
Published: October 2006

The site at which tubulin enters into polymer in the neuritic process is a very important datum in terms of our understanding of the mechanism of transport of the microtubular cytoskeleton out the axon. If the form of tubulin being transported out the axon is the microtubule, then assembly of tubulin into microtubules should occur at or near the cell body; if, however, the form of tubulin transported is free tubulin dimer, then assembly can occur at any free microtubule end out the neurite. We have injected a fluorescent analog of tubulin into differentiated PC12 cells and used differential extraction protocols to extract free dimer but not microtubules. We have imaged these cells before and after extraction by low‐light‐level video fluorescence microscopy and have used image analysis to examine the sites of tubulin incorporation into polymer or other unextracted components as a function of time. We find that tubulin in the distal reaches of the neurite is found initially as monomer and that its appearance in the unextracted component occurs later. This pattern of appearance of fluorescent tubulin initially in the soluble fraction and later in the unextractable component is qualitatively similar to that reported by other workers for biotinylated tubulin, but we see a larger gap between the rates of appearance in soluble fraction and in polymer. Quantitative analysis of fluorescence intensities in the two compartments with distance out the neurite reveals substantial variation between different neurites: In some neurites, the pattern of variation of unextracted/total tubulin suggests that tubulin enters into the unextracted component primarily near the cell body and that this unextracted component moves out the neurite with time, and in other neurites it suggests that monomer adds onto microtubule ends staggered out the neurite. In no case do we see a pattern suggesting that distal addition predominates. These analyses of fluorescence intensities in extracted and unextracted neurites suggest that both transport of polymerized microtubules and monomer addition onto staggered microtubule ends occur in PC12 neurites and that in individual neurites one or the other of these two behaviors may predominate.
Conservation of the Carboxyl Terminal Epitope of Myelin Proteolipid Protein in the Tetrapods and Lobe‐Finned Fish
Authors: Linington C. , Waehneldt T. V.
Published: October 2006

Immunochemical analysis of the myelin proteolipid protein (PLP) has identified the carboxyl terminal amino acid phenylalanine276 as the only PLP epitope conserved between the PLP components of rat and lungfish, species representing the phylogenetically most widely separated groups that synthesise typical CNS myelin. Immunoblotting using a rabbit antiserum raised against the carboxyl terminal sequence of rat PLP (residues 257–276) identified this epitope on the PLP components of both tetrapod (rat, chicken, lizard, and frog) and lobe‐finned fish (coelacanth and lungfish) CNS myelin, including the DM‐20 isoform of PLP, which is restricted to rat, chicken, and lizard CNS myelin. The conservation of the carboxyl terminus of PLP during evolution suggests this structure may play an important role in maintaining the organisation and function of PLP in the myelin membrane.
Gold plating on spectacle frames
Authors: Kenny Ian , Mitchell John W. C. , Walsh Glyn
Published: October 2006

An investigation was carried out into the thickness and standard of application of the plating and lacquer coatings applied to three metal spectacle frames. All conform to BS 6625 (1991) for plating thickness, but there was considerable variation in regularity and porosity.
A Method of Obtaining Renal Venous Blood in Unanesthetized Persons with Observations on the Extraction of Oxygen and Sodium Para-Amino Hippurate
Authors: Warren James V. , Brannon Emmett S. , Merrill Arthur J.
Published: October 2006

Catheterization of the renal vein offers a safe and relatively simple method of obtaining blood as it leaves the kidney in the resting unanesthetized human subject.

In preliminary observation on 8 subjects the renal arteriovenous oxygen difference varied from 1.9 to 2.8 volumes per cent., averaging 2.3 volumes per cent. Sodium para-amino hippurate, at low plasma levels, was 88 per cent. extracted during a single circulation through the kidney.
The Antibacterial Action of Penicillin Against Gram Negative Organisms
Authors: Hobby Gladys L.
Published: October 2006

It is apparent that penicillin exerts an antibacterial action against Gram negative as well as Gram positive organisms. This property of penicillin becomes more apparent in high potency preparations. It is possible that a form of penicillin showing greater activity against Gram negative organisms may exist. Studies on the nature of such a substance and on the in vivo action of penicillin against Gram negative organisms are in progress.
A Cylinder Guide for Use in Plate Assay of Penicillin
Authors: Oswald Elizabeth J. , Randall William A.
Published: October 2006

A guide or template is described which facilitates placing the cylinders on the agar surface in the plate assay of penicillin.

The general plan of conducting the assay is discussed and several advantages of the use of the guide are noted.

We wish to thank Mr. Albert G. Sterling, instrument maker, for fabricating the guide and the aluminum cylinders.
A Quantitative Study of the Fibrinolysin-Antifibrinolysin Reaction
Authors:   Commission on Acute Respiratory Diseases, Kaplan Melvin H.
Published: October 2006

The results of a study of the streptococcal fibrinolysin reaction and its inhibition by sera containing specific antibody are presented. It was possible to devise a quantitative antifibrinolysin test by controlling the various factors influencing the reaction. In man, the antifibrinolysin response was found to vary according to the strain of streptococcus responsible for the infection.
Effect of Merthiolate (Lilly) on Certain Specific Precipitation Reactions
Authors: Pressman David , Grossberg Allan L.
Published: October 2006

Merthiolate (Lilly), a substituted benzoic acid, has been shown to interfere, in concentrations usually used for preservation, with the specific precipitation of antisera against beef serum or sheep serum coupled with diazotized
p
-aminobenzoic acid,
p
-arsanilic acid, or
p
-(
p
-aminophenylazo) phenylarsonic acid. In some systems increased precipitation was observed, while in others decreased precipitation was observed.
The Changes in Rat Kidney Cocarboxylase Associated with the Injurious Effects of
dl
-Serine
Authors: Fishman William H. , Govier Wm. M.
Published: October 2006

When rats on either a stock or a vitamin B deficient diet are given the amino acid
dl
-serine by stomach tube, there is a fall in the cocarboxylase concentration of the kidney. This organ exhibits marked damage. There is, also, a lessened tendency for cocarboxylase to disappear from the tissue homogenate upon anaerobic incubation
in vitro.
In animals on the stock diet, only, there is a recovery of the normal cocarboxylase level. The probable relation of these findings to the mechanism of the production of the injurious action of serine is discussed.

We would like to acknowledge the helpful criticisms of Dr. C. Artom.
The Rhesus Gene and the Effect of Consanguinity
Authors: Stern Curt , Charles Donald R.
Published: October 2006

It is pointed out that consanguinity among the parents of children affected by erythroblastosis foetalis should be less than in the general population, unless other loci than the R locus are involved in the disease.