bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and characterization of Cs+‐permeable K+ channel current in mouse cerebellar Purkinje cells in lobules 9 and 10 evoked by molecular layer stimulation
Authors: Ishii Hiroshi , Nakajo Koichi , Yanagawa Yuchio , Kubo Yoshihiro
Published: July 2010

The mouse cerebellum consists of 10 lobules, which are distinguishable by their anatomical and functional properties. However, the differences in the slow postsynaptic currents (sPSCs) of Purkinje cells between lobules have not been well studied. We recorded the sPSCs of lobules 3, 9 and 10 evoked by tetanic stimulation of the molecular layer in cerebellar slices, and found a novel outward sPSC mediated by the GABAB receptor in loblues 9 and 10 but hardly at all in lobule 3. We showed that the lobule‐specific difference is at least partly attributable to differences in the density of GABAergic neurons (higher in lobule 10 than in lobules 3 and 9), and the functional expression level of postsynaptic GABAB receptor currents (larger in lobules 9 and 10 than in lobule 3). The G‐protein‐coupled inward rectifying K+ channel (GIRK) is known to be activated by GABAB receptors; however, the outward sPSC was not blocked by a GIRK blocker, was not sensitive to Cs+ block, and was observed when Cs+ was used as a charge carrier. These results suggest that a K+ channel other than GIRK could be activated by GABAB receptors. KCNK13 is a Cs+‐permeable K+ channel that shows intense expression of mRNA in Purkinje cells. KCNK13 current was enhanced by co‐expression of Gβγ subunits and was observed when Cs+ was used as a charge carrier in heterologous expression systems, and the amino acids critical for these features were identified by mutagenesis. Taken together, these results show that KCNK13 is a legitimate candidate for the Cs+‐permeable K+ channel activated by GABAB receptors, presumably via Gβγ subunits in Purkinje cells.
Efficacy of palatal block for analgesia following palatoplasty in children with cleft palate
Authors: JONNAVITHULA NIRMALA , DURGA PADMAJA , MADDURI VAMSIKRISHNA , RAMACHANDRAN GOPINATH , NUVVULA RAMBABU , SRIKANTH R. , DAMALCHERUVU MUKUNDA R.
Published: July 2010

Background:  This prospective randomized controlled study was undertaken to evaluate the efficacy of palatal block i.e. blocking of naso palatine, greater and also lesser palatine nerves in children with cleft palate undergoing palatoplasty by evaluating its effects on intraoperative anesthetic requirement, postoperative analgesia and parental satisfaction.

Methods:  Forty‐five pediatric patients aged below five undergoing cleft palate repair were randomly allocated to three groups of 15 each. After tracheal intubation, Group NB received no block for control, group S received 0.5 ml of normal saline and group B received 0.5 ml of 0.25% bupivacaine for palatal block. Postoperative pain score, the time to first demand of analgesia and number of rescue analgesic demands were noted. Finally, the parental satisfaction was graded.

Results:  The block had no anesthetic sparing effect. The mean pain scores were significantly lower in patients who received block than in the group NB. The mean area under curve for FLACC score in group NB was 29 with 95% CI of 25–32, group S was 15 with 95% CI of 8.9–22.3 and in group B, it was 10 with 95% CI of 6–14. The time to first demand of analgesia was 6 [4.5–6] h in group NB, 18 [6–18] h in group S and 18 [18–18] h in group B (P‐0.000). The number of demands of rescue analgesia was significantly less in group B 0 [0–0.25], 0 [0–2] in S group compared to group NB 3 [3–3] (P‐0.000). The parental satisfaction was good in patients who received block and poor in group NB.

Conclusion:  Palatal block is technically simple, safe and effectively provides postoperative analgesia with good parental satisfaction. Injection of saline also produced palatal nerve block; however, the effect was not consistent.
A novel isotonic‐balanced electrolyte solution with 1% glucose for perioperative fluid management in children‐ an animal experimental preauthorization study
Authors: WITT LARS , OSTHAUS WILHELM A. , BÜNTE CHRISTOPH , TEICH NATASCHA , HERMANN ELVIS J. , KASKE MARTIN , KOPPERT WOLFGANG , SÜMPELMANN ROBERT
Published: July 2010

Background:  The recommendations for perioperative maintenance fluid in children have been adapted from hypotonic to isotonic electrolyte solutions with lower glucose concentrations (1–2.5% instead of 5%) to avoid hyponatremia or hyperglycemia.

Objective:  The objective of this prospective animal study was to determine the margin of safety of a novel isotonic‐balanced electrolyte solution with 1% glucose (BS‐G1) in comparison with normal saline with 1% glucose (NS‐G1) in the case of accidental hyperhydration with a focus on acid–base electrolyte balance, glucose concentration, osmolality and intracranial pressure in piglets.

Methods:  Ten piglets (bodyweight 11.8 ± 1.8 kg) were randomly assigned to receive either 100 ml·kg−1 of BS‐G1 or NS‐G1 within one hour. Before, during and after fluid administration, electrolytes, lactate, hemoglobin, hematocrit, glucose, osmolality and acid–base parameters were measured.

Results:  Unlike BS‐G1, administration of NS‐G1 produced mild hyperchloremic acidosis (base excess BS‐G1 vs NS‐G1, baseline 1.9 ± 1.7 vs 2.9 ± 0.9 mmol·l−1, study end 0.2 ± 1.7 vs −2.7 ± 0.5 mmol·l−1, P < 0.05, chloride BS‐G1 vs NS‐G1 baseline 102.4 ± 3.4 vs 102.0 ± 0.7 mmol·l−1, study end 103.4 ± 1.8 vs 109.0 ± 1.4 mmol·l−1P < 0.05). The addition of 1% glucose led to moderate hyperglycemia (P < 0.05) with a concomitant increase in serum osmolality in both groups (P < 0.05).

Conclusion:  Both solutions showed a wide margin of safety in the case of accidental hyperhydration with less acid–base electrolyte changes when using BS‐G1. This novel solution could therefore enhance patient’s safety within the scope of perioperative volume management.
Fieldwork among the Dong national minority in Guizhou, China: Practicalities, obstacles and challenges
Authors: Cornet Candice
Published: July 2010

The People's Republic of China (PRC) is increasingly open to foreigners undertaking social science fieldwork; yet obstacles remain. Working with ethnic minorities adds further complexities because of the sensitive topics such research may raise. Based on recent fieldwork among the Dong in southeast Guizhou, as the first foreign researcher to ask for and gain official permission to work in the region, this article exposes some of the challenges, both practical and methodological, of conducting research in the PRC. Gaining access to my field site was a long trek through the hierarchic maze of Chinese administration. While reflecting upon this process, I detail my negotiations with local authorities. I then examine how I found reliable statistical data, was able to access the voices of peasants, acted to protect the anonymity of dissident informants, and negotiated working with local research assistants once in the field. These aspects, in turn, highlighted the importance of considering positionality in the field. Although each person's experiences and routes to fieldwork are unique, there are recurrent issues that shape the research process in the PRC. I reflect upon a number of these here, in the hope that this can smooth the way for future researchers.
Conducting fieldwork with Tarieng communities in southern Laos: Negotiating discursive spaces between neoliberal dogmas and Lao socialist ideology
Authors: Daviau Steeve
Published: July 2010

Based on research with ethnic minorities in Laos aimed at understanding how they cope with and negotiate political and economic ‘double domination’, this article examines the experiences of prolonged fieldwork in a remote Tarieng area in the Annam Range, southern Laos. After briefly reviewing Lao ethnographical policy and practice regarding ethnic minorities, I introduce the Tarieng people. I detail how I initially gained access to these local communities via long‐term engagement with a range of development project initiatives. Then, after eight years of conducting such fieldwork in a Tarieng area ‘below the radar of the state’, I managed to obtain official authorisations to continue research as a graduate student. In this new position, I accessed the field via different negotiations with central, provincial and local official bureaucracies. After detailing this process, back in the field I reveal my strategies to create a discursive space that has allowed me to access dissident Tarieng voices and agency. Finally, I highlight four central elements that have continued to shape my field research: language proficiency, working with research assistants, awareness of political relations and cultural sensitivity, and ethical concerns. These have emerged while the possibilities and constraints of political engagement with the Tarieng people are explored.
THE EFFECT OF HYPOXIA ON THE PENTOSE PHOSPHATE PATHWAY IN BRAIN
Authors: Hakim A. M. , Moss G. , Gollomp S. M.
Published: July 2010

—The role of the pentose phosphate pathway in brain glucose metabolism was studied in hypoxic rats. The method of directly labelling the 6‐phosphogluconate pool was used in both in vivo and in vitro experiments. Results indicate that there is significantly more glucose metabolized via this pathway in brains of hypoxic rats than in the controls.
THE REGULATION OF PYRUVATE DEHYDROGENASE IN BRAIN IN VIVO
Authors: Jope R. , Blass J. P.
Published: July 2010

—The activity of pyruvate dehydrogenase in the brains of mice frozen in liquid nitrogen was 14·0 nmol/min per mg protein. It rose to 23·8 nmol/min per mg protein after incubation of the brain homogenate with 10mm‐MgCl2 to activate (dephosphorylate) the enzyme, indicating that approx 60% of the enzyme was originally in the active form. Treatment with amobarbital or pentobarbital halved the proportion of pyruvate dehydrogenase in the active form. The proportion of pyruvate dehydrogenase in the active form increased during ischemia, activation being complete within one min. Anesthesia with amobarbital slowed the activation during ischemia but did not alter the total amount of pyruvate dehydrogenase activity. The concentration of ATP, the ATP/ADP ratio and the adenylate energy charge increased as the proportion of pyruvate dehydrogenase in the active form decreased during barbiturate anesthesia, and they decreased as the proportion of pyruvate dehydrogenase in the active form increased during ischemia. After treatment with insulin, the proportion of pyruvate dehydrogenase in the active form increased by 30%. but the energy charge did not change. Treatment of mice with ether, morphine, ethanol, or diazepam did not change the proportion of pyruvate dehydrogenase in the active form although these treatments have been reported to alter pyruvate oxidation in brain in vivo. Treatments which altered pyruvate oxidation in the brain did not consistently alter the proportion of pyruvate dehydrogenase in the active form, unless they also altered energy charge.
MICROHETEROGENEITY AND PHOSPHOAMINO ACIDS IN THE CARBOXY‐TERMINAL HALF OF MYELIN BASIC PROTEIN
Authors: Martenson R. E. , Kramer A. J. , Deibler Gladys E.
Published: July 2010

—Three chromatographically distinct peptic peptides (F80‐1, F80‐2 and F80‐3) derived from the C‐terminal half of the bovine and guinea‐pig myelin basic proteins were characterized. The three peptides of each animal species had the same N‐terminal residue (phenylalanine) and essentially the same amino acid composition, but they differed in electrophoretic mobility at alkaline pH. The least basic peptide (F80‐3) differed from the others in showing a deficit of C‐terminal arginine residues and in containing phosphorus, 0·37 and 0·46 g‐atom/mol of bovine and guinea‐pig peptides, respectively. Other peptic peptides. derived from the N‐terminal half of the basic protein, were essentially phosphorus‐frcc. Analyses of partial acid hydrolyzates of peptide F80‐3 by high voltage electrophoresis showed the presence of both phosphoserine and phosphothreonine. After incubation with E. coli alkaline phosphatase (EC 3.1.3.1). 34 and 40% of the bovine and guinea‐pig F80‐3 peptides. respectively, were converted to peptide F80‐1. This reaction involved the loss of 2 net negative charges, and its extent corresponded to loss of essentially all of the phosphate originally present in the peptide. This result indicated that the phosphorylated species of peptide F80‐3 contained one phosphate group per molecule.
TRANSMITTER METABOLIZING ENZYMES AND FREE AMINO ACID LEVELS IN SENSORY AND MOTOR NERVES AND GANGLIA OF THE SHORE CRAB (CARCINUS MAENAS)
Authors: Emson P. C. , Bush B. M. H. , Joseph M. H.
Published: July 2010

—The distribution of ChAT (choline acetyltransferase), GAD (glutamate decarboxylase) and acetylcholinesterase in some sensory and motor nerves of the shore crab, Carcinus maenas, has been investigated using micro‐assay techniques. ChAT was concentrated in the afferent nerve fibres of the thoracic‐coxal muscle receptor as well as in the coxo‐basal chordotonal receptor nerve and other leg sensory fibres. GAD was found in leg motor nerves including the promotor and remotor muscle nerves, being undetectable in the sensory nerves. Acetylcholinesterase was found in similar levels in both sensory and motor nerves assayed.

Amino acid analysis using a micro‐dansylation technique showed that sensory nerves had low GABA levels, whereas the leg nerve including motor fibres had substantially higher GABA concentrations. GAD and GABA were also found in low amounts in the leg promoter mucle, which is consistent with GABA being a neuromuscular transmitter.
DETERMINATION OF MONOAMINE OXIDASE IN RAT BRAIN BY ELECTRON SPIN RESONANCE TECHNIQUES
Authors: Huang Rosa H. , Eiduson S. , Shih J. C.
Published: July 2010

—The recent development of spin‐labeling techniques has permitted its application to the measurement of the concentration of rat brain mitochondrial monoamine oxidase (MAO) and detection of possible multiple forms of this enzyme. Spin‐labeled p‐hydroxyamphetamine (SHA) was used as a probe of the active site of the enzyme. The binding of SHA to MAO was monitored by measuring the intensity of the × band ESR spectra obtained with a Varian V‐4502 ESR spectrometer. The measured volume of each sample was about 0·02 ml and time required for each measurement was 5 min. In the presence of MAO, a linear relationship was observed between spectral intensity and the concentration of the spin‐label in a range of 4 × 10−6m to 10−3m. A plot of the amount of enzyme‐inhibitor complex formed against the concentration of SHA suggested the existence of at least three different affinities of SHA to the possible multiple forms of MAO. Measurements made using a constant concentration of the spin‐label (3·21 × 10−5m) resulted in a calibration curve with two plateaus when the concentration of the enzyme‐inhibitor complex was plotted against the apparent concentration of MAO. These data also suggested that spin‐labeled hydroxyamphetamine had distinctly different affinities toward the multiple forms of MAO that were present in the partially purified preparation employed. Measurements made with constant concentrations of SHA and MAO but with varying temperatures (4‐56°C) resulted in a curve with two plateaus which also suggested the existence of at least three different binding affinities of the enzyme preparation for the inhibitor. This ESR technique is simple, rapid, accurate and fairly sensitive. It was also applied to the measurement of MAO in human platelets.
Expression of a Plasma Membrane Proteolipid During Differentiation of Neuronal and Glial Cells in Primary Culture
Authors: Shea Thomas B. , Fischer Itzhak , Sapirstein Victor
Published: July 2010

Abstract: Plasma membrane proteolipid protein (PMPLP) synthesis was examined in embryonic rat neurons and neonatal rat glial cells during differentiation in culture. Glial cultures were treated with 1 mM N6,O2, dibutyryl cyclic adenosine monophosphate (dbcAMP) following confluency to induce differentiation, which resulted in the elaboration of long cellular processes. However, no changes in the biosynthetic level of PMPLP was observed during the differentiation of these cells. Neurons differentiated spontaneously in culture, forming cellular aggregates immediately following plating and elaborating a network of neurites over 7 days. The differentiation of neurons was accompanied by a sevenfold increase in PM‐PLP synthesis with increases in biosynthetic rate observed betvyeen days 1 and 3 and between days 3 and 7 in culture. Ultrastructural examination of neurons indicated that the Golgi apparatus was also developing during this period of time, with an increase in both the number of lamellae and generation of vesicles. The transport of PM‐PLP to the plasma membrane was therefore examined in neurons at day 7 in culture by pulse labeling experiments with monensin and colchicine. Monensin (1 μM) was found to inhibit the appearance of radiolabeled PM‐PLP in the plasma membrane by 63%, indicating that a functional Golgi apparatus is required for transport of PM‐PLP to its target membrane. Colchicine (125 μM) also inhibited the appearance of newly synthesized PM‐PLP in the plasma membrane by >40%, suggesting that microtubules may also be required for PM‐PLP transport to the plasma membrane.
Na+‐Dependent “Binding” of D‐Aspartate in Brain Membranes Is Largely Due to Uptake into Membrane‐Bounded Saccules
Authors: Danbolt Niels C. , Storm‐Mathisen Jon
Published: July 2010

Abstract: Na+‐dependent “binding” of acidic amino acids in brain plasma membranes was examined by procedures similar to those employed in earlier studies, using the metabolically inert D‐[3H]aspartate as a probe. The “binding” showed characteristics similar to those described before in terms of affinity (KD, 400 nM). density of sites (Bmax, 300 pmol/mg protein), sensitivity to D,L‐threo‐3‐hydroxyaspartate. and requirement for Na+. It turned out that the “binding” represents uptake into membrane‐bounded saccules (which according to the inulin and H2O spaces constituted 3.4 μ/mg protein and comprised about 50% of the volume of the sedimented membranes), rather than binding to the transport carrier. This conclusion is based on the observations that the “binding” of D‐aspartate (1) was released by osmotic shock; (2) was abolished by thorough washing of membranes in H2O prior to assay, Which removed endogenous contents of amino acids, and could be recovered by loading the washed membranes with glutamate; (3) was reduced by prior freezing and thawing; (4) was low on incubation at 0°C; (5) had a bell‐shaped time course similar to that reported for uptake; and (6) had a slow rate of reversal compared to the apparent KD. True binding would have considerably lower apparent Bmax than the carrier‐mediated uptake. This and its likely rapid rate of dissociation would make binding to the carrier difficult to detect by the methods used up to now.
Isolation of Undegraded Free and Membrane‐Bound Polysomal mRNA from Rat Brain
Authors: Rademacher Bruce E. , Steele William J.
Published: July 2010

Abstract: A new procedure is described for the isolation of undegraded free and membrane‐bound polysomal mRNA from rat brain in which polysomes are simultaneously separated from soluble components of the cell and slowly sedimenting ribosome species and concentrated by rate‐zonal centrifugation on short linear sucrose gradients. This avoids the problem encountered in conventional procedures of pelleting polysomes along with membranous materials that are not solubilized by detergents and that appear to contain traces of nucleases. It also permits a more thorough analysis of the mRNA populations actively engaged in protein synthesis, since both polyadenylated and nonpolyadenylated mRNAs are isolated together. Moreover, the likelihood of sedimenting nonpolysomal mRNP particles along with polysomes is reduced by using rate‐zonal rather than pelleting centrifugation. The translational activity in vitro of free and membrane‐bound polysomal RNA prepared in this way is high and is about 1.5 times that of RNA prepared by a conventional pelleting technique. The difference is attributable to better preservation of large mRNAs, as inferred from two‐dimensional gel electrophoretic analysis of translation product abundance. The recovery of both classes of polysomal RNA is about 90%. The method is simple, efficient, and adapted for isolation of small amounts of polysomal RNA.
Pantothenic Acid Transport Through the Blood‐Brain Barrier
Authors: Spector Reynold , Sivesind Cindy , Kinzenbaw Dale
Published: July 2010

Abstract: The unidirectional influx of d‐pantothenic acid (PA) across cerebral capillaries, the anatomical locus of the blood‐brain barrier, was measured with an in situ rat brain perfusion technique using [3H]d‐PA (1.1 Ci/mmol). PA was transported across the blood‐brain barrier by a saturable system that could be described by a MichaelisMenten transport model with a half‐saturation concentration and maximal influx rate of 19 μM and 0.21 nmol/g of brain/min, respectively. PA (0.3 μM) transport through the blood‐brain barrier was significantly inhibited by probenecid, nonanoic acid, and biotin (all 0.25 mM), but not by penicillin G, pyruvate, β‐hydroxybutyrate. l‐leucine (all 1 mM), or poly‐l‐lysine HBr (1 mg/ml). Probenecid (0.25 mM), nonanoic acid (0.5 mM), and PA (1.0mM) did not inhibit [3H]l‐leucine transport through the blood‐brain barrier, whereas 30 μM‐l‐leucine inhibited [3H]leucine transport to 23% of control values. Thus, PA is transported through the blood‐brain barrier by a lowcapacity, saturable transport system with a half‐saturation concentration ‐10 times the plasma PA concentration. Although involved in the transfer of PA from blood into brain, this system does not play an important regulatory role in the synthesis of CoA from PA in brain.
Calcium‐Activated Neutral Proteinase of Human Brain: Subunit Structure and Enzymatic Properties of Multiple Molecular Forms
Authors: Vitto Anthony , Nixon Ralph A.
Published: July 2010

Abstract Calcium‐activated neutral proteinase (CANP) was purified 2,625‐fold from postmortem human cerebral cortex by a procedure involving chromatography on diethylaminoethyl (DEAE)‐cellulose, phenyl‐Sepharose, Ultrogel AcA‐44, and DEAE‐Biogel A. The major active form of CANP exhibited a molecular weight of 94–100 kilodaltons (Kd) by gel filtration on Sephacryl 300 and consisted of 78‐Kd and 27‐Kd subunits. Two‐dimensional gel electrophoresis resolved the small subunit into two molecular species with different isoelectric points. CANP degraded most human cytoskeletal proteins but was particularly active toward fodrin and the neurofilament protein subunits (145 Kd > 200 Kd > 70 Kd). The enzyme required 175 μMCa2+ for half‐maximal activation and 2 mM Ca2+ for optimal activity toward [methl‐14C]azocasein. Other divalent metal ions were poor activators of the enzyme, and some, including copper, lead, and zinc, strongly inhibited the enzyme. Aluminum, a neurotoxic ion that induces neurofilament accumulations in mammalian brain, inhibited the enzyme 47% at 1 mM and 100% at 5 mM A second CANP form lacking the 27‐Kd subunit was partially resolved from the 100‐Kd heterodimer during DEAE‐Biogel A chromatography. The 78‐Kd monomer exhibited the same specific activity, calcium ion requirement, pH optimum, and specificity for cytoskeletal proteins as the 100‐Kd heterodimer, suggesting that the 27‐Kd subunit is not essential for the major catalytic properties of the enzyme. The rapid autolysis of the 27‐Kd subunit to a 18‐Kd intermediate when CANP is exposed to calcium may explain differences between our results and previous reports, which describe brain mCANP in other species as a 76‐80‐Kd monomer or a heterodimer containing 76‐80‐Kd and 17‐20‐Kd subunits. The similarity of the 100‐Kd human brain CANP to CANPs in nonneural tissues indicates that the heterodimeric form is relatively conserved among various tissues and species.
Prevention of the Nigrostriatal Toxicity of 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine by Inhibitors of 3,4‐Dihydroxyphenylethylamine Transport
Authors: Mayer Roberta A. , Kindt M. Victoria , Heikkila Richard E.
Published: July 2010

Abstract The 3,4‐dihydroxyphenylethylamine (DA, dopamine) uptake inhibitors GBR 13,069, amfonelic acid, WIN‐35,065‐2, WIN‐35,428, nomifensine, mazindol, cocaine, McN‐5908, McN‐5847, and McN‐5292 were effective in preventing [3H]DA and [3H]l‐methyl‐4‐phenylpyridinium (MPP+) uptake in rat and mouse neostriatal tissue slices. These DA uptake inhibitors also were effective in attenuating the MPP+‐induced release of [3H]DA in vitro. 1‐Methyl‐4‐phenyl‐l,2,3,6‐tetrahydropyridine (MPTP) administration to mice (6 ± 25 mg/kg i.p.) resulted in a large (70–80%) decrement in neostriatal DA. WIN‐35,428 (5 mg/kg), GBR 13,069 (10 mg/kg), McN‐5292 (5 mg/kg), McN‐5908 (2 mg/ kg), and amfonelic acid (2 mg/kg), when administered intraperitoneally 30 min prior to each MPTP injection, fully protected against MPTP‐induced neostriatal damage. Other DA uptake inhibitors showed partial protection in vivo at the doses selected. Desmethylimipramine did not prevent [3H]MPP+ uptake or MPP+‐induced release of [3H]DA in vitro, and did not protect against MPTP neurotoxicity in vivo. These results support the hypothesis put forth previously by others that the active uptake of MPP+ by dopaminergic neurons is necessary for toxicity.
Pitfalls in the Assay of Cyclic AMP‐Dependent Protein Kinase Activity in Microsacs from Torpedo marmorata
Authors: Eriksson Håkan , Salmonsson Robert , Liljeqvist Gisela , Heilbronn Edith
Published: July 2010

Abstract Endogenous phosphorylation of the nicotinic acetylcholine receptor (nAChR) in microsacs from Torpedo marmorata was found to be affected by several reagents commonly used in the preparation of cyclic AMP (cAMP)‐dependent protein kinases and in its activity determination. The presence of a Na+,K+‐ATPase inhibitor is essential to avoid a rapid depletion of ATP, even when a membrane fraction highly enriched in the nAChR is used. The presence of the thiol reducing agent dithiothreitol was found to abolish the cAMP dependence of nAChR phosphorylation, whereas the less potent reagent 2‐mercaptoethanol did not affect the assay. Concentrations in the millimolar range of the chelators EDTA and EGTA were found to inhibit nAChR phosphorylation effectively. This inhibition was not due to a withdrawal of Ca2+ by the chelators, but rather to a reversible inhibition by the Mg2+ complexes. These observations may explain some of the discrepancies found in the literature concerning endogenous and exogenous nAChR phosphorylation.
Parvalbumin in Cat Brain: Isolation, Characterization, and Localization
Authors: Stichel Christine C. , Kägi Urs , Heizmann Claus W.
Published: July 2010

Because of the increasing evidence that Ca2+‐binding proteins have important regulating functions in nerve cells and because of the indications that there are species differences in the structures of these proteins, parvalbumin was purified from cat brain and muscle. Brain and muscle parvalbumins were found to be indistinguishable from each other in their biochemical and im‐munological properties. However, cat parvalbumin differs from all other mammalian parvalbumins by its apparently lower Mr on sodium dodecyl sulfate‐polyacryl‐amide gel electrophoresis of 10–11K (compared to rat parvalbumin, 12K), and a lower pI of 4.6 (rat parvalbumin, 4.9), in the tryptic peptide maps, and in the immu‐nological properties, indicating a distinct primary structure. With the purified parvalbumin as antigen, polyclonal antibodies were raised in rabbits and these were subsequently used for immunohistochemical localizations of parvalbumin in the cat brain. In the visual cortices of adult cats immunoreactive neurons were present throughout layers II and IV. In cerebellar cortex. Pur‐kinje, basket, and stellate cells were immunoreactive. Comparison with staining patterns obtained with anti‐serum against rat parvalbumin revealed some cross‐reactivity but confirmed the existence of species differences in the antigenic structure of rat and cat parvalbumin.
Identification and Initial Characterization of Adipokinetic Hormone‐Like Immunoreactive Peptides of Rat Origin
Authors: Schueler Paula A. , Elde Robert P. , Herman William S. , Mahoney Walter C.
Published: July 2010

An antiserum was raised to adipokinetic hormone (AKH), a 10‐amino‐acid‐residue peptide found in the arthropod Locusta migratoria. The antiserum demonstrated not only immunocytochemical reaction with some other arthropod species, but also stained many areas of the rat CNS, certain islet cells of the pancreas, and some anterior pituitary cells. The pattern of staining was unlike that for any known rat neuropeptide or hormone. With the antiserum used as the detection system, HPLC and high‐voltage electrophoresis yielded two peptides that were purified to homogeneity from rat hypothalamic median eminence. These peptides have unique amino acid compositions, indicating they may be heretofore unknown rat neuropeptides.
Exogenous Glutamate Is Metabolized to Glutamine and Exported by Rat Primary Astrocyte Cultures
Authors: Waniewski Robert A. , Martin David L.
Published: July 2010

Rat cortical astrocytes in primary culture were examined for their capacity to transport and metabolize exogenous l‐[U‐l4C]glutamate. After incubation for time periods up to 120 min, cells and incubation media were analyzed for labelled and endogenous glutamate and its metabolic products by HPLC coupled with fluorescence detection and liquid scintillation counting. Glutamine was the major labelled metabolite after 120 min, accounted for 38% of the original glutamate label, and was found primarily in the incubation medium. A further 13.5% of the label was recovered in deaminated metabolites of glutamate, 1.2% was associated with aspartate, 23% remained in glutamate, and 10.2% was found in an acid‐precipitated cell fraction. More than 84% of the label was recovered in these fractions, suggesting that the maximum possible formation and loss of I4CO2 was 16%. The rate of total glutamine synthesis was 1.1 nmol ± mg protein‐1 x min‐1 when 9 μM exogenous glutamate was present. The total amount of glutamine synthesized greatly exceeded the consumption of glutamate, indicating that a substantial proportion of glutamine was synthesized from other carbon sources. Almost all of the newly formed glutamine was exported into the medium. These results indicate that astrocytes in primary culture, by accumulating glutamate, producing glutamine, and exporting it, are capable of carrying out the glial component of the glutamine cycle.
Phosphorylation of Synaptic Membrane Glycoproteins: The Effects of Ca2+ and Calmodulin
Authors: Kearney Krystyna A. , Gurd James W.
Published: July 2010

Synaptic membranes were incubated with [γ‐32P]ATP, and glycoproteins were isolated by affinity chromatography on concanavalin A agarose. Glycoproteins accounted for 1.5–2.5% of the total 32P incorporated into synaptic membrane proteins. Ca2+ and calmodulin enhanced the phosphorylation of synaptic membrane glycoproteins approximately threefold. In the presence of Ca2+ and calmodulin, the rate of glycoprotein dephosphorylation was also increased three‐ to fourfold. Gel electrophoretic analysis identified several synaptic membrane glycoproteins that incorporated 32P, with the most highly labeled glycoprotein under basal phosphorylating conditions having an apparent Mr of 205,000 (gpiii). Ca2+ and calmodulin produced a marked increase in the phosphorylation of a glycoprotein with an apparent Mr of 180,000 (gpiv) and lesser increases in the labeling of three other glycoproteins. Membranes that had been labeled with [γ‐32P]ATP were extracted with Triton X‐100 under conditions that yield a detergent‐insoluble residue enriched in postsynaptic structures. The Triton X‐100 insoluble residue accounted for 20–25% of the 32P associated with synaptic membrane glycoproteins. Gpiv and other glycoproteins, the phosphorylation of which was stimulated by calmodulin, were located exclusively in the Triton X‐100 insoluble residue, whereas gpiii and other calmodulin‐insensitive glycoproteins partitioned predominantly into the Triton X‐100‐soluble fraction. Phosphopeptide maps and phosphoamino acid analysis of gpiv isolated from synaptic membranes and a postsynaptic glycoprotein of apparent Mr of 180,000 (gp180) isolated from synaptic junctions indicated that the former protein was identical to the previously identified postsynapticspecific gp180. In addition to phosphoserine and phosphothreonine, gpiv also contained phosphotyrosine, identifying it as a substrate for tyrosine‐protein kinase as well as for Ca2+/calmodulin‐dependent protein kinase.
Adrenal Medullary Tropomyosins: Purification and Biochemical Characterization
Authors: Côté A. , Doucet J.‐P. , Trifaró J.‐M.
Published: July 2010

Tropomyosins have been isolated from bovine adrenal medulla. Purified from a heat‐stable extract, the adrenal medulfary tropomyosins show the same chromatographic patterns as platelet tropomyosin components purified under very similar conditions on ion‐exchange (DEAE‐Sephacel) and hydroxylapatite columns. When analyzed by polyacrylamide gel electrophoresis, the purified fraction, reduced and denatured, yielded three polypeptides with apparent molecular weights of 38,000, 35,500, and 32,000. The molar ratio of the two major polypeptides (38 kd and 32 kd) was 2:1. The predominant form of 38 kd is different from other nonmuscle tropomyosins previously isolated and with which an apparent molecular weight of 30,000 is normally associated. The three adrenal medullary tropomyosins have similar isoelectric points of about 4.7. When adrenal tropomyosins were subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the presence of 8 M urea, each form showed a shift to a higher molecular weight, which is a characteristic of muscle tropomyosin. The 38,000 adrenal medullary tropomyosin exhibits a stronger affinity for F‐actin than the other forms. Peptide profiles obtained after limited proteolytic digestion show some similarity between the two predominant tropomyosins of the bovine adrenal medulla and also between these and the α and β forms of bovine skeletal muscle tropomyosin.
Transglutaminase and the Neuronal Cytoskeleton in Alzheimer's Disease
Authors: Miller Christopher C. J. , Anderton Brian H.
Published: July 2010

Transglutaminase [EC 2.3.2.13, (R)‐glutaminyl‐peptide:amine γ‐glutamyltransferase], an enzyme that catalyzes the introduction of glutamine‐lysine cross‐links into proteins, was purified. Neurofilament and microtubule proteins were substrates for this enzyme but the insoluble neurofibrillary tangles (NFT) isolated from Alzheimer's disease brain were not substrates. In vitro cross‐linking of neurofilaments and microtubules by the enzyme did not produce paired helical filaments (PHF), which are the major ultrastructural component of NFT. These results make it unlikely that PHF are formed by the straightforward cross‐linking of neurofilaments or microtubules.
Differential Effects of Tetanus Toxin on Inhibitory and Excitatory Neurotransmitter Release from Mammalian Spinal Cord Cells in Culture
Authors: Williamson Lura C. , Fitzgerald Sandra C. , Neale Elaine A.
Published: July 2010

Abstract: The effect of tetanus toxin on depolarization‐evoked and spontaneous synaptic release of inhibitory and excitatory neurotransmitters was examined in murine spinal cord cell cultures. Toxin action on the release of radiolabeled glycine and glutamate was followed over time intervals corresponding to the early phase of convulsant activity through the later phase of electrical quiescence. Tetanus toxin inhibited potassium‐evoked release of [3H]glycine and [3H]glutamate in a time‐ and dose‐dependent manner. Ninety minutes after the application of toxin (6 × 10−10M), the stimulated release of [3H]glycine was blocked completely, whereas stimulated release of [3H]glutamate was not blocked completely until 150–210 min after toxin application. Fragment C, the binding portion of the tetanus toxin molecule, had no effect on stimulated release of either transmitter. The spontaneous synaptic release of [3H]glycine was blocked totally within 90 min of toxin exposure. In contrast, the spontaneous release of [3H]glutamate, in toxin‐exposed cultures, was elevated to nearly twice that of control cultures at this time. Thus, toxin‐induced convulsant activity is characterized by a reduction in the spontaneous synaptic release of inhibitory neurotransmitter with a concomitant increase in the release of excitatory neurotransmitter, as well as the more rapid onset of blockade of depolarization‐evoked release of inhibitory versus excitatory neurotransmitter.
Muscarinic Acetylcholine Receptor Enhances Phosphatidylcholine Hydrolysis via Phospholipase D in Bovine Chromaffin Cells in Culture
Authors: del Carmen García María , López Manuela G. , García Antonio G. , Crespo Mariano Sánchez
Published: July 2010

Abstract: Although it is well‐established that inositol‐containing lipids serve as precursors of intracellular second messenger molecules in chromaffin cells, we describe some findings that show the formation of diacylglycerol from phosphatidylcholine in response to agonist‐mediated stimulation. Stimulation of chromaffin cells by acetylcholine produced a high turnover of phosphatidylcholine, as suggested by the release of [3H]choline derived from [3H]‐phosphatidylcholine in experiments performed with [3H]choline chloride‐prelabeled cells. An enhanced breakdown of phosphatidylcholine was also inferred from the finding of an increased formation of [3H]diacylglycerol in chromaffin cells prelabeled with [3H]glycerol. The diacylglycerol mass that accumulated after stimulation showed a distinct temporal course and seemed to exceed the mass that has been reported to be derived from phosphatidylinositol. In keeping with the purported origin from phosphatidylcholine, diacylglycerol showed a high content in [3H]oleate molecular species. Phospholipase D activity measurements and experiments performed in the presence of propranolol (an inhibitor of phosphatidic acid:phosphohydrolase) suggested that phosphatidylcholine is hydrolyzed by a phospholipase D activity, producing phosphatidic acid, which is subsequently degraded to diacylglycerol, rather than by a phospholipase C. Incubation of chromaffin cells in the presence of atropine before addition of acetylcholine showed complete inhibition of the increased formation of [3H]‐diacylglycerol, whereas d‐tubocurarine failed to do so. Taken together, these results suggest that acetylcholine activates phosphatidylcholine breakdown and diacylglycerol formation in chromaffin cells via a muscarinic‐type receptor.
Amyloid Precursor Protein Is Synthesized by Retinal Ganglion Cells, Rapidly Transported to the Optic Nerve Plasma Membrane and Nerve Terminals, and Metabolized
Authors: Morin Peter J. , Abraham Carmela R. , Amaratunga Anil , Johnson Robin J. , Huber Glenn , Sandell Julie H. , Fine Richard E.
Published: July 2010

Abstract: We have investigated the synthesis, axonal transport, and processing of the β‐amyloid precursor protein (APP) in in vivo rabbit retinal ganglion cells. These CNS neurons connect the retina to the brain via axons that comprise the optic nerve. APP is synthesized in retinal ganglion cells and is rapidly transported into the optic nerve in small transport vesicles. It is then transferred to the axonal plasma membrane, as well as to the nerve terminals and metabolized with a f1/2 of less than 5 h. A significant accumulation of C‐terminal amyloidogenic or nonamyloidogenic fragments is seen in the optic nerve 5 h after [35S]‐ methionine, [35S]cysteine injection, which disappears by 24 h. The major molecular mass species of APP in the optic nerve is ∼110 kDa, and is an APP isoform that does not contain a Kunitz protease inhibitor domain. Higher molecular mass species containing this sequence are seen mostly in the retina. A protease(s) that can potentially cleave APP to generate an amyloidogenic fragment is present in the same optic nerve membrane compartment as APP.
Comparison Between the Effects of Botulinum Toxin‐Induced Paralysis and Denervation on Molecular Forms of Acetylcholinesterase in Muscles
Authors: Sketelj Janez , Črne‐Finderle Neva , Sket Dušan , Dettbarn Wolf‐D. , Brzin Miro
Published: July 2010

Abstract: Velocity sedimentation analysis of acetylcholinesterase (AChE) molecular forms in the fast extensor digitorum longus muscle and in the slow soleus muscle of the rat was carried out on days 4, 8, and 14 after induction of muscle paralysis by botulinum toxin type A (BoTx). The results were compared with those observed after muscle denervation. In addition, the ability of BoTx‐paralyzed muscles to resynthesize AChE was studied after irreversible inhibition of the preexistent enzyme by diisopropyl phosphorofluoridate. Major differences were observed between the effects of BoTx treatment and nerve section on AChE in the junctional region of the muscles. A precipitous drop in content of the asymmetric A12 AChE form was observed after denervation, whereas its decrease was much slower and less extensive in the BoTx‐paralyzed muscles. Recovery of junctional AChE and of its A12 form after irreversible inhibition of the preexistent AChE in BoTx‐paralyzed muscles was nevertheless very slow. It seems that a greater part of the junctional A12 AChE form pertains to a fraction with a very slow turnover that is rapidly degraded after denervation but not after BoTx‐produced muscle paralysis. The postdenervation decrease in content of junctional A12 AChE is therefore not primarily due to muscle inactivity. The extrajunctional molecular forms of AChE seem to be regulated mostly by muscle activity because they undergo virtually identical changes both after denervation and BoTx paralysis. The differences observed in this respect between the fast and slow muscles after their inactivation must be intrinsic to muscles.
Rapid Brain Uptake of Manganese(II) Across the Blood‐Brain Barrier
Authors: Rabin Olivier , Hegedus Lajos , Bourre Jean‐Marie , Smith Quenlin R.
Published: July 2010

Abstract: 54Mn2+ uptake into brain and choroid plexus from the circulation was studied using the in situ rat brain perfusion technique. Initial uptake from blood was linear with time (30 s to 6 min) and extrapolated to zero with an average transfer coefficient of ∼6 × 10‐5 ml/s/g for brain and ∼7 × 10‐3 ml/s/g for choroid plexus. Influx from physiologic saline was three‐ to fourfold more rapid and exceeded that predicted for passive diffusion by more than one order of magnitude. The lower uptake rate from blood could be explained by plasma protein binding as the free fraction of 54Mn2+ in rat plasma was ≤30%. Purified albumin, transferrin, and α2‐macroglobulin were each found to bind 54Mn2+ significantly and to restrict brain 54Mn2+ influx. The results demonstrate that 54Mn2+ is readily taken up into the CNS, most likely as the free ion, and that transport is critically affected by plasma protein binding. The results support the hypothesis that Mn2+ transport across the blood‐brain barrier is facilitated by either an active or a passive mechanism.
GABA‐Mediated Synaptic Interaction Between the Visual and Vestibular Pathways of Hermissenda
Authors: Alkon D. L. , Anderson M. J. , Kuzirian A. J. , Rogers D. F. , Pass D. M. , Collin C. , Nelson T. J. , Kapetanovic I. M. , Matzel L. D.
Published: July 2010

Abstract: The synaptic convergence of the eyes and the vestibular hair cells in the nudibranch mollusc Hermissenda has been shown previously to mediate the learning of simple visual‐vestibular associations. The neurotransmitter mediating this interaction between the visual and vestibular organs was characterized. HPLC chromatography, confirmed by mass spectroscopic analysis, demonstrated endogenous GABA in the statocysts, in a concentration approximately 150 times greater than in the whole CMS. Additional confirmation was provided by immunocytochemical localization of GABA in hair cell axons and branches that converge with photoreceptor terminal branches. Depolarization of the hair cells in the caudal region of the statocyst in response to positive current injection or vibratory stimulation caused a hyperpolarization and a cessation of the type B photoreceptor impulse activity. The inhibition of the B cell was unaffected by addition to the artificial sea water bath of the adrenergic antagonist yohimbine (250 μM), the cholinergic antagonist atropine (250 μM), and the serotonergic antagonist imipramine (50 μM). In contrast, the GABAA antagonist bicuculline (250 μM) significantly reduced the inhibitory interaction. Moreover, the GABA reuptake inhibitor guvisine (250 μM)M) increased the hyperpolarization. Pressure microapplication of GABA (12.5 or 25 μM) onto the terminal branches of the B cell resulted in a concentration‐dependent hyperpolarization and cessation of spikes in the B cell. Depolarization of the caudal hair cell, or direct GABA application, decreased input resistance across the B cell soma membrane. Moreover, removal of chloride from the extracellular solution reduced inhibition of the B cell induced by GABA application or hair cell stimulation. Furthermore, application of the GABAB agonist baclofen hyperpolarized the type B cell and reduced or eliminated spontaneous impulse activity at the resting membrane potential. The reversal potentials for inhibition induced in all three procedures ranged from −70 to −80 mV and were consistent with mixed Cl‐ and K+ conductances. These results implicate GABA as the endogenous neurotransmitter mediating visual‐vestibular interactions in this animal, and suggest a possible role of GABA in visual‐vestibular associative learning.
Calcium Regulation of Vasoactive Intestinal Polypeptide mRNA Abundance in SH‐SY5Y Human Neuroblastoma Cells
Authors: Adler E. M. , Fink J. Stephen
Published: July 2010

Abstract: Second messenger regulation of gene expression provides a mechanism by which neurons can transduce environmental stimuli into long‐term changes in the expression of molecules involved in neuronal signaling. We have investigated calcium‐dependent induction of vasoactive intestinal polypeptide (VIP) mRNA and compared it with induction of VIP mRNA by cyclic AMP. Depolarization with 60 mM KCI or exposure to the calcium ionophore A23187 increases VIP mRNA levels in SH‐SY5Y cells. The increase in VIP mRNA content in response to Ca2+ mobilization is slow, independent of adenylate cyclase activation, and requires de novo protein synthesis. The increase in VIP mRNA content in response to elevation of cyclic AMP levels by forskolin/isobutylmethylxanthine is independent of Ca2+ influx and does not require new protein synthesis. The mRNA for the transcription factors ATF‐3, c‐fos, c‐jun, junB, and zif/268 is induced by A23187. Of these, ATF‐3 showed the greatest relative induction by A23187 compared with induction by forskolin/isobutyl‐methylxanthine.