bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuritic Plaque Amyloid in Alzheimer's Disease Is Highly Racemized
Authors: Shapira Raymond , Austin Garth E. , Mirra Suzanne S.
Published: December 2006

Abstract: Neuritic plaque core amyloid protein in Alzheimer's disease brain tissue was investigated for the extent of amino acid racemization. Long‐lived human proteins exhibit racemization of certain amino acids over the course of a human lifetime. Purified core amyloid was found to contain relatively large proportions of D‐aspartate and D‐serine, suggesting that neuritic plaque amyloid is derived from a long‐lived precursor protein. Alternatively, racemization of protein amino acids may be abnormally accelerated in Alzheimer's disease.
Purification of Choline Acetyltransferase from the Locust Schistocerca gregaria and Production of Serum Antibodies to This Enzyme
Authors: Lutz E. M. , Lloyd S. J. , Tyrer N. M.
Published: December 2006

Abstract: Choline acetyltransferase (ChAT; EC 2.3.1.6) was purified from the heads of Schistocerca gregaria to a final specific activity of 1.61 μmol acetylcholine (ACh) formed min−l mg−1 protein. The molecular mass of the enzyme as determined by gel filtration is 66,800 daltons. The final enzyme preparation showed one major band at 65,000 daltons on sodium dodecyl sulfate (SDS)‐polyacrylarnide gel electrophoresis, which corresponds with the native molecular mass of the enzyme, a band at 56,000 daltons, and two bands at 40,500 and 38,000 daltons. Antibodies raised against ChAT in rabbit react only with the active band on native gel after Western blotting. They strongly react with the 65,000‐dalton polypeptide band on Western blots of SDS gel separation of pure preparation of enzyme and with both the 65,000‐ and 56,000‐dalton bands after SDS gel separation of crude extract.
Glutamate Transport in Large Muscle Fibres of Balanus nubilus
Authors: Revest P. A. , Baker P. F.
Published: December 2006

Abstract: The uptake of glutamate and other acidic amino acids into barnacle single muscle fibres has been characterized. The uptake of glutamate consists of two components, one Na‐independent and one Na‐dependent. The Na‐dependent uptake is saturable (half‐maximal at 250 μM external glutamate) and is inhibited by a variety of analogues of which L‐cysteate and D‐ and L‐aspartate are the most potent. These amino acids are also transported into the muscle in a Na‐dependent manner. The excitatory agonists kainate, quisqualate, and N‐methyl‐D‐aspartate do not inhibit or affect uptake in any way. Progressive replacement of external Na by choline reduces uptake with very little effect on the apparent affinity for glutamate, suggesting that Na and glutamate bind to the transporter independently. The kinetics of activation are consistent with a requirement for at least two Na ions. Na activation of glutamate uptake can be inhibited by guanidinium with kinetics that are consistent with competitive inhibition at the Na binding site. Studies on the efflux of L‐glutamate and other analogues have shown that efflux rates are only slightly increased by the removal of Na and do not seem to be affected in any clear manner by external levels of acidic amino acids.
[3H]Neurotensin(8–13) Binds in Human Brain to the Same Sites as Does [3H]Neurotensin but with Higher Affinity
Authors: Kanba Kiyoko S. , Kanba Shigenobu , Nelson Albert , Okazaki Haruo , Richelson Elliott
Published: December 2006

Abstract: The binding of [3H]neurotensin(8–13) to membranes from human frontal cortex at 0°C was time dependent, specific, saturable, and reversible. Saturation isotherms provided an equilibrium dissociation constant (KD) of 0.52 nM, and the maximal number of binding sites (B max) was 3.5 pmol/g original wet weight of tissue. Scat‐chard analysis yielded a straight line, and the Hill coefficient was equal to 1, a result indicating that [3H]‐neurotensin(8–13) bound to single, noncooperative sites. The KD values of several analogs of neurotensin determined in competition with [3H]neurotensin(8–13) were similar to those previously determined in competition with [3H]‐neurotensin. The regional distribution of binding sites for [3H]neurotensin(8–13) was also similar to that for [3H]‐neurotensin. These results suggest that [3H]neurotensin(8–13) binds to the same sites as [3H]neurotensin and that [3H]neurotensin(8–13) has a higher affinity than [3H]‐neurotensin for these sites in human brain.
Activators of Protein Kinase C Act at a Postreceptor Site to Amplify Cyclic AMP Production in Rat Pinealocytes
Authors: Sugden David , Klein David C.
Published: December 2006

Abstract: Activation of α1‐adrenoceptors appears to amplify β‐adrenergic stimulation of cyclic AMP (CAMP) accumulation in rat pinealocytes severalfold by a mechanism involving activation of a Ca2+‐, phospholipid‐dependent protein kinase (protein kinase C). The mechanism of action of protein kinase C was investigated in this report using intact cells. Activation of protein kinase C with 4β‐phorbol 12‐myristate 13‐acetate (PMA; 10−7M) or the α1‐adrenergic agonist phenylephrine (PE; 10−6M) did not inhibit cAMP efflux in β‐adrenergically stimulated cells. The amplification of the β‐adrenergic cAMP response by these agents also occurred in the presence of isobutylmethylxanthine (10−3M) and Ro 20–1724 (104‐M), an observation suggesting that inhibition of cAMP phosphodiesterase activity is not the mechanism of action. Furthermore, although PMA (107‐ M) caused a sixfold increase in the magnitude of the cAMP response to isoproterenol, it did not alter the EC50 of the response (1.7 × 10−8M), a result indicating that protein kinase C activation does not alter β‐adrenoceptor sensitivity. The cAMP response following cholera toxin pretreatment (60–120 min) was rapidly and markedly enhanced by a,‐adrenergic agonists (cirazoline > PE > methoxamine), by phorbol esters (PMA > 4β‐phorbol 12,13,‐dibutyrate >> 4α‐phorbol 12,13‐didecanoate), and by synthetic diacylglycerols (1,2‐dioctanoylglycerol > 1‐oleoyl 2‐acetylglycerol >> diolein). The cAMP response to forskolin (10−5M) was also increased by PE (3 × 10−6M) and PMA (10−7M). Together, these observations indicate that protein kinase C activation amplifies β‐adrenergic stimulation of pinealocyte cAMP production at a site beyond the receptor, perhaps on a regulatory guanine nucleotide binding protein or adenylyl cyclase itself.
Possible Involvement of Glutathione S‐Transferases in the Cell Growth of C6 Astroglioma Cells
Authors: Senjo Masanori , Ishibashi Teruo
Published: December 2006

Abstract: The changes of glutathione S‐transferase activity were investigated using rat brain astroglioma C6 cells that were synchronized at different phases of the cell cycle. The enzyme showed two significant activity peaks at G2 and G1 phases. Furthermore, when C6 glioma cells were exposed to a culture medium supplemented with specific glutathione S‐transferase inhibitors, ethacrynic acid and caffeic acid, cell growth was remarkably suppressed. These results suggest that glutathione S‐transferases may be closely related to the mechanism of cell proliferation.
Production of Polyclonal Antisera to Choline Acetyltransferase Using a Fusion Protein Produced by a cDNA Clone Victor
Authors: Muñoz‐Maines J. , Slemmon J. Randall , Panicker Mitradas M. , Neighbor Noel , Salvaterra Paul M.
Published: December 2006

Abstract: A fusion protein containing a Drosophila choline acetyltransferase (ChAT) cDNA insert was purified from a λ gtll lysate of Escherichia coli. The cDNA insert, which contained a 728‐amino acid coding region for ChAT, was used for immunizing rabbits. Three different antisera were produced that could recognize native Drosophila ChAT with low titer. In addition, all three antisera stained enzyme polypeptides using the Western blot technique at high titers. The antisera recognized ChAT polypeptides with molecular masses of 67 and 54 kilodaltons in Western blots of partially purified enzyme; these polypeptides had previously been identified using monoclonal anti‐ChAT antibodies and are the major components of completely purified enzyme. It was surprising that when these antisera were used to stain Western blots of Drosophila head homogenates, the major immunoreactive band had a molecular mass of 75 kilodaltons. The relationship of this 75‐kilodal‐ton polypeptide to ChAT activity was investigated by fractionating fresh fly head homogenates using rapid HPLC gel filtration chromatography. Analysis of column fractions for enzyme activity and immunoreactive polypeptides indicated that the 75‐ and 67‐kilodalton polypeptides can be resolved and are both enzymatically active. In addition, a correlation was observed between the relative immunostaining intensities of both the 75‐ and 67‐kilodalton bands and ChAT activity when supernatants from fresh fly head homogenates were autolyzed at 37°C. Our results indicate that ChAT is present in fresh Drosophila heads primarily as an active enzyme with a molecular mass of 75 kilodaltons. The previously reported molecular mass of 67 kilodaltons for native Drosophila ChAT as well as ChAT polypeptides in partially purified enzyme preparations may have resulted from proteolysis of the 75‐kilodalton form of the enzyme. Recombinant DNA technology provides a good approach to raise high‐titered, sequence‐directed polyclonal antibodies to ChAT without the difficulties encountered in purifying the enzyme directly from Drosophila.
DARPP‐32 and Phosphatase Inhibitor‐1, Two Structurally Related Inhibitors of Protein Phosphatase‐1, Are Both Present in Striatonigral Neurons
Authors: Nairn Angus C. , Hemmings Hugh C. , Walaas S. Ivar , Greengard Paul
Published: December 2006

Abstract: DARPP‐32 (dopamine‐ and cyclic AMP‐regulated phosphoprotein of Mr= 32,000) and phosphatase inhibitor‐1, two previously characterized inhibitors of protein phosphatase‐1, were identified in both the neostriatum and the substantia nigra. Phosphatase inhibitor‐1 was partially purified from bovine caudate nucleus and found to be distinct from DARPP‐32 in some of its biochemical properties. The neuronal localization of DARPP‐32 and phosphatase inhibitor‐1 within the rat neostriatum and substantia nigra was investigated by studying the effects of kainic acid. Injection into the neostriatum of kainic acid, which destroys striatonigral neurons and striatonigral fibers, decreased the amounts of DARPP‐32 and phosphatase inhibitor‐1 to the same extent, both in the lesioned neostriatum and in the ipsilateral substantia nigra. The specific activity of protein phosphatase‐1 in the neostriatum was unaffected by kainic acid. The results indicate that, in rat brain, DARPP‐32 and phosphatase inhibitor‐1 are both present in striatal neurons and in striatonigral fibers, and that they probably coexist in at least a subpopulation of striatonigral neurons. In contrast, protein phosphatase‐1 does not appear to be enriched in any specific neuronal subpopulation in the neostriatum.
Benzodiazepine Enhancement of γ‐Aminobutyric Acid‐Mediated Chloride Ion Flux in Rat Brain Synaptoneurosomes
Authors: Morrow A. Leslie , Paul Steven M.
Published: December 2006

Abstract: Benzodiazepine agonists such as Ro 11–6896 [B10(+)], diazepam, clonazepam, and flurazepam were found to enhance muscimol‐stimulated 36Cl− uptake into rat cerebral cortical synaptoneurosomes. The rank order of potentiation was B10(+) > diazepam > clonazepam > flurazepam. These bedazepines had no effect on 36Cl− uptake in the absence of muscimol. Further, the inactive en‐antiomer, Ro 11–6893 [B10(‐)], and the peripheral benzodiazepine receptor ligand Ro 5–4864 did not potentiate muscimol‐stimulated 36Cl− uptake at concentrations up to 10 μM. In contrast, the benzodiazepine receptor inverse agonists ethyl‐β‐carboline‐3‐carboxylate and 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylic acid methyl ester Benzodiazepines and β‐carbolines altered the apparent K0.5 of muscimol‐stimulated 36Cl− uptake, without affecting the Vmax. The effects of both benzodiazepine receptor agonists and inverse agonists were reversed by the benzodiazepine antagonists Ro 15–1788 and CGS‐8216. These data further confirm that central benzodiazepine receptors modulate the capacity of γ‐aminobutyric acid receptor agonists to enhance chloride transport and provide a biochemical technique for studying benzodiazepine receptor function in vitro.
Novel Regulatory Role of Phosphorylated Clathrin Light Chain β in Bovine Brain Coated Vesicles
Authors: Hanson Veneta G. , Schook William J. , Puszkin Saul
Published: December 2006

Abstract: The 50‐kilodalton (kDa) assembly polypeptide of bovine brain clathrin coated vesicles (CCVs) is phosphorylated in a cyclic nucleotide‐ and Ca2+‐independent manner and is dephosphorylated by a Mg2+‐ATP‐dependent CCV phosphatase. This report provides evidence for modulation of the phosphorylation reaction of the 50‐kDa assembly polypeptide by phosphorylated clathrin light chain β (pLCβ). In vitro, phosphorylated LCβ inhibits phosphorylation of the 50‐kDa polypeptide in CCVs. Furthermore, incubation of previously phosphorylated 50‐kDa polypeptide in CCVs with phosphorylated LCβ results in a rapid dephosphorylation of the 50‐kDa assembly polypeptide. Both phenomena are time and concentration dependent. Monoclonal antibodies to LCβ prevent the modulatory effect of phosphorylated LCβ on the 50‐kDa assembly polypeptide phosphorylation in CCVs. The results obtained indicate for the first time, to our knowledge, that phosphorylated LCβ has a modulatory role in CCVs. The data also suggest that phosphorylated LCβ promotes activation of a coated vesicle phosphatase.
Two Isoenzymes of Na+, K+‐ATPase Have Different Kinetics of K+ Dephosphorylation in Normal Cat and Human Brain Cortex
Authors: Guillaume D. , Grisar T. , Delgado‐Escueta A. V. , Laschet J. , Bureau‐Heeren M.
Published: December 2006

Abstract: Analysis of purified Na+, K+‐ATPase from cat and human cortex by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis reveals two large catalytic subunits called α(‐) (lower molecular weight) and α(+) (higher molecular weight). Differences in K+ dephosphorylation of these two molecular forms have been investigated by measuring the phosphorylation level of each protein after their separation on sodium dodecyl sulfate gels. In the presence of Na+, Mg2+, and ATP, both subunits are phosphorylated. Increasing concentrations (from 0 to 3 mM) of K+ induce progressive dephosphorylation of both α‐subunits, although the phosphoprotein content of α(‐) is decreased significantly less than that of α(+). Ka values of α(‐) for K+ are 40% and 50% greater in cat and human cortex, respectively, than values of α(+), α(‐) and α(+) are thought to be localized in specific cell types of the brain: α(‐) is the exclusive form of nonneuronal cells (astrocytes), whereas α(+) is the only form of axolemma. Our results support the hypothesis that glial and neuronal Na+, K+‐ATPases are different molecular entities differing at least by their K+ sensitivity. Results are discussed in relation to the role of glial cells in the regulation of extracellular K+ in brain.
Effect of Long‐Lasting Diabetes Mellitus on Rat and Human Brain Monoamines
Authors: Lacković Zdravko , Šlković Melita , Kuci Zyrafete , Relja Maja
Published: December 2006

Abstract: Experimental alloxan‐ or streptozotocin‐produced diabetes in rats was accompanied by an increase in the levels of norepinephrine, dopamine, and serotonin, whereas the contents of metabolites, i.e., 5‐hydroxyindoleacetic acid and homovanillic acid, in the whole brain gradually decreased with the duration of diabetes. Among the striatum, thalamus, and hypothalamus of alloxan diabetic rats, monoamine alterations were observed only in the hypothalamus; after 1 week an increase of norepinephrine content and after 13 weeks an increase of norepinephrine and dopamine contents were found. Tissues of 11 brain regions of 10 diabetic and 12 control patients post mortem were investigated for monoamine concentrations. Patients were all male, of similar age and interval between death and autopsy. Diabetic patients had an increase in the content of serotonin in the medial and lateral hypothalamus. The content of dopamine increased in the medial hypothalamus, putamen, and medial and lateral pallidus. In diabetic patients, the content of norepinephrine increased in the lateral pallidus and decreased in the nucleus accumbens and claustrum. Thus, it seems that diabetes mellitus in rats, as well as in humans is associated with regionally specific changes in brain monoamines.
Development of Haloperidol‐Induced Dopamine Release in the Rat Striatum Using Intracerebral Dialysis
Authors: Walters D. E. , Chapman C. D. , Howard S. G.
Published: December 2006

Abstract: Haloperidol‐induced dopamine (DA) release and metabolism were studied in the rat striatum at 10–11, 21–22, and 35–36 days of age using intracerebral dialysis and HPLC with electrochemical detection. There was an age‐related increase in basal DA release and extracellular levels of 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), with the greatest increases occurring between 10–11 and 21–22 days of age. Haloperidol (0.1 mg/kg, i.p.) significantly increased DA release at each age compared to control. Also, haloperidol produced a significantly greater increase in DA release at 10–11 days than at 21–22 or 35–36 days of age when expressed as percentage of predrug release. Haloperidol increased DA release over 60 min to 235%, 138%, and 158% above baseline at 10–11, 21–22, and 35–36 days of age, respectively, after which time the levels remained relatively constant. Haloperidol significantly increased extracellular DOPAC and HVA levels at each age compared to controls, but there were no significant differences in DOPAC or HVA levels between ages in response to haloperidol. The results indicate that, at 10 days of age, DA release in the striatum is physiologically functional and that the regulatory feedback control of DA release and metabolism in the striatum develops prior to 10 days of age.
Phosphorylation of Microtubule Proteins in Rat Brain at Different Developmental Stages: Comparison with That Found in Neuronal Cultures
Authors: Díaz‐Nido Javier , Serrano Luis , Hernandez María A. , Avila Jesús
Published: December 2006

Abstract: The phosphorylation of rat brain microtubule protein on intracranial injection of labeled phosphate has been analyzed. The major microtubule protein components phosphorylated in vivo in rat brain are the high‐molecular‐weight microtubule‐associated proteins (MAPs) MAP‐1A, MAP‐1B, and MAP‐2. A slight phospholabeling of β‐tubulin, which corresponds to the phosphorylation of a minor neuronal β‐tubulin isotype, is also observed. Whereas MAP‐1B, MAP‐2, and β‐tubulin are phosphorylated in the brain of 5‐day‐old rat pups, when most neurons of the CNS are extending processes, MAP‐1A phosphorylation is observed only after neuronal maturation takes place. The phosphorylation of MAP‐1A, MAP‐1B, and β‐tubulin may be due mainly to casein kinase II or a related enzyme, whereas MAP‐2 appears to be modified by other enzymes such as the cyclic AMP‐dependent protein kinase (protein kinase A) and the calcium/ phospholipid‐dependent protein kinase (protein kinase C). Microtubule protein phosphorylation has also been studied in neuronal cultures. In differentiated neuroblastoma cells, only MAP‐1B and β‐tubulin are phosphorylated in a manner coupled to neurite outgrowth. In primary cultures of fetal rat brain neurons, the pattern of microtubule protein phosphorylation resembles that found in vivo in rat pup brain. As phosphorylated MAP‐1A and MAP‐1B are present mainly on assembled microtubules, whereas the phosphorylation of MAP‐2 decreases its interaction with microtubules, a role can be suggested for the phosphorylation of these proteins in the regulation of microtubule assembly and disassembly during neuronal development.
Paralysis of Innervated Cultured Human Muscle Fibers Affects Enzymes Differentially
Authors: Martinuzzi Andrea , Askanas Valerie , Engel W. King
Published: December 2006

Abstract: Increased accumulation of muscle‐specific isozyme (MSI) of creatine kinase (CK), lactate dehydrogenase (LDH), glycogen phosphorylase (GP), and phosphoglycerate mutase (PGAM) occurs with development and indicates muscle fiber maturation. The expression of MSIs of those four enzymes is greatly enhanced in innervated‐contracting as compared to noninnervated and noncontracting cultured human muscle fibers. We have now studied the effect of contractile activity on developmental accumulation of MSIs in innervated‐contracting, innervated‐paralyzed (2 μM tetrodotoxin for 30 days), and noninnervated‐noncontracting cultured human muscle fibers. Muscle acetylcholinesterase (AChE) and total enzyme activities were also studied under the same conditions. We observed a different dependency on contractile activity between total enzymatic activities of CK., LDH, and AChE, which were substantially reduced after paralysis, and GP and PGAM, which were unchanged. The expression of MSIs of CK, GP, PGAM, and LDH was always significantly increased in innervated as compared to noninnervated fibers. While the expression of MSIs of GP and PGAM was the same in contracting‐innervated and paralyzed‐innervated muscle fibers, the expression of MSIs of CK and LDH in paralyzed‐innervated muscle fibers was very slightly decreased as compared to their contracting‐innervated controls. Our studies demonstrate that in human muscle: (1) total enzymatic activities and the expression of MSIs of GP and PGAM are regulated by neuronal effect(s); (2) total enzymatic activities of CK, LDH, and AChE depend mainly on muscle contractile activity; and (3) MSIs of CK and LDH are regulated predominantly by neuronal factors and to a much lesser degree by muscle contractile activity.
M1 Muscarinic Acetylcholine Receptor in Cultured Rat Neostriatum Regulates Phosphoinositide Hydrolysis
Authors: Akins Paul T. , Surmeier D. James , Kitai S. T.
Published: December 2006

Abstract: : Muscarinic acetylcholine receptor expression and function in cultured rat neostriatal neurons were examined. All experiments were performed on intact neurons grown in vitro for 12‐14 days. The muscarinic antagonist N‐[3H]methylscopolamine ([3H]NMS) binds to a single site in cultures with a KD of 89 pM and a Bmax of 187 fmol/mg of protein, or 32,000 sites/neuron. Competition studies using [3H]NMS were performed to determine what receptor sur > types were present. Nonlinear analysis of competition curves was best described with a single binding site for atropine, pirenzepine, and AF‐DX 116 {11‐[[2‐[(diethylamino)‐methyl]‐1‐piperidinyl]acetyl]‐5,11‐dihydro‐6H‐pyrido[2,3‐b][1,4]benzodiazepine‐6‐one}, with Ki values of 0.6, 62, and 758 nM, respectively. These results indicate that the muscarinic receptors present in neostriatal cultures are of the M1subtype, having high affinity for pirenzepine and low affinity for AF‐DX 116. In contrast with antagonists, carbachol displaced [3H]NMS from two sites with Ki values of 6.5 and 147 μM, with the higher‐affinity form predominant (83% of sites). The M1 receptor subtype was linked to phosphoinositide turnover. Carbachol stimulated the formation of phosphoinositides with an EC50 of 37 μM and was antagonized by atropine. At equimolar doses, pirenzepine was more potent than AF‐DX 116 at antagonizing the response.
Effects of Axotomy on Distribution and Concentration of Elements in Rat Sciatic Nerve
Authors: LoPachin Richard M. , LoPachin Vicki R. , Saubermann Albert J.
Published: December 2006

Abstract: X‐ray microprobe analysis was used to determine the effects of axotomy on distribution and concentration (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in frozen, unfixed sections of rat sciatic nerve. Elemental concentrations were measured in axoplasm, mitochondria, and myelin at 8, 16, and 48 h after transection in small‐, medium‐, and large‐diameter fibers. In addition, elemental composition was determined in extraaxonal space (EAS) and Schwann cell cytoplasm. During the initial 16 h following transection, axoplasm of small fibers exhibited a decrease in dry weight concentrations of K and Cl, whereas Na and P increased compared to control values. Similar changes were observed in mitochondria of small axons, except for an early, large increase in Ca content. In contrast, intraaxonal compartments of larger fibers showed increased dry weight levels of K and P, with no changes in Na or Ca concentrations. Both Schwann cell cytoplasm and EAS at 8 and 16 h after injury had significant increases in Na, K, and Cl dry weight concentrations, whereas no changes, other than an increase in Ca, were observed in myelin. Regardless of fiber size, 48 h after transection, axoplasm and mitochondria displayed marked increases in Na, Cl, and Ca concentrations associated with decreased K. Also at 48 h, both Schwann cell cytoplasm and EAS had increased dry weight concentrations of Na, Cl, and K. The results of this study indicate that, in response to nerve transection, elemental content and distribution are altered according to a specific temporal pattern. This sequence of change, which occurs first in small axons, precedes the onset of Wallerian degeneration in transected nerves.
Identification of ω‐Conotoxin Binding Sites on Adrenal Medullary Membranes: Possibility of Multiple Calcium Channels in Chromaffin Cells
Authors: Jan Chung‐Ren , Titeler Milt , Schneider Allan S.
Published: December 2006

Abstract: Binding of 125I‐ω‐conotoxin GVIA and [3H]nitrendipine to membranes from bovine adrenal medulla was investigated to test for the presence of N‐ and L‐type Ca2+ channels in adrenal chromaffin cells. Saturable, high‐affinity binding sites for 125I‐ω‐conotoxin and [3H]nitrendipine were detected in a membrane fraction from adrenal medulla. [3H]Nitrendipine binding sites were found to have a KD of 500 ± 170 pM and a Bmax of 26 ± 11 pmol/g of protein. 125I‐ω‐Conotoxin binding sites had a KD of 215 ± 56 pM and a Bmax of 105 ± 18 pmol/g of protein, about four times the number of sites found for [3H]nitrendipine. 125I‐ω‐Conotoxin binding was potently inhibited by unlabeled toxin and Ca2+ but was unaffected by dihydropyridines, verapamil, and diltiazem. [3H]Nitrendipine binding was not affected by ω‐conotoxin, whereas it was inhibited by other dihydropyridines. Bay K 8644 potentiated K+‐evoked cytosolic Ca2+ transients measured by fura‐2 fluorescence, and this potentiation was completely blocked by nifedipine. In contrast, ω‐conotoxin had no effect on Bay K 8644‐evoked Ca2+ transients. Thus, the binding sites for ω‐conotoxin and for nitrendipine appear to be different. The results confirm the presence of L‐type Ca2+ channels and open the possibility of N‐type Ca2+ channels as the ω‐conotoxin binding sites in chromaffin cell membranes.
Cellular Localization of Transforming Growth Factor‐α mRNA in Rat Forebrain
Authors: Seroogy Kim B. , Lundgren Kerstin H. , Lee David C. , Guthrie Kathleen M. , Gall Christine M.
Published: December 2006

Abstract: The cellular localization of transforming growth factor‐α (TGFa) mRNA in juvenile and adult rat forebrain was examined using in situ hybridization with a 35S‐labeled cRNA probe. TGFα cRNA‐labeled neuronal perikarya were distributed across many forebrain regions including the olfactory bulb, caudate‐putamen, nucleus accumbens, olfactory tubercle, ventral pallidum, amygdala, hippocam‐pal stratum granulosum and CA3 stratum pyramidale, and piriform, entorhinal, and retrosplenial cortices. TGFα cRNA‐hybridizing cells were also localized to several thalamic nuclei and to the suprachiasmatic, dorsomedial, and ventromedial nuclei of the hypothalamus. In addition, labeled cells were present in regions of white matter including the corpus callosum, anterior commissure, internal and external capsules, optic tract, and lateral olfactory tract. Thus, both neurons and glia appear to synthesize TGFα in normal brain. Hybridization densities were greater in neuronal fields at 2 weeks of age compared with the adult, suggesting a role for TGFα in the development of several forebrain systems. Our results demonstrating the prominent and widespread expression of TGFα mRNA in forebrain, combined with the extremely low abundance of epidermal growth factor mRNA in brain, support the argument that TGFα is the principal endogenous ligand for the epidermal growth factor receptor in normal brain.
Lindane Administration to the Rat Induces Modifications in the Regional Cerebral Binding of [3H]Muscimol, [3H]‐Flunitrazepam, and t‐[35S]Butylbicyclophosphorothionate: An Autoradiographic Study
Authors: Solà Carme , Martínez Emili , Camón Lluïsa , Pazos Angel , Rodríguez‐Farré Eduard
Published: December 2006

Abstract: The effect of lindane administration on the specific binding of ligands to different sites on the GABAA receptor‐ionophore complex was studied in the rat brain by receptor mapping autoradiography. [3H]Muscimol (Mus), [3H]flunitrazepam (Flu), and t‐[35S]butylbicyclophosphorothionate (TBPS) were used as specific ligands of GABA, benzodiazepine, and picrotoxinin binding sites, respectively. Rats received a single oral dose of 30 mg/kg lindane and they were classified into two groups according to the absence or presence of convulsions. Vehicle‐treated groups acted as controls. The effect of the xenobiotic on ligand binding was measured in different brain areas and nuclei 12 min or 5 h after its administration. Lindane induced a generalized decrease in [35S]TBPS binding, which was present shortly after dosing. In addition, [3H]Flu binding was increased in lindane‐treated animals, this modification also appearing shortly after administration but diminishing during the studied time. Finally, lindane induced a decrease in [3H]Mus binding, which became more evident over time. These modifications were observed both in the presence and in the absence of convulsions. However, an increase in [3H]‐Mus binding was detected shortly after lindane‐induced convulsions. The observed decrease in [35S]TBPS binding is in agreement with the postulated action of lindane at the picrotoxinin binding site of the GABAA receptor chloride channel. The effects observed on the binding of [3H]Flu and [3H]Mus may be secondary to the action of lindane as an allosteric antagonist of the GABAA receptor.
Locally Synthesized Phosphatidylcholine, but Not Protein, Undergoes Rapid Retrograde Axonal Transport in the Rat Sciatic Nerve
Authors: Padilla Stephanie , Freeman Elizabeth B. , Tandon Pushpa , Wilson Valerie Z.
Published: December 2006

Abstract: Retrograde axonal transport of phosphatidylcholine in the sciatic nerve has been demonstrated only after injection of lipid precursors into the cell body region. We now report, however, that after microinjection (1 μl) of [methyl‐3H]choline chloride into the rat sciatic nerve (35‐40 mm distal to the L4 and L5 dorsal root ganglia), time‐dependent accumulation of 3H‐labeled material occurred in dorsal root ganglia ipsilateral, but not contralateral, to the injection site. The level of radioactivity in the ipsilateral dorsal root ganglia was minimal at 2 h after isotope injection but was significantly increased at 7, 24, 48, and 72 h after intraneural isotope injection (n = 3–8 per time point); at these time points, all of the radiolabel in the chloroform/methanol extract of the ipsilateral dorsal root ganglia was present in phosphatidylcholine. The radioactivity in the water‐soluble fraction did not show a time‐dependent accumulation in the ipsilateral dorsal root ganglia as compared with the contralateral DRGs, ruling out transport or diffusion of precursor molecules. In addition, colchicine injection into the sciatic nerve proximal to the isotope injection site prevented the accumulation of radiolabel in the ipsilateral dorsal root ganglia. Therefore, this time‐dependent accumulation of radiolabeled phosphatidylcholine in the ipsilateral dorsal root ganglia is most likely due to retrograde axonal transport of locally synthesized phospholipid material. Moreover, 24 h after injection of both [3H]choline and [35S]‐methionine into the sciatic nerve, the ipsilateral/contralateral ratio of radiolabel was 11.7 for 3H but only 1.1 for 35S. indicating that only locally synthesized choline phospholipids, but not protein, were retrogradely transported.
Norepinephrine Suppresses Inducible Nitric Oxide Synthase Activity in Rat Astroglial Cultures
Authors: Feinstein Douglas L. , Galea Elena , Reis Donald J.
Published: December 2006

Abstract: Exposure of primary rat astrocyte cultures to bacterial endotoxin lipopolysaccharide (LPS) causes expression of a Ca2+‐in‐dependent form of nitric oxide synthase (NOS). In these cells, the presence of norepinephrine (NE) caused a dose‐dependent inhibition of the LPS induction of NOS activity, with an IC50 value of 100 nMand significant suppression at 100 pAf. Short incubations (5‐40 min) with NE were as effective as 24‐h continuous exposure, and inhibition was observed up to the longest incubation period measured (56 h). In contrast, previously induced NOS activity was not affected by exposure to NE. The effects of NE were mediated primarily by binding to β‐adrenergic receptors (β‐ARs) because (a) the β‐AR antagonist propranolol, but not the n‐AR antagonist phentol‐amine, could reverse the effects of NE; (b) the β‐AR agonist isoproterenol. but not the a‐AR agonist phenylephrine, was as effective as NE in blocking the effects of LPS; and (c) incubation with the cyclic AMP analogue dibutyryl cyclic AMP replicated the effects of NE. In contrast to astroglial cultures, LPS induction of NOS activity in RAW 264.7 macrophage cells was not affected by NE or dibutyryl cyclic AMP. These results indicate that in brain, inducible NOS in astrocytes can be regulated by neurotransmitter binding to glial receptors.
Angiotensin II Type 1 Receptor mRNA Levels in the Brains of Normotensive and Spontaneously Hypertensive Rats
Authors: Raizada Mohan K. , Sumners Colin , Lu Di
Published: December 2006

Abstract: The type 1 angiotensin II (All) receptor (AT1‐R) has been implicated in the physiological actions mediated by All in the brain. In view of the reported hyperactivity of the brain All system in the spontaneously hypertensive rat (SHR), we compared the expression of AT,‐R mRNAs in the brains of normotensive [Wistar Kyoto (WKY)] and SHR animals. Northern blot analysis showed about three‐ and ∼20‐fold increases in the levels of AT1‐R mRNAs from the hypothalamus and brainstem areas, respectively, of the SHR compared with the WKY rat brain. This was attributable to greater levels of both AT,1A‐ and AT,1B‐R mRNA subtypes in these areas from the SHR. These observations suggest that increased All receptor levels in SHR brain may, in part, be a result of increased expression of the AT1‐R gene.
Calcium/Calmodulin‐Stimulated Protein Kinase II Is Present in Primary Cultures of Cerebral Endothelial Cells
Authors: Deli Mária A. , Joó Ferenc , Krizbai István , Lengyel Imre , Nunzi Maria G. , Wolff Joachim R.
Published: December 2006

Abstract: Calcium/calmodulin‐stimulated protein kinase II (CaMPK II). a major kinase in brain, has been established to play an important role in neurotransmitter release and organization of postsynaptic receptors, and it is known to be involved in long‐term potentiation and memory. Less is known about the function of this enzyme in nonneural cells. Here we report on the production, presence, and phosphorylation of the α‐subunit of CaM‐PK II in primary cultures of cerebral endothelial cells. These results raise the possibility that α‐CaM‐PK II can act as one of the key enzymes of calcium‐mediated intracellular signaling in the cerebral endothelial cells and suggest that α‐CaM‐PK II may participate in such basic cellular processes as permeability in physiological and pathological conditions.
Effect of Meniscal Release on Rate of Subsequent Meniscal Tears and Owner‐Assessed Outcome in Dogs with Cruciate Disease Treated with Tibial Plateau Leveling Osteotomy
Authors: THIEMAN KELLEY M. , TOMLINSON JAMES L. , FOX DEREK B. , COOK CRISTI , COOK JAMES L.
Published: December 2006

Objective— To determine and compare rates of meniscal tears after tibial plateau leveling osteotomy (TPLO) among 3 groups of dogs based on treatment method: arthrotomy with meniscal release (openR), arthrotomy without meniscal release (openNR), arthroscopy without meniscal release (scopeNR), and compare long term owner‐assessed outcomes for the same groups.

Study Design— Retrospective cohort study.

Sample Population— Stifles (n=254) of dogs that had TPLO.

Methods— The three groups were compared for significant (P<.05) differences in rate of subsequent tears using a χ2 test. Odds ratios for likelihood of subsequent meniscal tears were determined. Data for signalment, outcome, time to peak function, and time to subsequent tear were compared for significant differences using ANOVA, t‐test, or rank sum test.

Results— Subsequent meniscal tears were diagnosed in 16 cases (6.3%). Of dogs with subsequent meniscal tears, 9 had openNR, 4 had openR, and 3 had scopeNR; the proportion of subsequent meniscal tears was significantly different (P=.035) among groups. Odds ratio indicated that subsequent meniscal tear was 3.8 times more likely to occur for openNR than openR or scopeNR. No significant differences among groups were noted for measures of outcome.

Conclusions— Meniscal release did not reduce the rate of subsequent meniscal tears when compared with cases treated arthroscopically or when compared with all cases combined, but may be advantageous when meniscal pathology cannot be comprehensively assessed in the cranial cruciate deficient stifle. Meniscal release had no effects on owner‐assessed outcome as determined in this study.

Clinical Relevance— The low rates of subsequent meniscal tears in conjunction with the relatively high and equivocal levels of owner‐assessed outcome and time to peak function for all 3 treatment groups suggest that any of these surgical management strategies can be considered acceptable. We suggest that a meniscal release be performed when complete and thorough exploration of the joint and meniscus cannot be, or are not, performed.
In Vivo Evaluation of Intra‐Articular Protection in a Novel Model of Canine Cranial Cruciate Ligament Mid‐Substance Elongation Injury
Authors: LOPEZ MANDI J. , ROBINSON SANDRA O. , QUINN MARGARET M. , HOSGOOD GISELLE , MARKEL MARK D.
Published: December 2006

Objectives—To evaluate the effects of intra‐articular protection (IAP) on the canine cranial cruciate ligament (CrCL) and stifle in a CrCL midsubstance elongation injury model.

Study Design—Experimental longitudinal cohort study.

Animals—Skeletally mature female mixed breed hounds (n=12; mean±SEM weight, 25.6±0.7 kg).

Methods—After CrCL elongation in 1 stifle of each dog, IAP was applied in 6 joints. In vivo assessment included radiographs, cranial–caudal joint translation, gait analysis, and synovial fluid levels of 3B3(−) (proteoglycan epitope) and C2C (collagen II neoepitope) up to 12 weeks after surgery. Joint translation and rotation were quantified at necropsy. CrCL midsubstance length was determined before and after elongation and at necropsy. CrCLs were subjectively assessed with light microscopy. Comparisons were made between stifles containing elongated CrCLs with and without IAP and unoperated controls.

Results—Four weeks after surgery, ground reaction forces were significantly decreased in operated limbs. Absolute C2C levels were significantly elevated in operated stifles 4 weeks post‐surgery. C2C and 3B3(−) levels normalized to total protein were significantly elevated in IAP+ stifles 8 weeks after surgery. Protected CrCLs appeared to have decreased granulation tissue and better collagen fiber alignment.

Conclusions—IAP has negligible effects on the canine stifle based on the response variables evaluated in this 12‐week study. Protection of elongated CrCLs may promote reduced, organized scar formation.

Clinical Relevance—These results support the healing capacity of the canine CrCL midsubstance following elongation injury and IAP application to potentially reduce cicatrix formation in elongated CrCLs.
Regulation of Protein Trafficking by Glycosylphosphatidylinositol Valence in African Trypanosomes1
Authors: SCHWARTZ KEVIN J. , BANGS JAMES D.
Published: December 2006

ABSTRACT. The structure, biosynthesis, and attachment of glycosylphosphatidylinositol (GPI) anchors were all first determined for the variant surface glycoprotein (VSG) of African trypanosomes, and all of the basic aspects of this work have been shown to be universal in eukaryotic organisms. However, the role of GPI anchors in protein trafficking within trypanosomes has lagged behind the more standard eukaryotic model systems such as yeast and polarized epithelial cells. Trypanosomes are also highly polarized cells in which all endocytosis and exocytosis intersect at a discrete domain of the plasma membrane, the flagellar pocket. Within these convergent pathways trafficking of GPI anchored proteins correlates strongly with valence: homodimeric VSG with two GPIs is stably incorporated into the cell surface coat, heterodimeric transferrin receptor with a single GPI is found in the flagellar pocket and is slowly delivered to the lysosome for degradation, and recombinant GPI minus VSG reporters are rapidly degraded in the lysosome. Here we summarize recent data confirming this correlation using a tool kit of recombinant GPI anchored reporters, including a reporter designed to be conditionally modulated between a GPI valence of one and two.
Determination of Precise Unit‐Cell Parameters of the α‐Tricalcium Phosphate Ca3(PO4)2 Through High‐Resolution Synchrotron Powder Diffraction
Authors: Yashima Masatomo , Kawaike Yoichi , Tanaka Masahiko
Published: December 2006

Unit‐cell parameters of the α‐tricalcium phosphate [TCP; Ca3(PO4)2] were investigated using high‐resolution synchrotron powder diffraction and the Rietveld method. The diffraction experiment was conducted at 29°C at the BL‐15XU experimental station of SPring‐8, Japan. Precise unit‐cell parameters of the α‐TCP were obtained; a=12.87271 (9), b=27.28034(8), c=15.21275(12) Å, α=γ=90°, and β=126.2078(4)°. The calculated density of α‐TCP (2.8677 g/cm3) is smaller than that of β‐TCP, indicating the “looser” structure of α‐TCP.
Formation of Silicone Carbide Membrane by Radiation Curing of Polycarbosilane and Polyvinylsilane and its Gas Separation up to 250°C
Authors: Wach Radoslaw A. , Sugimoto Masaki , Yoshikawa Masahito
Published: December 2006

Silicon carbide (SiC) ceramic coating was developed from precursor polymer blend of polycarbosilane and polyvinylsilane on porous alumina substrate by radiation curing. The polymers were crosslinked with oxygen present in the atmosphere during irradiation and pyrolyzed at 850°C in order to convert the polymer into SiC ceramics. Fabricated SiC film was used as a membrane for gas separation, achieving high separation ratios of 206 for H2 and 241 for He over the nitrogen at 250°C.
Fabrication of Three‐Dimensional Micro Photonic Crystals of Resin‐Incorporating TiO2 Particles and their Terahertz Wave Properties
Authors: Chen Weiwu , Kirihara Soshu , Miyamoto Yoshinari
Published: December 2006

Three‐dimensional (3D) photonic crystals with a diamond structure made from 40 vol% TiO2–acrylate dielectric composites were formed by means of a CAD/CAM micro‐stereolithography system. The lattice constant of the diamond unit cell was 500 μm and the forming accuracy was 10 μm. The photonic band gap in the Γ–X 〈100〉 direction measured by terahertz‐time‐domain spectroscopy appeared at 280–360 GHz, which agreed fairly well with the band gap calculated by the plane wave expansion method.
Mineralizing Effect of Li2B4O7 and Na2B4O7 on Magnesium Aluminate Spinel Formation
Authors: Bhattacharya G. , Zhang S. , Jayaseelan D. D. , Lee W. E.
Published: December 2006

Lithium borate (Li2B4O7) and sodium borate (Na2B4O7) mineralize spinel formation from stoichiometric MgO and Al2O3 between 1000° and 1100°C. Mineralization with both compounds is shown to be mediated by B‐containing liquids which form glass on cooling. However, the liquid compositions depend on the type of mineralizer and temperature, suggesting that templated grain growth or dissolution–precipitation mechanisms are operating, one dominating over the other under certain conditions. Na2B4O7‐mineralized compositions show predominantly templated grain growth at 1000°C, which changes to dissolution–precipitation at 1100°C, whereas Li2B4O7‐mineralized compositions show dissolution–precipitation from 1000°C. Li2B4O7 is a stronger mineralizer as spinel formation is complete with 3 wt% Li2B4O7 at 1000°C and with ≥1.5 wt% addition at 1100°C, whereas Na2B4O7‐mineralized compositions are found to retain some unreacted corundum even at 1100°C.
Corrosion of CVD Silicon Carbide in 500°C Supercritical Water
Authors: Barringer E. , Faiztompkins Z. , Feinroth H. , Allen T. , Lance M. , Meyer H. , Walker L. , Lara‐Curzio E.
Published: December 2006

A high‐purity CVD β‐SiC showed a relatively low corrosion rate in deoxygenated supercritical water at 500°C. The corrosion rate was lower than that previously reported for CVD SiC in 360°C water and much lower than that reported for sintered and reaction‐bonded SiC. The present study confirmed that CVD SiC was preferentially attacked at the grain boundaries. Analytical examinations did not reveal the presence of a measurable oxide scale. As a result, it is believed that corrosion of the high‐purity SiC occurred via hydrolysis to hydrated silica species at the surface that were rapidly dissolved into the supercritical water.
Sintering Behavior and Dielectric Properties of Bi3NbO7 Ceramics Prepared by Mixed Oxides and High‐Energy Ball‐Milling Methods
Authors: Zhou Di , Wang Hong , Yao Xi
Published: December 2006

The sintering behavior and dielectric properties of Bi3NbO7 ceramics prepared by the high‐energy ball milling (HEM) method and conventional mixed oxides method with V2O5 addition were investigated. All the samples were sintered between 840° and 960°C. For the ceramics prepared by the mixed oxides method, the pure tetragonal Bi3NbO7 phase formed without any cubic phase. With changing sintering temperature, the dielectric constant ɛr lies between 79 and 92, while the Q×f values are between 300 and 640 GHz. The samples sintered at 870°C have the best microwave dielectric properties with ɛr=79, Q×f=640 GHz, and the temperature coefficients of resonant frequency τf between 0 and −20 ppm/°C. For the ceramics prepared by the HEM, a pure cubic phase was obtained. The ɛr changes between 78 and 80 and Q×f were between 200 and 290 GHz.
Onset of Cone Cracking in Ion‐Exchanged Glass
Authors: Smail Jacob W. , Green David J. , Abrams Matthew B.
Published: December 2006

A simple fracture mechanics approach is used to predict the critical crack size required to initiate Hertzian cracks in a brittle material in the absence and presence of a residual surface stress. To test the approach, the load and location of ring/cone cracks are measured in a silicate glass before and after ion exchange. The crack sizes estimated by this technique are, however, different before and after the ion‐exchange process. It is concluded that the ion‐exchange process changes the nature of the surface flaws, which is a key assumption in previous theoretical analyses in using Hertzian contacts as a means to estimate surface stress.