bluuebunny/crossref_metadata_2025_split huggingface dataset:
Calmodulin and Ca2+‐ and Calmodulin‐Dependent Protein Kinase in Rat Anterior Pituitary Gland
Authors: Hatada Yasuyuki , Munemura Masahide , Fukunaga Kohji , Yamamoto Hideyuki , Maeyama Masao , Miyamoto Eishichi 
Published: October 2006

Abstract:Calmodulin and Ca2+‐ and calmodulin‐dependent protein kinase were identified in the rat anterior pituitary gland. The concentration of calmodulin was 1.18 ± 0.11 μg/mg protein (n = 7) in the cytosol fraction. The calmodulin of the anterior pituitary gland co‐migrated with brain calmodulin on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The Ka value of the partially purified enzyme for Ca2+ was 3.3 μ.M in the presence of 0.30μ.M calmodulin. Trifluoperazine and chlorpromazine, calmodulin‐interacting agents, inhibited enzyme activity, with Ki values of 1.3 and 2.6 x 10‐5M, respectively. The enzyme was resolved into two peaks of activity, with sedimentation coefficients of 5.5 S and 16.5 S, by sucrose density gradient centrifugation. At least nine proteins were phosphorylated by the enzyme in a Ca2+‐ and calmodulin‐dependent manner. In light of these results, the possibility that calmodulin and the calmodulin‐activatable protein kinase system are involved in the mediation of the Ca2+ effect on hormone release from the anterior pituitary gland must be given consideration.
Regulation of Rat Pineal Hydroxyindole‐O‐Methyltransferase in Neonatal and Adult Rats
Authors: Sugden David , Klein David C. 
Published: October 2006

Abstract:The relative importance of neural, and some nonneural, mechanisms in the control of pineal hydroxyindole‐O‐methyltransferase (HIOMT) activity during development and in the adult rat was studied. In neonatal rats, guanethidine‐treatment, bilateral superior cervical ganglionectomy (SCGX), or exposure to constant light did not prevent the initial appearance of HIOMT activity, indicating that neural stimulation of the gland is not essential for the development of HIOMT activity. In adult rats, decentralization or removal of the SCG led to a slow fall in HIOMT activity, to about 30% of control activity, indicating that the enzyme is largely under neural control. Additionally, adrenalectomy or hypophysectomy had no effect on HIOMT activity, refuting the suggestion that adrenal and/or gonadal steroids are of major importance in the regulation of this enzyme. The fall in activity of the enzyme after SCGX or exposure to constant light probably does not represent a shift in the Km of the enzyme nor the selective disappearance of a distinct molecular species. Similar changes in HIOMT activity and cyclic GMP responsiveness occur in response to alterations in the length of the daily dark period, adding further evidence to our earlier speculation that there may be a functional relationship between these two.
The Nerve Growth Factor Receptor on PC 12 Cells: Interconversion Between Two Forms with Different Binding Properties
Authors: Block Timothy , Bothwell Mark 
Published: October 2006

Abstract:PC 12 cells possess two classes of nerve growth factor (NGF) receptors on their surfaces which can be distinguished by kinetic criteria. The majority class binds and releases 125I‐NGF at a relatively rapid rate and has been called fast. The second class of receptors has been called slow because of relatively slower rates of binding and release of 125I‐NGF, and also may be distinguished from fast receptors by their cytoskeletal association and resistance to trypsin. PC 12 cell plasma membranes were prepared and shown to have only the fast class of receptors. These membranes were fused to receptorless 3T3 cells with polyethylene glycol. The resultant fused cells were shown to possess NGF receptors, essentially all of which behave like slow receptors. Immunofluorescence microscopy was used to monitor the introduction of PC 12 cell membrane and NGF receptorsinto 3T3 cells. Results obtained with C10‐2, a monoclonal antibody specific for a major PC12 cell‐surface antigen, show that up to 90% of 3T3 cells receive PC 12 membrane and that the PC 12 membrane becomes integrally incorporated into the 3T3 cell plasma membrane. It is suggested that an association of receptors with cytoskeleton may be involved in the conversion of fast to slow receptor behavior, and that the differing proportion of fast and slow NGF receptors in PC 12 and 3T3 cells reflects the differing cytoskeletal organization of these cells.
The Effect of the Shiverer Mutation on Myelin Basic Protein Expression in Homozygous and Heterozygous Mouse Brain
Authors: Barbarese E. , Nielson M. L. , Carson J. H. 
Published: October 2006

Abstract:We report (a) that the shiverer mutation has pleiotropic phenotypic effects on myelin basic protein expression in the CNS of homozygous (shi/shi) mice and (b) that each of the effects of the shiverer allele is expressed co‐dominantly with the wild‐type allele in heterozygous (+/shi) animals. First, the total amount of myelin basic protein, as determined by radioimmunoassay, that accumulates in the CNS is approximately 0.1% of the wildtype amount in shi/shi animals and approximately 50% in +/shi animals. Second, the four major forms of myelin basic protein, with molecular weights of 21,500, 18,500, 17,000, and 14,000, that are present in wild‐type mouse CNS are undetectable in either whole brain or purified myelin of shi/shi animals, and each of the four proteins is reduced commensurately in brain and myelin of +shi animals. Third, the small amount of myelin basic protein‐related material that does accumulate in the shi/shi brain consists of several polypeptides, with molecular weights ranging from 25,000 to 100,000, the pattern of which is different from that found in wild‐type brain. The pattern of myelin basic protein‐related polypeptides in +lshi brain is a composite of the wild type and the shiverer mutant. Fourth, messenger RNA from shi/shi brain, when translated in vitro, encodes a set of myelin basic protein‐related polypeptides qualitatively similar to that encoded by wild‐type messenger RNA, except that the 18,500 and 14,000 translation products are greatly reduced, while other myelin basic protein‐related translation products are spared. The pattern of myelin basic protein‐related translation products for +/shi messenger RNA is intermediate between the patterns for +/+ and shi/shi messenger RNAs. The results suggest that the genetic lesion in the shiverer mutation impinges on the structural gene (or genes) encoding myelin basic protein or on a cis‐acting regulatory element controlling that gene (or genes).
Purification and Molecular Characterization of the Brain Synaptic Membrane Glutamate‐Binding Protein
Authors: Michaelis Elias K. , Michaelis Mary L. , Stormann Thomas M. , Chittenden W. Leroy , Grubbs Robert D. 
Published: October 2006

Abstract:A glutamate‐binding protein from rat brain synaptic plasma membranes has been purified to apparent homogeneity. This protein has a Mr of 14,300 based on amino acid and carbohydrate analyses. The protein is enriched with tryptophan residues, which contribute substantially to its hydrophobic nature. It also has a relatively high content of acidic amino acids, which determine its low isoelectric point (4.82). The protein exhibits either a single, high‐affinity class of sites for L‐[3H]glutamate binding (KD= 0.13 μM) when binding is measured at low protein concentrations, or two classes of sites with high (KD= 0.17 μM) and low affinities (KD= 0.8 μM) when binding is measured at high protein concentrations. These observations suggest preferential binding of L‐glutamate to a self‐associating form of the protein. The displacement of protein‐bound L‐[3H]glutamic acid by other neuroac‐tive amino acids has characteristics similar to those observed for displacement of L‐glutamate from membrane binding sites. Chemical modification of the cysteine and arginine residues results in an inhibition of glutamate binding activity. The possible function of this protein in the physiologic glutamate receptor complex of neuronal membranes is discussed.
Alterations of the Eicosanoid Synthetic Capacity of Rat Brain Microvessels Following Ischemia: Relevance to Ischemic Brain Edema
Authors: Koide Tohru , Gotoh Osamu , Asano Takao , Takakura Kintomo 
Published: October 2006

Abstract:To know the mechanism underlying ischemic brain edema, a time‐course analysis of the eicosanoid synthetic capacity of brain microvessels was carried out using unilateral, middle cerebral artery (MCA)‐occluded rats. Concomitant with the development of brain edema the synthetic capacity of all products, including cyclooxygenase and lipoxygenase products, increased significantly. Next the effects of 15‐hydroperoxyarachidonic acid (15‐HPAA) on the synthetic capacity of microvessels were examined. The drug caused a generalized increase of each product, the profile of which was similar to that obtained with ischemic hemispheres, although the ratios of each product differed somewhat among them. The enhanced synthesis of eicosanoids by 15‐HPAA was markedly suppressed by radical scavengers such as α‐tocopherol, hydroquinone, and 1,2‐bis(nicotineamide)‐propane. Furthermore, the evolution of brain edema was virtually suppressed by the systemic administration of 1,2‐bis(nicotineamide)‐propane. The above result suggests that the enzyme activity of the arachidonic acid (AA) cascade of microvessels is stimulated by its own products. Such a mechanism will form a vicious cycle that accelerates the accumulation of free radicals within microvessels and thus may play a role in the progressing disruption of the blood‐brain barrier (BBB) following ischemia.
Two‐Step Immunoaffinity Purification of Acetylcholinesterase from Rabbit Brain
Authors: Mintz K. P. , Brimijoin S. 
Published: October 2006

Abstract:Acetylcholinesterase (AChE; EC 3.1.1.7) extracted in 1% Triton X‐100 from rabbit brain was purified 2,000‐fold by chromatography on agarose conjugated with a monoclonal antibody directed against human red blood cell cholinesterase. After elution from the immunoadsorbent with pH 11 buffer, the preparation was purified further by affinity chromatography on phenyltrimethylammonium‐Sepharose 4B with decamethonium elution. Overall yield of purified enzyme was 37% of the AChE originally solubilized, with a specific activity of 2,950 units/mg protein. Electrophoresis under reducing conditions in 7.5% sodium dodecyl sulfate polyacrylamide gels revealed only one silver‐staining polypeptide band. A streamlined purification procedure enabled the isolation of electrophoretically homogenous AChE to be completed in fewer than 7 days, at yields exceeding 50%. Electrophoretic analysis of purified AChE indicated an apparent MW of 71,000 for the monomeric subunit. Gel filtration and sucrose density gradient centrifugation in the presence of Triton X‐100 showed little difference between the properties of the native and the purified enzyme. The molecular mass of the main species was estimated from the gel filtration and sedimentation data to be 280,000 daltons. Kinetic parameters of the purified protein (Km= 0.16 ± 0.01 mM) were close to those of the native enzyme (Km= 0.12 ± 0.01 mM) when examined with acetylthiocholine iodide as substrate. The two‐step immunopurification procedure presented in this communication offers a convenient route to homogenous neural AChE in quantities useful for detailed biochemical and immunochemical study.
Allosteric Modulation of Flunitrazepam Binding to Rat Brain Benzodiazepine Receptors by Methyl β‐Carboline‐3‐Carboxylate
Authors: Chiu Ted H. , Rosenberg Howard C. 
Published: October 2006

Abstract:The inhibition of flunitrazepam (FNP) binding to rat brain benzodiazepine (BZ) receptors by methyl β‐carboline‐3‐carboxylate (MCC) was studied. Biphasic dissociation was observed for [3H]FNP and [3H]MCC in cerebral cortex, cerebellum, and hippocampus, although the dissociation of [3H]MCC was much faster. The dissociation rate of [3H]FNP was increased by MCC in the cerebellum, but was not altered in cerebral cortex or hippocampus. [3H]FNP binding stimulated by γ‐aminobutyric acid was enhanced in the presence of MCC in all three regions examined. These results indicate that MCC exerts these effects by interacting with allosteric sites that are different from the FNP recognition sites on the BZ receptors.
Separation of Neuropeptides by HPLC: Evaluation of Different Supports, with Analytical and Preparative Applications to Human and Porcine Neurophysins, β‐Lipotropin, Adrenocorticotropic Hormone, and β‐Endorphin
Authors: Richter W. O. , Schwandt P. 
Published: October 2006

Abstract:The separation of 38 hypophyseal or hypothalamic peptides and proteins by reversed‐phase HPLC is described. The efficacy of several supports is discussed: large‐pore reversed‐phase silica supports turned out to be most effective in the separation of neuropeptides and proteins. The use of high sodium phosphate and phosphoric acid in the eluent resulted in high yields and better separation. On the basis of this procedure, β‐lipotropin, β‐endorphin, and adrenocorticotropic hormone were prepared from porcine pituitary glands, and human neurophysins from human pituitary posterior lobes. Moreover, this system is used for the analysis of the perifusate of pituitary posterior lobes, showing the release of microheterogeneous neurophysins.
Halothane‐Induced Alterations of Glucose and Pyruvate Metabolism in Rat Cerebra Synaptosomes
Authors: Johnson G. V. W. , Hartzell Charles R. 
Published: October 2006

Abstract:Synaptosomes isolated from rat cerebra were used to study the effects of the inhalational anesthetic, halothane, on cholinergic processes. To identify possible mechanisms responsible for the depression of acetylcholine synthesis, we examined the effects of halothane on precursor metabolite metabolism involved with supplying the cytosol with acetyl‐CoA for acetylcholine synthesis. Three percent halothane/air (vol/vol) depressed 14CO2 evolution from labeled pyruvate and glucose. Steadystate 14CO2 evolution from [1‐14C]glucose was depressed 84% by halothane, while 14CO2 evolution from [6‐14C]glucose and [3,4‐14C]glucose was decreased 67 and 52%, respectively, when compared with control conditions. Halothane inhibited the activities of both pyruvate dehydrogenase (14% depression) and ATP‐citrate lyase (32% depression). Total synaptosomal acetyl‐CoA concentrations were unaffected by halothane. Three percent halothane/air (vol/vol) caused a 77% increase in medium glucose depletion rate from 1.38 nmol (mg protein)−1 min−1 to 2.44 nmol (mg protein)−1 min−1. Production of lactate by the synaptosomes in the presence of halothane increased by 231% from a control rate of 1.44 nmol (mg protein)−1 min−1 to 4.77 nmol (mg protein)−1 min−1. Lactate production rate from pyruvate was also enhanced by 56% in the presence of halothane. These data lend support to the concept that the NAD+/NADH potential may be involved in the halothane‐induced depression of acetylcholine synthesis.
Composite Technique for Regional Neurochemical Studies: Measurement of Energy and Neurotransmitter Metabolites in Single Tissue Sample
Authors: Djuricic Bogdan M. , Ueki Yasuyuki , Spatz Maria 
Published: October 2006

Abstract:A combined method is described for the determination of various metabolites from a single tissue sample of the brain. It comprises a quick inactivation of cerebral enzymes by microwave irradiation, easy separation of the desired brain regions, and perchloric acid extraction of tissue substances, which are assayed either by specific enzymatic techniques or by HPLC with electrochemical detection. The obtained values of most energy and neurotransmitter metabolites in the brain are in agreement with those reported using other methods. However, this technique, in contrast to the brain freezing in vitro or freeze‐blowing, provides a more efficient procedure for rapid arrest of cerebral metabolism even in the deep brain structures and is therefore suitable for detection of early changes particularly those occurring in experimental pathological conditions such as ischemia.
Protein Carboxyl Methylation in Synaptic Membrane of Rat Brain: The Possible Presence of Adenosine‐Bound S‐Adenosyl‐L‐Homocysteine Hydrolase in the Membrane
Authors: Miyake Masaharu , Innami Toshihiko 
Published: October 2006

AbstractThe effects of some neurotransmitters, adenosine (Ad), and homocysteine (Hcys) on protein carboxyl methylation in synaptic plasma membranes from rat cerebral cortex were examined. Neither any of the neurotransmitters nor Ad had a detectable effect. Incubation of the membrane with DL‐Hcys alone (5 × 10‐5M), the combination of both Ad (5 × 10‐5) and DL‐Hcys (5 × 10‐5), or S‐adenosyl‐L‐homocysteine (SAH) (1 × 10‐6) strongly decreased the methyl ester formation. The inhibitory effect of the combination of both compounds may be interpreted in terms of the increased SAH concentration due to the presence of SAH hydrolase in the membrane. The inhibitory effect of Hcys alone was blocked by preincubation with Ad deaminase or Neplanocin A, a potent inhibitor of SAH hydrolase, suggesting the presence of Ad‐bound SAH hydrolase in the synaptic membrane. Ad‐bound SAH hydrolase activity estimated by the inhibition of methylation in the presence of Hcys was located in the membrane fractions including synaptosomes, myelin, and microsomes (about 70%), but the SAH hydrolase activity estimated on the basis of the inhibitory effect of the combination of both Ad and Hcys was localized exclusively in the soluble fraction (about 90%). The distribution of the latter activity is coincident with that of SAH hydrolase reported to date. Incubation of the synaptic membrane with Hcys alone and with the combination of both Ad and Hcys markedly increased the SAH concentration. The stimulatory effect of Hcys alone was blocked by Ad deaminase.
Phosphorylation Characteristics of Brain Clathrin‐Coated Vesicle Endogenous Proteins
Authors: Schook William J. , Parker Carlo , Silva Walter I. , Kohtz D. Stave , Puszkin Saul 
Published: October 2006

AbstractClathrin‐coated vesicles purified from bovine brain express protein kinase activity on two principal endogenous vesicle‐associated substrates: a 50,000‐Mr poly‐peptide (pp50) and clathrin‐associated protein2 (CAP2; the faster‐migrating clathrin light chain). Various exogenous substrates, e.g., casein, phosvitin, histone II, and histone III, also are phosphorylated. The pp50 protein kinase activity of clathrin‐coated vesicles is not modulated by Ca2+, calmodulin, phosphatidylserine, or cyclic AMP. On the other hand, phosphorylation of the other endogenous substrates requires certain activators, including histone, polylysine, polyarginine, or polyethylenimine. Phosphate incorporation into pp50 was sensitive to divalent cations that inhibit sulfhydryl‐dependent enzymes in the following order of potency: Zn2+ > Hg2+ > Cd2+, Cu2+, and Pb2+. Phosphate incorporation into CAP2 with polylysine present was insensitive to divalent cations. The alkylating agents dithiodini‐trobenzene, phenacyl bromide, and N‐ethylmaleimide inhibited phosphate incorporation into pp50 up to 90% without affecting incorporation into the other substrates. Vanadium pentoxide inhibited phosphorylation of CAP2 but had a minimal effect on pp50. CAP2 kinase activity was separated from the coated vesicle membrane and from disassembled clathrin triskelions, coeluting with the assembly polypeptide complex on a Sepharose 4B column. It retained phosphorylation properties similar to those of intact vesicles. These data imply that clathrin‐coated vesicle kinases are elements of the coat proteins and may be involved in the assembly/disassembly of clathrin triskelions or interactions of coated vesicles with other cellular components.
Inactive Monomeric Acetyleholinesterase in the Low‐Salt‐Soluble Extract of the Electric Organ from Torpedo marmorata
Authors: Stieger S. , Brodbeck U. , Witzemann V. 
Published: October 2006

AbstractProteolytic fragmentation of (3H]diisopropylflu‐orophosphate‐labelled catalytic subunits of different molecular forms of acetylcholinesterase demonstrates that all forms extracted from the electric organ from Torpedo marmorata are true acetylcholinesterases. This is supported by immunochemical results showing that the radiolabelled polypeptides are readily recognized by specific anti‐acetyl‐cholinesterase antibodies. Although distinct structural differences exist, all forms contain a similar peptide carrying the serine hydroxyl of the esteratic subsite. Dimeric, detergent‐soluble acetylcholinesterase is present in the low‐salt‐soluble extract (Mr of the catalytic subunit 66,000) together with a monomeric form (apparent Mr 76,000). This monomeric polypeptide is hydrophilic, enzymatically inactive, and might represent a precursor of the asymmetric forms of acetylcholinesterase.
Effect of Postmortem Factors on Muscarinic Receptor Subtypes in Rat Brain
Authors: Burke Robert E. , Greenbaum Daniel 
Published: October 2006

AbstractAlthough prior studies have supported the validity of measuring total muscarinic receptor binding in postmortem brain, there has not been a study of postmortem effects on muscarinic receptor subtypes, M1 and M2, defined by high and low affinity for pirenzepine, respectively. We have examined in rat brain the effect of postmortem delay at room temperature, storage at 4°C and‐20°C, and multiple freeze/thaw cycles on total muscarinic binding, measured with [3H]quinuclidinylbenzilate ([3H]QNB) and on M1 muscarinic binding, measured with [3H]pirenzepine ([3H]‐Pir). We found that delay at room temperature up to 4 h, or storage at 4°C for 24 h or at‐20°C for 4 weeks, or 3 freeze/thaw cycles had no effect on [3H]QNB or [3H]Pir binding. Exposure of brain to room temperature for 15 h, however, led to an increase in [3H]QNB binding, without change in [3H]Pir. Scatchard analysis showed an increase in binding sites without a change in affinity. We conclude that [3H]‐QNB and [3H]Pir are valid measures of total and M1 muscarinic binding, respectively, under these circumstances, but that caution must be used in the interpretation of indirect measures of M2 binding.
Induction of N‐Glycosylation Activity in Cultured Embryonic Rat Brain Cells
Authors: Bhat Narayan R. , Waechter C. J. 
Published: October 2006

AbstractDevelopmental changes in protein N‐glycosylation activity have been studied using cultures of dissociated fetal rat brain cells as an in vitro model system. These cultures undergo an initial phase of neurite outgrowth and cell proliferation (4–6 days in culture), followed by a period of cellular differentiation. N‐Glycosylation activity has been measured by assaying the incorporation of [2‐3H]‐mannose into dolichol‐linked oligosaccharides and glyco‐protein over a period of 1–25 days in culture. This study revealed a marked induction of N‐glycosylation activity beginning at approximately 1 week of culture. [2‐3H]‐Mannose incorporation into the oligosaccharide‐lipid intermediate fraction and glycoprotein reached maximal values between 12 and 16 days of culture and declined thereafter. The major dolichol‐linked oligosaccharide labeled by the brain cell cultures was shown to be Glc3Man9GlcNAc2 by HPLC analysis. Parallel incorporation studies with [3H]leucine showed that the increase in protein N‐glycosylation was relatively higher than a concurrent increase in cellular protein synthesis observed during the induction period. Maximal labeling of glycoprotein corresponded to the period of glial differentiation, as indicated by a sharp rise in the marker enzymes, 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (an oligodendroglial marker) and glutamine synthetase (an astroglial marker). The results describe a developmental activation of the N‐glycosylation pathway and suggest a possible relationship between N‐linked glycoprotein assembly and the growth and differentiation of glial cells.