bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal Antibodies to Rabbit Brain Acetylcholinesterase: Selective Enzyme Inhibition, Differential Affinity for Enzyme Forms, and Cross‐Reactivity with Other Mammalian Cholinesterases
Authors: Mintz Keith P. , Brimijoin Stephen
Published: October 2006

Abstract: Eleven unique monoclonal IgG antibodies were raised against rabbit brain acetylcholinesterase (AChE, EC 3.1.1.7), purified to electrophoretic homogeneity by a two‐step procedure involving immunoaffinity chromatography. The apparent dissociation constants of these antibodies for rabbit AChE ranged from about 10 nM to more than 100 nM (assuming one binding site per catalytic subunit). Species cross‐reactivity was investigated with crude brain extracts from rabbit, rat, mouse cat, guinea pig, and human. One antibody bound rabbit AChE exclusively; most bound AChE from three or four species; two bound enzyme from all species tested. Identical, moderate affinity for rat and mouse brain AChE was displayed by two antibodies; two others were able to distinguish between these similar antigens. Nine of the antibodies had lowered affinity for AChE in the presence of 1 M NaCl, but two were salt resistant. Analysis of mutual interferences in AChE binding suggested that certain of the antibodies were competing for nearby epitopes on the AChE surface. One antibody was a potent AChE inhibitor (IC50= 10−8M), blocking up to 90% of the enzyme activity. Most of the antibodies were less able to bind the readily soluble AChE of detergentfree brain extracts than the AChE which required detergent for solubilization. The extreme case, an antibody that was unable to recognize nearly half of the “soluble” AChE, was suspected of lacking affinity for the hydrophilic enzyme form.
Muscarinic and Nicotinic Cholinergic Binding Sites in the Terminal Abdominal Ganglion of the Cricket (Acheta domesticus)
Authors: Meyer Mark R. , Reddy G. Rajarami
Published: October 2006

Abstract: Cricket (Acheta domesticus) terminal abdominal ganglia (TG) contain high concentrations (∼2 pmol/ mg protein) of muscarinic and nicotinic cholinergic binding sites, based on the capacity of TG to bind specifically the labelled ligands L‐[3H]quinuclidinyl benzilate ([3H]QNB) and [125I]α‐bungarotoxin ([125I]α‐BGT) with high affinity. For both ligands, binding is saturable and reversible. Competitive displacement experiments indicate that the [3H]QNB and [125I]α‐BGT binding sites probably represent pharmacologically distinct classes of putative TG acetylcholine receptors (AChRs). Results from physiological recording and autoradiographic localization experiments demonstrate that a portion of the putative nicotinic AChRs is localized in synaptic regions of the well‐characterized cercal sensory‐giant interneuron pathway in the TG, where they are likely to serve as functional synaptic AChRs. Unlike nicotinic ligands, muscarinic agents do not appear to be pharmacologically active in this pathway. Therefore, in the insect CNS, putative muscarinic and nicotinic AChRs coexist at high density, but can be pharmacologically distinguished from one another on the basis of criteria derived from both ligand binding and physiological methods.
Glycerol Phosphate Dehydrogenase Activity of Oligodendrocytes Isolated from Adult Pig Brain: Its Inducibility by Hydrocortisone
Authors: Montz Hildegard P. M. , Althaus Hans H. , Gebicke‐Haerter Peter J. , Neuhoff Volker
Published: October 2006

Abstract: Developing oligodendrocytes cultured in vitro express glycerol phosphate dehydrogenase (GPDH; EC 1.1.1.8) and are known to respond to glucocorticoid treatment by increased activity of GPDH. We present evidence that GPDH is enriched in white matter and oligodendrocytes of adult pig brain. Bulk‐isolated oligodendrocytes maintained in culture for several weeks exhibit an almost constant level of GPDH activity. Furthermore, a 4‐day stimulation with hydrocortisone induces GPDH specific activity of long‐term cultured oligodendrocytes from adult pig brain.
Differences in L‐[3H]Glutamate Accumulation and Endogenous L‐Glutamate Content in Synaptic Vesicles from Mice Selectively Bred for Differences in Ethanol Sensitivity
Authors: Disbrow Jeffrey K. , Ruth James A.
Published: October 2006

Abstract: The ATP‐stimulated accumulation of L‐[3H]glutamate by whole brain synaptic vesicle preparations from long‐sleep and short‐sleep mice, lines selectively bred for difference in sleep time response to acute ethanol administration, was examined. L‐[3H]Glutamate accumulation in vesicles from short‐sleep mice was approximately twice that observed in vesicles from longsleep mice at three glutamate loading concentrations. The vesicular content of endogenous L‐glutamate in crude and enriched vesicle preparations from short‐sleep mice was approximately 1.5‐fold higher than in vesicles from long‐sleep mice. The data suggest that L‐glutamate associated with synaptic vesicles may serve a role in glutamate neurosecretion.
Intraventricular 5,7‐Dihydroxytryptamine Increases Thyrotropin‐Releasing Hormone Content in Regions of Rat Brain
Authors: Manaker Scott , Engber Thomas M. , Knight Pamela B. , Winokur Andrew
Published: October 2006

Abstract: Rats received intraventricular (i.v.t.) injections of 5,7‐dihydroxytryptamine (5,7‐DHT) (100–600 μg). Some animals also received intraperitoneal injections of the 5‐hydroxytryptamine uptake blocker fluoxetine (FX) (20 mg/kg) or the norepinephrine uptake blocker desmethylimipramine (DMI) (48 mg/kg) 30–90 min prior to i.v.t. 5,7‐DHT. Rats were killed between 2 and 35 days following i.v.t. 5,7‐DHT, brains were dissected, and regions were assayed for thyrotropin‐releasing hormone (TRH) by radioimmunoassay. Dose‐dependent increases in TRH content following i.v.t. 5,7‐DHT were noted in the brainstem and hippocampus. DMI pretreatment blocked the increase in hippocampal TRH, but not in brainstem TRH. FX pretreatment was ineffective in blocking any increases in TRH content. These results suggest differential regulation of regional TRH content by interactions with specific neurotransmitter systems.
Radioactive N,N‐Dimethylphenylethylamine: A Selective Radiotracer for In Vivo Measurement of Monoamine Oxidase‐B Activity in the Brain
Authors: Inoue Osamu , Tominaga Toshiyoshi , Yamasaki Toshio , Kinemuchi Hiroyasu
Published: October 2006

N‐[methyl‐14C]N,N‐dimethylphenylethylamine (DMPEA) was synthesized and its availability as a selective radiotracer for in vivo measurement of mouse brain monoamine oxidase (MAO) activity was examined. Relatively high incorporation of labelled DMPEA into brain (about 10% of the injected dose/per gram of brain) was observed just after its injection; however, radioactive dimethylamine, a metabolite produced from labelled DMPEA in the brain 1 h after DMPEA injection, was reduced in a dose‐dependent manner by pretreatment with various doses of a specific MAO‐B inhibitor, 1‐deprenyl, but was not reduced appreciably by pretreatment with a specific MAO‐A inhibitor, clorgyline. Pretreatment with 1‐deprenyl did not affect significantly the rate of incorporation of the radiotracer DMPEA into the brain, suggesting that reduction of the radioactivity in brain by this compound might be due to a decrease in the rate of production of the radioactive metabolite dimethylamine by brain MAO‐B. The amount of the radioactive metabolite trapped in the brain was found to be proportional to the brain MAO‐B activity remaining after pretreatment with 1‐deprenyl. In vitro deamination of DMPEA by mouse brain MAO showed a higher sensitivity to inhibition by 1‐deprenyl than that by clorgyline. These results indicate that DMPEA is a selective substrate for mouse brain MAO‐B both in vivo and in vitro and that the positron emitter [11C]DMPEA might be used instead of [14C]DMPEA as a radiotracer for in vivo measurement of MAO‐B activity in human brain.
Effects of Anti‐Glutamate‐Binding Protein Antibodies on Synaptic Membrane Ion Flux, Glutamate Transport and Release, and l‐Glutamate Binding Activities
Authors: Roy S. , Galton N. , Michaelis E.
Published: October 2006

Antibodies (Abs) raised against the l‐glutamate‐binding protein (GBP) purified from bovine brain were used to define the possible physiologic activity of GBP in synaptic membranes. Three processes were examined for their sensitivity to the Abs: the excitatory amino acid stimulation of thiocyanate (SCN−) flux, the transport of l‐glutamic acid across the synaptic membrane, and the depolarization‐induced release of l‐glutamate. Only the amino acid‐induced changes in ion flux were inhibited by the anti‐GBP Abs. The change in membrane potential produced by exposure of synaptic membranes to excitatory amino acids was measured as the increase in the uptake of the lipophilic anion SCN−. The l‐glutamate‐induced SCN− influx was 40 times more sensitive to inhibition by the anti‐GBP Abs than the stimulation of ion flux by kainate, and 60 times more sensitive than that produced by quisqualate. The anti‐GBP Abs did not inhibit the activation of ion flux produced by N‐methyl‐d‐aspartate. The inhibition of glutamate‐stimulated ion fluxes by the Abs was complete, whereas the inhibition of l‐glutamate binding to either the rat or bovine brain GBP was not. The results obtained indicated that although the majority of the anti‐GBP Abs were not directed against the glutamate recognition site of the GBP and of presumed synaptic membrane receptors, they were effective in blocking the activation of receptor‐associated ion channels. Thus, the GBP may be considered a component of some excitatory amino acid receptor complexes.
Novel Opioid Peptide Amidorphin: Characterization and Distribution of Amidorphin‐Like Immunoreactivity in Bovine, Ovine, and Porcine Brain, Pituitary, and Adrenal Medulla
Authors: Liebisch D. C. , Seizinger B. R. , Michael G. , Herz A.
Published: October 2006

Abstract: We have recently isolated from bovine adrenal medulla a novel C‐terminally amidated opioid peptide, amidorphin, which derives from proenkephalin A. Amidorphin revealed a widespread distribution in bovine, ovine, and porcine tissue. Particularly high concentrations of amidorphin immunoreactivity were detected in adrenal medulla, posterior pituitary, and striatum, similar to the major gene products of proenkephalin A. In the adrenal medulla of each species. authentic amidorphin was the predominant immunoreactive form. Pituitary and brain, however, contained predominantly putative N‐terminally shortened fragments of amidorphin of a slightly lower molecular weight and shorter retention times on HPLC. In addition, in ovine adrenal medulla, a putative high‐molecular‐weight form of amidorphin was detected. These findings are indicative of a tissue‐specific processing of the proenkephalin A precursor, leading predominantly to authentic amidorphin in the adrenal medulla and further processing to smaller C‐terminal fragments in the brain and pituitary.
Possible Involvement of Inhibitory GTP Binding Regulatory Protein in α2‐Adrenoceptor‐Mediated Inhibition of Adenylate Cyclase Activity in Cerebral Cortical Membranes of Rats
Authors: Kitamura Yoshihisa , Nomura Yasuyuki , Segawa Tomio
Published: October 2006

Abstract: Influences of α2‐adrenoceptor stimulation on adenylate cyclase activity were investigated in cerebral cortical membranes of rats. Pretreatment of the membranes with islet‐activating protein and NAD resulted in a significant increase in basal activity as well as in GTP‐or forskolin/GTP‐induced elevation of adenylate cyclase activity. Strong activation of adenylate cyclase was also caused in membranes pretreated with cholera toxin together with NAD in comparison to that in control membranes, suggesting that adenylate cyclase activity is perhaps regulated by stimulatory and inhibitory GTP binding regulatory protein existing in synaptic membranes. In addition, adrenaline (with propranolol) or clonidine significantly reduced adenylate cyclase activity stimulated by pretreatment with forskolin and GTP. The inhibitory effects of adrenaline were also observed in membranes pretreated with cholera toxin and NAD. Moreover, the inhibition by adrenaline or clonidine was completely abolished by treatment with (a) yohimbine or (b) islet‐activating protein and NAD. It is suggested that α‐receptor stimulation causes inhibitory influences on adenylate cyclase activity mediated by the inhibitory GTP binding regulatory protein in synaptic membranes of rat cerebral cortex.
Antisera Against the Indolealkylamines: Tryptophan, 5‐Hydroxytryptophan, 5‐Hydroxytryptamine, 5‐Methoxytryptophan, and 5‐Methoxytryptamine Tested by an Enzyme‐Linked Immunosorbent Assay Method
Authors: Geffard Michel , Dulluc Josette , Rock Anne‐Marie
Published: October 2006

Abstract: Antisera were raised against tryptophan, 5‐hydroxytryptophan, 5‐hydroxytryptamine, 5‐methoxytryptophan, and 5‐methoxytryptamine, by conjugating each molecule to bovine serum albumin and to human serum albumin via glutaraldehyde, in such a way as to preserve the original part. Antibody specificity was tested with the enzyme‐linked immunosorbent assay method. The specificity of each anti‐indolealkylamine‐glutaraldehyde antibody was established with competition experiments by using an adsorbed immunogenic conjugate and indolealkylamines either free or conjugated with poly‐L‐lysine. The nonconjugated compounds were poorly recognized. In the same way, the nonreduced conjugates always appeared less immunoreactive than the reduced ones. Calculated from the specificity study of each antiserum, the cross‐reactivity ratios were found to be smallest for the most immunoreactive conjugates. Thus, a specific immune response was defined for each compound belonging to the same metabolic pathway.
Carnosine Synthesis in Olfactory Tissue During Ontogeny: Effect of Exogenous β‐Alanine
Authors: Margolis E. L. , Grill M. , Kawano T. , Farbman A. I.
Published: October 2006

Abstract: Carnosine has now been demonstrated by chemical analysis to be present in rat olfactory mucosa on day 16 of gestation. The tissue content of this dipeptide then increases progressively during fetal and postnatal life. Radioactive carnosine can be isolated from cultured embryonic rat olfactory mucosa incubated with [14C]β‐alanine as early as 13–14 days of gestation. The amount of incorporation also increases progressively with the initial age of the explant and with time in culture indicating in vitro maturation of the carnosine synthesis capability of olfactory tissue. To test whether the level of β‐alanine was limiting the synthesis of carnosine, we evaluated the effect of elevated β‐alanine levels on tissue carnosine content. Exogenous β‐alanine caused an increase in the tissue content of carnosine at several ages in vivo and in vitro. In adult animals this increase was observed in olfactory bulb, olfactory muscosa, and skeletal muscle. However, there was no associated alteration in carnosine synthetase activity. In addition, the different half‐lives of carnosine in olfactory tissue and muscle seemed unaltered, arguing against any effect on degradative enzymes. Thus, tissue carnosine levels are regulated, at least in part, by substrate availability. The early appearance of carnosine synthetic capacity during prenatal development indicates that this enzyme activity should be a valuable aid in studying early events in olfactory neuron maturation.
Regulation of Anterior Pituitary D2 Dopamine Receptors by Magnesium and Sodium Ions
Authors: Watanabe Masayuki , George Susan R. , Seeman Philip
Published: October 2006

Abstract: At D2 3,4‐dihydroxyphenylethylamine (dopamine) receptors in anterior pituitary tissue, magnesium ions shifted receptors to agonist high‐affinity states, but decreased the affinity of the antagonist [3H]spiperone. Conversely, sodium ions shifted the receptors to agonist low‐affinity states, but increased the affinity of [3H]spiperone. Magnesium is proposed to stabilize the hormone–receptor–guanine nucleotide regulatory protein complex, whereas sodium appears to destabilize this ternary complex. Thus, magnesium and sodium appear to mediate their regulatory effects via a common component at the D2 dopamine‐receptor ternary complex.
Quantitation of the Myelin‐Associated Glycoprotein in Human Nervous Tissue from Controls and Multiple Sclerosis Patients
Authors: Johnson David , Sato Shuzo , Quarles Richard H. , Inuzuka Takashi , Brady Roscoe O. , Tourtellotte Wallace W.
Published: October 2006

Abstract: Myelin‐associated glycoprotein (MAG) was measured by radioimmunoassay in the human CNS and peripheral nervous system (PNS). The level of MAG, expressed as ng/μg of total protein, was ∼20‐fold higher in whole homogenates of cerebral white matter (4.7 ± 0.60) than of peripheral nerve (0.12–0.28). MAG concentrations were only slightly higher in the isolated myelin fractions from these tissues: CNS myelin, 5.6 ng/μg; PNS myelin, 0.37 ng/μg. The levels of MAG were measured in nine plaques, periplaque regions, and areas of macroscopically normal‐appearing white matter (NAWM) from six separate multiple sclerosis brains and compared with the levels of other myelin proteins in the same samples. MAG and other myelin proteins were reduced to very low levels in plaques. The levels of MAG and basic protein (BP) and the activity of 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP) in periplaque areas were significantly lower than those in control white matter, and MAG and BP levels were also significantly reduced in NAWM. In a periplaque region and NAWM from the most rapidly progressing case of multiple sclerosis examined, the MAG content was between 30 and 35% of the control level, whereas BP and PLP levels and CNP activity were between 50 and 85% of control values. The reduction of MAG content in periplaque regions from all nine multiple sclerosis plaques examined was significantly greater than the reductions of BP level and CNP activity. In NAWM samples, the mean reduction of MAG content was also greater than the reductions of BP level and CNP activity, but the difference was only statistically significant in comparison to CNP. These quantitative biochemical studies support earlier immunocytochemical observations suggesting that the MAG level is reduced more than levels of other myelin proteins at the periphery of some multiple sclerosis plaques.
Release of Previously Incorporated γ‐[3H]Aminobutyric Acid in Rabbit Caudate Nucleus Slices
Authors: Limberger N. , Späth L. , Starke K.
Published: October 2006

Abstract: The release of γ‐aminobutyric acid (GABA) was studied in slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused. Aminooxyacetic acid was present throughout. Both the tritium in the slices and that in the superfusate consisted practically entirely of [3H]GABA. Stimulation for 2 min by electrical field pulses of 3 ms width and 9 V/cm voltage drop (36 mA current strength) at 5 or 20 Hz elicited an overflow of [3H]GABA that amounted to 0.23 or 0.47% of the tritium content of the tissue, respectively, and was diminished by 85% in the presence of tetrodotoxin. At higher current strength, less of the stimulation‐evoked overflow was tetrodotoxin‐sensitive. cis‐1,3‐Aminocyclohexane carboxylic acid diminished the uptake of [3H]GABA into the tissue but did not change the percentage released by electrical stimulation. Ca2+ withdrawal greatly accelerated basal [3H]GABA efflux and almost abolished the response to stimulation. Nipecotic acid 10–1,000 μM enhanced both the basal and (up to eightfold) the stimulation‐evoked overflow. The method described allows us to elicit electrically a quasiphysiological, i.e., Ca2+‐dependent and tetrodotoxin‐sensitive, neuronal release of [3H]GABA. Nipecotic acid diverts released [3H]GABA from reuptake to overflow.
Brain Dialysis: In Vivo Metabolism of Dopamine and Serotonin by Monoamine Oxidase A but Not B in the Striatum of Unrestrained Rats
Authors: Kato Takeshi , Dong Bei , Ishii Kayoko , Kinemuchi Hiroyasu
Published: October 2006

Abstract: A dialysis cannula was implanted into rat striatum while the animals were anesthetized, and the area was perfused with Ringer solution while the animals were unanesthetized after at least 3 days following surgery. Concentrations of the metabolites of 3,4‐dihydroxy‐phenylethylamine (DA) and 5‐hydroxytryptamine (5‐HT) in the perfusate were determined by HPLC with electrochemical detection. Levels of the DA metabolites 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the perfusate significantly decreased after pargyline administration (50 mg/kg i.p.), which may inhibit not only monoamine oxidase (MAO)‐B but also MAO‐A in these high doses. The level of the 5‐HT metabolite 5‐hydroxyindoleacetic acid (5‐HIAA) also decreased after pargyline treatment, although change in the relative level of 5‐HIAA was less than that of DOPAC or HVA. To clarify the mechanisms for the metabolism of monoamines in rat striatum, highly specific MAO‐A and ‐B inhibitors were used in the following experiments. Treatment with l‐deprenyl (10 mg/kg), a specific inhibitor for MAO‐B, did not cause any statistically significant change in DOPAC, HVA, and 5‐HIAA levels. No significant change was found in rat striatal homogenates at 2 h after the same treatment with l‐deprenyl. In contrast, low‐dose treatment (1 mg/kg) with clorgyline, a specific inhibitor for MAO‐A, caused a significant decrease in levels of these three metabolites in both the perfusates and tissue homogenates. In addition to the above three metabolites, the level of 3‐methoxytyramine, which is an indicator of the amount of DA released, greatly increased after treatment with a low dose (1 mg/kg) of clorgyline. These results suggest that DA and 5‐HT in rat striatum may be preferentially deaminated by MAO‐A in vivo.
Regulation of Glycerol Phosphate Dehydrogenase and Lactate Dehydrogenase Activity by Forskolin and Dibutyryl Cyclic AMP in the C6 Glial Cells
Authors: Montiel Fatima , Aranda Ana , Villa Aida , Pascual Angel
Published: October 2006

Abstract We have compared the effects of norepinephrine, forskolin, and dibutyryl cyclic AMP (Bt2cAMP) on the regulation of the cytosolic enzyme glycerol phosphate dehydrogenase (GPDH) in the C6 rat glioma cell line. Forskolin and Bt2cAMP elicit a dose‐dependent increase in the levels of the enzyme that was, however, unaffected by norepinephrine. The half‐maximal effect of forskolin was obtained at 7–8 μM. and the effect was maximal at 30 μM. Dexametha‐sone at a 50 nM concentration produced a two‐ to sixfold induction of GPDH after 48 h. The combination of dexa‐methasone with forskolin or Bt2cAMP leads to an elevation in GPDH levels that is higher than that produced by one of the compounds alone. This potentiation is found when both agents are added together with or after the glucocorticoid. The increase in uninduced and dexamethasone‐induced GPDH activity was blocked by cycloheximide and actino‐mycin D, indicating that de novo protein and RNA synthesis are required. The activity of cytosolic lactate dehydrogenase activity did not change after incubation with dexa‐methasone, but increased with forskolin or Bt2cAMP.
Myelin‐Associated Glycoprotein Shares an Antigenic Determinant with a Glycoprotein of Human Melanoma Cells
Authors: Noronha Antonio B. , Harper John R. , Ilyas Amjad A. , Reisfeld Ralph A. , Quarles Richard H.
Published: October 2006

Abstract A sulfated 100K‐dalton glycoprotein has been shown to be released into the culture medium of melanoma cells. Monoclonal antibodies 10C5 and 11B5, which were raised to human melanoma cells, as well as HNK‐1 bind to this glycoprotein. It is shown here that mouse anti‐myelin‐associated glycoprotein (MAG) carbohydrate antibodies raised to human MAG and a human IgM paraprotein associated with neuropathy also bind to the same 100K molecule. However, anti‐MAG antibodies recognizing peptide epitopes do not appear to react with this glycoprotein of melanoma cells, a result suggesting that its similarity to MAG is restricted to shared carbohydrate moieties. The anti‐melanoma antibodies (10C5 and 11B5) resemble HNK‐1 in binding to MAG and to some 19–28K‐dalton glycoproteins and sulfated, glucuronic acid‐containing sphingoglycolipids of the peripheral nervous system (PNS). In addition, the anti‐melanoma antibodies cross‐react with neural cell adhesion molecule (N‐CAM), an observation emphasizing the shared antigenicity between MAG and other adhesion molecules. The results demonstrate that the anti‐melanoma antibodies fall into a class of monoclonal antibodies (including HNK‐1, human IgM paraproteins associated with neuropathy, anti‐human MAG antibodies, and L2 antibodies) that are characterized by reactivity against related carbohydrate determinants shared by human MAG, N‐CAM, and several protein and lipid glyco‐conjugates of the PNS.
Complexity of Nuclear and Polysomal RNA from Squid Optic Lobe and Gill
Authors: Capano Carla Perrone , Gioio Anthony E. , Giuditta Antonio , Kaplan Barry B.
Published: October 2006

Abstract: The sequence complexity of nuclear and polysomal RNA from squid optic lobe and gill was measured by RNA‐driven hybridization reactions with single‐copy [3H]DNA. At saturation, brain nuclear and polysomal RNAs were complementary to 22.8 and 7.9% of the DNA probe, respectively. Assuming asymmetric transcription, the complexity of nuclear and polysomal RNA was equivalent to 2.5 × 108 and 8.8 × 107 nucleotides, respectively. Approximately 80–85% of the sequence complexity of brain total polysomal RNA was found in the polyadenylated RNA fraction. In contrast to these findings, nuclear and polysomal RNAs from gill hybridized to 9.1 and 2.9%, respectively, of the single‐copy DNA, values that were 2.5‐fold lower than those obtained in the CNS. Taken together, the results focus attention on the striking diversity of gene expression in the squid CNS and extend to the cephalopod mollusks the observation that nervous tissue expresses significantly more genetic information than other somatic tissues or organs.
Possible Hydrophobic Region in Myelin Basic Protein Consisting of an Orthogonally Packed β‐Sheet
Authors: Martenson Russell E.
Published: October 2006

Abstract:Theoretical analysis was carried out to determine how the ∼20% of β‐structure observed in the 18.5 kilodalton (kDa) myelin basic protein (MBP) could be organized into a relatively stable β‐sheet. The β‐sheet is presumed to consist of the five most hydrophobic segments of polypeptide chain, which have β‐structure potential. These correspond approximately to sequences 15–21, 37–45, 84–92, 106–112, and 148–154 (rabbit MBP sequence numbering) and constitute β‐strandsa,b,c,d, ande, respectively. A number of constraints are imposed upon the sheet; e.g., it should have the same topology in all MBP forms (21.5, 18.5, 17, and 14 kDa); strandeshould lie at the sheet edge; strandsb, c, anddshould be ordered sequentially; the sheet formed by strandsa, b, c, anddshould be antiparallel; a maximum of the nonpolar surface area should be removed from the aqueous milieu; and charged side chains should be solvent‐accessible. On the basis of these constraints it is possible to propose six orthogonally packed β‐sheets having different topologies. If strandeis restricted to an antiparallel alignment, the number of different sheets is reduced to four. Each of these sheets can form a relatively compact hydrophobic globular region. Two of the strands (aande) can undergo transitions to α‐helix without disrupting the structure of the remaining sheetbcdor producing major topologic rearrangements of the polypeptide chain.
Affinity Labeling of Adenosine A1 Binding Sites
Authors: Martini C. , Lucacchini A.
Published: October 2006

Abstract: The adenosine A1 receptors of sheep brain membranes have been identified by the specific binding of radio‐labeled cyclohexyl [3H]adenosine ([3H]CHA). Pretreatment of membranes with periodate‐oxidized CHA causes a doseand time‐dependent decrease in the number of binding sites. No decrease occurs when membranes are pretreated with CHA. Binding of [3H]CHA to the remaining sites occurs with the same characteristics as binding to the untreated receptor population.
Threshold Requirements for Oxygen in the Release of Acetylcholine from, and in the Maintenance of the Energy State in, Rat Brain Synaptosomes Ian R. Park.
Authors: Thorn Michael B. , Bachelard Herman S.
Published: October 2006

Abstract: An oxystat was designed to enable maintenance of very low, predetermined oxygen tensions (below 1 μM) in incubated suspensions of synaptosomes. The oxygen thresholds for the energy state (ATP and creatine phosphate levels), for lactate production, and for acetylcholine release were compared. The approximate thresholds (μM O2) in veratridine‐stimulated preparations were: oxygen consumption, 10; ATP, 10; creatine phosphate, 15; lactate release, 20; and acetylcholine release, 25. The results for release of total acetylcholine and of the acetylcholine newly synthesized from [14C]glucose were indistinguishable. The results from this study are discussed in relation to hypoxia and to reported in vivo observations.
Composition of Gangliosides and Neutral Glycosphingolipids of Brain in Classical Tay‐Sachs and Sandhoff Disease: More Lyso‐GM2 in Sandhoff Disease?
Authors: Rosengren Birgitta , Månsson Jan‐Eric , Svennerholm Lars
Published: October 2006

Abstract: The ganglioside composition of the brain from an individual with classical Tay‐Sachs disease and from an individual with Sandhoff disease was examined using our new quantitative methods for ganglioside content determination and compared with that of age‐matched control brains. The concentration of GM2 was found to be 12.2 and 13.0 μmol/ g of fresh tissue in Tay‐Sachs disease and in Sandhoff disease cerebral gray matter, respectively. GM2 was 86 and 87% respectively, of total gangliosides. The concentration of GM1 and, in particular, GM3 ganglioside was also found to be increased, whereas the concentration of the major di‐ and trisialogangliosides (GD1a, GD1b, and GT1b) had diminished markedly. There was no significant increase in level of any other ganglioside than lyso‐GM2. Its concentration was 12 and 16 nmol/g in cerebral gray matter of two Tay‐Sachs disease brains and 43 nmol/g in Sandhoff disease brain. The SandhofF disease brain also differed from the classical Tay‐ Sachs disease brain by having a much higher concentration of gangliotriaosylceramide and globotetraosylceramide. The structures of relevant gangliosides and neutral glycolipids were established by fast atom bombardment‐mass spectrometry and permethylation studies.
Solubilization of Calcium Channel Antagonist Binding Sites from Rat Brain
Authors: Finkel Mitchell S. , Tallman John F.
Published: October 2006

Abstract: Calcium antagonist binding sites were solubilized from rat brain membranes using the detergent 3‐[(3‐cholamidopropyl)dimethylammonio] 1 ‐propanesulfonate (CHAPS). CHAPS‐solubilized [3H]nitrendipine binding sites are saturable over a range of 0.05–4 nM, and Scatchard analysis reveals a single, high‐affinity (KD= 0.49 × 0.10 nM), low‐capacity (Bmax= 56 × 4 fmol/mg of protein) binding site. Reversible ligand competition experiments using solubilized binding sites demonstrated appropriate pharmacologic specificity, with dihydropyridines (nifedipine = nitrendipine < Bay K 8644) completely displacing binding, verapamil partially displacing binding, and diltiazem enhancing binding, as previously described in membrane preparations. Lyophilized Crotalus atrox venom was purified by ion exchange chromatography followed by gel filtration to a single peptide band on sodium dodecyl sulfate‐ polyacrylamide gel electrophoresis. This fraction of molecular weight 60,000 competitively inhibits [3H]nitrendipine binding to both membrane and soluble preparations with an IC50 of 5 μg/ml. This polypeptide should serve as a useful ligand for future efforts in purifying the dihydropyridine calcium channel binding site in brain.
Isolated Brain Microvessels as In Vitro Equivalents of the Blood‐Brain Barrier: Selective Removal by Collagenase of the A‐System of Neutral Amino Acid Transport
Authors: Cardelli‐Cangiano P. , Fiori A. , Cangiano C. , Barberini F. , Allegra P. , Peresempio V. , Strom R.
Published: October 2006

On treatment with collagenase, brain microvessels, together with several protein components, lose some enzymatic activities such as alkaline phosphatase and γ‐glutamyltranspeptidase, whereas no change occurs in the activities of 5′‐nucleotidase and glutamine synthetase. The energy‐requiring „A‐system” of polar neutral amino acid transport is also severely inactivated, whereas the L‐system for the facilitated exchange of branched chain and aromatic amino acids is preserved. In the collagenase‐digested microvessels, this leads to loss of the transtimulation effect of glutamine on the transport of large neutral amino acids, because such transtimulation is due to a cooperation between the A‐ and L‐systems. By contrast, NH4+ maintains (and even enhances) its ability to stimulate the L‐system of amino acid transport, presumably through glutamine synthesis within the endothelial cells.
Axonal Transport of Slow Component a in Sciatic Nerves of Hypo‐and Hyperthyroid Rats
Authors: Sidenius Per , Nagel Poul , Larsen JØrn Rolighed , Boye Niels , Laurberg Peter
Published: October 2006

: Axonal transport of slow component a was studied in dorsal root afferents of the sciatic nerves of hypo‐and hyperthyroid rats. Three experimental groups of rats were made hypothyroid at the age of 12 weeks by the administration of 131I. From the age of 22 weeks to the end of the study, the groups were treated with daily subcutaneous injections of thyroxine in various doses to make them hypo‐(0 ug/100 g), normo‐(1 ug/100 g), and hyperthyroid (6 ug/100 g), respectively. The hypothyroid group had a moderate thyroid hormone deficiency (a serum triiodothyro‐nine level of 0.19 ± 0.10 nmol/L and a heart/body weight ratio of 1.87 ± 0.09 g/kg at time of killing compared with 0.60 ± 0.09 nmol/L and 2.18 ± 0.06 g/kg, respectively, for the control group). The hyperthyroid group was severely deranged, with serum triiodothyronine being 3.30 ± 0.37 nmol/L and a heart/body weight ratio of 3.11 ± 0.16 g/kg.

The hypothyroid rats shov/ed a reduction in mean velocity for the transport of slow component a (0.80 ± 0.07 mm/day compared with 0.91 ± 0.05 mm/day in the controls). The width of the wave of activty was smaller for the hyperthyroid group than for the control group (6.6 ± 0.7 mm compared with 8.1 ± 1.2 mm), suggesting an increased clearance of the axonally transported activity in the proximal axon. A decrease in transport of slow component a in hypo‐thyroidism may be the explanation of peripheral neuropathy with axonal degeneration occasionally seen in patients with severe myxoedema.
A Micromethod for the Isolation of Large and Small Microvessels from Frozen Autopsied Human Brain
Authors: Tsuji Teruyuki , Mimori Yasuyo , Nakamura Shigenobu , Kameyama Masakuni
Published: October 2006

Microvessels were isolated from autopsied human brain using a simple procedure involving disruption, sieving, and centrifugation on a sucrose density gradient. The present procedure is characterized by isolation, from frozen autopsied brain, of materials either from the cerebral cortex or white matter, and subsequent separation of the capillary fraction from the large vessel fraction. The preparation appears highly purified under phase‐contrast microscopic examination. The purity was also established by the enrichment of γ‐glutamyl transpeptidase activity and by the nearly negligible cerebroside content in the vessel fractions as compared to the brain homogenate.
Identification of the 5‐HT1A Receptor Binding Subunit in Rat Brain Membranes Using the Photoaffinity Probe [3H]8‐Methoxy‐2‐[N‐n‐Propyl, N‐3‐(2‐Nitro‐4‐Azidophenyl)Aminopropyl]Aminotetralin
Authors: Emerit M. B. , Mestikawy S. El , Gozlan H. , Cossery J. M. , Besselievre R. , Marquet A. , Hamon M.
Published: October 2006

The synthesis of a tritiated derivative of the 5‐HT1A photoaffinity probe 8‐methoxy‐2‐[N‐n‐propyl, N‐3‐(2‐nitro‐4‐azidophenyl)aminopropyl]aminotetralin ([3H]8‐methoxy‐3′‐NAP‐amino‐PAT) allowed the use of this probe for attempting the irreversible labeling of specific binding sites in rat brain membranes. Sodium dodecyl‐sulfate‐polyacrylamide gel electrophoresis of proteins solubilized from hippocampal microsomal membranes that had been incubated with 20 nM [3H]8‐methoxy‐3′‐NAP‐amino‐PAT under UV light revealed a marked incorporation of 3H label into a 63‐kilodalton protein termed PI . As expected of a possible correspondence between P1 and 5‐HT1A receptor binding sites, 3H labeling by the photoaffinity probe could be prevented by selective 5‐HT1A ligands such as 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin, ipsapirone, buspirone, and gepirone and by N‐ethylmaleimide, but not by the 5‐HT2 antagonist ketanserin, noradrenaline‐and dopamine‐related drugs, monoamine oxidase inhibitors, and chlorimipramine. Furthermore, the regional and subcellular distributions of P1 were identical to those of specific 5‐HT1A binding sites. These results indicated that the binding subunit of the 5‐HT1A receptor is a 63‐kilodalton protein with a functionally important sulfhydryl group(s).
Calmodulin‐Dependent Phosphatases of PC12, GH3, and C6 Cells: Physical, Kinetic, and Immunochemical Properties
Authors: Farber Len H. , Wilson Frank J. , Wolff Donald J.
Published: October 2006

Calmodulin‐dependent phosphoprotein phosphatase (CaMDP) activity has been found in each of three cultured cell lines: rat pheochromocytoma (PC 12), glioma (C6), and pituitary adenoma (GH3) cells. These CaMDP activities bind to immobilized calmodulin in the presence of Ca2+ and are eluted by EGTA. Sucrose density centrifugation revealed that the phosphatase activities exhibited sedimentation coefficients of 4.37, 4.23, and 4.59 for proteins derived from C6, GH3, and PC 12 cells, respectively. The Stokes radii measured for the PC 12 and C6 activities were 41.8 and 40.0 A, respectively. The estimated molecular weights calculated for the enzymes from these data are 79, 100 and 72, 200. The phosphatase activities required the presence of divalent cations such as Ca2+ or Mn2+ for expression of activity, which was optimal only in the presence of calmodulin. The apparent Km for phosphorylated myelin basic protein substrate was 8 μM. Affinity‐purified antibodies to the B subunit of bovine brain CaMDP were found by immunoblot (Western blot) to cross‐react with a single protein among proteins extracted from PC 12, C6, and GH3 cells that had been resolved by two‐dimensional electrophoresis. In each case, the cross‐reacting protein exhibited an Mr of 16,000 and an isoelectric point of 4.7, values virtually identical to those reported previously for the B subunit of bovine brain CaMDP (sometimes called calcineurin). This cross‐reacting protein was found among cellular proteins eluted from immobilized calmodulin by EGTA. Immuno‐cytochemical localization of the cross‐reacting protein in undifferentiated PC 12 cells or in cells differentiated in response to nerve growth factor revealed its presence diffusely throughout the cytoplasm. These experiments support the contention that each of these cell lines contains a calmodulin‐regulated phosphatase homologous physically and kinetically, and immunologically related to bovine brain CaMDP.
Alkylation of Free Sulfhydryls Fortifies Electroplax Subsynaptic Structures
Authors: Gysin Reinhard , Ranagan Steven D.
Published: October 2006

The cysteine‐rich 43,000‐dalton peripheral membrane protein, v1, is localized at the cytoplasmic face of electroplax and muscle cholinergic synapses, where it is thought to play an important role in the endplate supramolecular structure. The peripheral membrane protein properties of v1 are inferred by its removal from nicotinic cholinergic membranes by the action of mild alkali or lithium diiodosa‐licylate. An interesting property of v1 is its high concentration of free sulfhydryl groups, whose exact role in synaptic structure is still largely unknown. Alkylation of free sulfhydryls with N‐ethylmaleimide (3 mM) has a profound effect on the association of v1 with synaptic membranes, rendering v1 unextractable by pH 11 treatment or by lithium diiodosalicylate and, concomitantly, decreasing v1 's electrophoretic mobility on sodium dodecyl sulfate‐polyacrylamide gels. lodoacetamide and iodoacetate have similar effects, but at concentrations 10‐to 100‐fold higher than required for N‐ethylmaleimide. Furthermore, sulfhydryl modification also stabilizes the association of nicotinic receptor subunits with the detergent‐insoluble cytoskeleton. N‐Ethylma‐leimide treatment increases the fraction of insoluble receptor molecules on extraction with Triton X‐100, sodium cholate, or octylglucoside. These results suggest an important role of sulfhydryl groups in the structural stability of the postsynaptic cholinergic membrane.
Nerve Growth Factor Receptors in Human Neuroblastoma Cells
Authors: Marchetti Dario , Perez‐Polo J. Regino
Published: October 2006

Receptors for the nerve growth factor protein (NGFR) present in the human neuroblastoma cell line LAN‐1 were characterized. LAN‐1 cells display high‐affinity (type I, with KD value of 5.9 × 10‐11M) and low‐affinity (type II, with KD value of 9.2 × 10‐9M) binding to NGF. NGFR were fractionated by preparative isoelectric focusing in a granulated gel (PEGG). High‐affinity binding was found in the 5.9–6.2 pH region of the PEGG, and low‐affinity binding in the 4.6–4.8 and 8.8–9.3 pH ranges. After further analysis by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) we observed both 92.5‐and 200‐kDa molecular species associated with NGF binding activity. The 200‐kDa protein was found in fractions displaying high‐affinity NGF binding and the 92.5‐kDa protein in fractions displaying low‐affinity NGF binding. Equilibrium binding analysis of NGF in PEGG fractions confirmed the presence of two specific saturable binding sites with KD values similar to those observed for whole dissociated cells. When NGFR II activity from the acidic region of the PEGG chromatogram was incubated with NGFR II from the basic region of the PEGG chromatogram, there was no change in NGF binding or in the number of apparent NGF receptors. However, incubation of these same fractions with a fraction having only NGFR I showed an apparent increase in high‐affinity NGF binding and a decrease in low‐affinity NGF binding. Immunoprecipitation of this “mixed” fraction and analysis on SDS‐PAGE under reduced and nonreduced conditions showed 200‐kDa and 92.5‐kDa proteins under nonreduced conditions and a 92.5‐kDa protein under reduced conditions. Our findings are consistent with the hypothesis that there are two distinct NGF receptors in NGF‐responsive cells. The interconvertibility of low‐and high‐affinity receptors and the possible existence of a modulator type protein or of “silent” type receptors are also in agreement with our findings.
Na+‐Dependent Transport of Taurine by Membrane Vesicles of Neuroblastoma ± Glioma Hybrid Cells
Authors: Yuasa Shigeki , Hamprecht Bernd
Published: October 2006

The transport of taurine into membrane vesicles prepared from neuroblastoma × glioma hybrid cells 108CC5 was studied. A great part of the taurine uptake by the membrane preparation is due to the transport into an osmotically sensitive space of membrane vesicles. Taurine uptake by membrane vesicles is an active transport driven by the concentration gradient of Na+ across the membrane (outside concentration > inside). The Km value of 36 μM for Na+‐dependent taurine uptake indicates a high‐affinity transport system. The rate of taurine transport by the membrane vesicles is enhanced by the K+ gradient (inside concentration > outside) and the K+ ionophore valinomycin. Taurine transport is inhibited by several structural analogs of taurine: hypotaurine, β‐alanine, and taurocyamine. All these results indicate that the taurine transport system of the membrane vesicles displays properties almost identical to those of intact neuroblastoma × glioma hybrid cells.
Cholecystokinin Stimulates Dopamine Synthesis in Synaptosomes by a Cyclic AMP‐Dependent Mechanism
Authors: Chowdhury Muhammad , Steardo Luca , Fillenz Marianne
Published: October 2006

The effects of different fragments of cholecystokinin (CCK) on dopamine synthesis were studied in synaptosomal preparations from the striatum, substantia nigra, and frontal cortex. In striatal synaptosomes, dopamine synthesis rate measured by dopamine accumulation was 12.5% lower than that measured by 3,4‐dihydroxyphenylalanine (DOPA) accumulation; however, K+‐accelerated synthesis was the same for both methods. Synthesis rate was independent of exogenous tyrosine levels. In the three regions studied, the combined stimulatory effects of 8‐Br‐cyclic AMP and high K+ were additive. CCK‐5, CCK‐3, CCK‐27–33, and CCK‐8 (sulphated) enhanced synthesis, CCK‐5 being the most potent fragment. The nonsulphated octapeptide had no effect. In all three regions, CCK‐5 and high K+ had an additive effect on dopamine synthesis; CCK‐5 and 8‐Br‐cyclic AMP together produced the same enhancement of synthesis as CCK‐5 alone. CCK‐5 produced similar dose‐dependent increases in dopamine synthesis and cyclic AMP accumulation in striatal synaptosomes, and both effects were blocked by the CCK antagonist proglumide.
Effects of Systemic Kainic Acid Administration on Regional Na+, K+‐ATPase Activity in Rat Brain
Authors: Sztriha L. , Joó F. , Dux L. , Böti Zsuzsanna
Published: October 2006

Abstract: Changes in the activity of Na+, K+‐ATPase and in the water, Na+, and K+ levels in the parietal cortex, hippocampus, and thalamus were investigated in rats 1, 3, 6, and 24 h following systemic kainic acid injection. An increase in Na+, K+‐ATPase activity was observed in all three regions 3 h after the treatment, with a subsequent decrease in enzyme activity. The elevation in Na+,K+‐ATPase activity was accompanied by an increase in the Na+ content and a decrease in the K+ content. These changes are presumed to occur because of repeated discharges and excessive prolonged depolarization in response to kainic acid. The decreases in Na+,K+‐ATPase activity 6 and 24 h following kainic acid treatment coincide with neuropathological damage and edema formation, mainly in the hippocampus and thalamus.
Diazepam Increases γ‐Aminobutyric Acid in Human Cerebrospinal Fluid
Authors: Loscher Wolfgang , Schmidt Dieter
Published: October 2006

Abstract: : In 11 neurological patients, levels of the inhibitory neurotransmitter‐γ‐aminobutyric acid (GABA) were determined in cerebrospinal fluid (CSF) before and 1,3, 5, and 8 min after intravenous injection of diazepam (2 or 5 mg). GABA levels increased progressively after intravenous injection of 5 but not 2 mg of the benzodiazepine, the differences from preinjection values being significant at 3, 5, and 8 min. Furthermore, when relative CSF GABA alterations determined after injection of diazepam were compared to those determined in sequential CSF aliquots of 10 patients without diazepam injection, mean GABA increases after di azepam were significantly different from controls in all CSF fractions. The data suggest that, in addition to its well‐known effects on postsynaptic GABA function, diazepam may exert effects on endogenous GABA concentrations and/or on GABA release in the human CMS as reflected by elevation of GABA levels in human CSF.