摘要:

Abstract:Somatostatin (SRIF) receptors (SRIF‐Rs) are transiently expressed in a germinative lamina of the rat cerebellum, the external granule cell layer. The appearance of SRIF‐Rs coincides with the expression of SRIF‐like immunoreactivity in the cerebellum. However, the cellular location of SRIF‐Rs does not overlap with the distribution of SRIF‐like immunoreactivity, with the latter being restricted to ascending fibers arising from the brainstem, to perikarya within the white matter, and to some Purkinje cells. The characterization of SRIF‐Rs in the immature (13–day‐old) rat cerebellum was conducted by means of binding experiments in membraneenriched preparations and autoradiography, using two radioligands, [125I‐Tyr0,D‐Trp8]SRIF‐14 ([125I‐Tyr0,d‐Trp8]S14) andI25I‐SMS 204–090. The pharmacological profile of cerebellar SRIF‐Rs was compared with that of adult cortical SRIF‐Rs. Saturation studies performed in 13–day‐old rat cerebellum showed that the A'Dvalues for [125I‐Tyr0,D‐Trp8]S14 and125I‐SMS 204–090 binding were 0.35 ± 0.04 and 0.39 ± 0.01 nM, respectively. The correspondingBmaxvalues were 52.7 ± 4.8 and 49.9 ± 5.3 fmol/mg of protein, a result indicating that radioligands with high specific radioactivity (2,000 Ci/mmol) bind to a single class of high‐affinity sites (SSI). Competition studies showed that different D‐Trp‐sub‐stituted analogs displaced [125I‐Tyr0,d‐Trp8]S14 binding with Hill coefficients>1, a finding indicating the existence of different subtypes of binding sites. When [Tyr0,d‐Trp8]S14 was used as a competitor, two sites were resolved by Scatchard analysis in both 13–day‐old cerebellum and adult cerebral cortex. The higher‐affinity sites correspond to the SSI subtype identified in saturation experiments, whereas the lower‐affinity sites most likely correspond to the SS2 subtype. Ionic supplementation studies showed that divalent cations were required to obtain maximal specific binding on the SSI sites. In particular, Mn2+was the most efficient cation for promoting binding of [125I‐Tyr0,d‐Trp8]S14. Addition of GTP to the incubation buffer induced a marked reduction of specific binding. The results obtained by membrane binding assays were similar to those obtained by quantitative autoradiography, a result indicating that the microenvironment of SRIF‐Rs was preserved in both types of tissue preparations. Receptors expressed in the developing rat cerebellum exhibited the sameKDand similar pharmacological profile as those observed in the adult rat cortex. These results show that SRIF‐binding sites transiently expressed in the external granule cell layer of the cerebellum of young rats are indistinguishable from adult rat brain SRIF‐Rs. The extremely high density of SRIF‐Rs found in the external granule cell layer in 13–day‐old rats suggests that SRIF may pla

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04552.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:As assessed by HPLC with electrochemical detection, 3‐hydroxyanthranilic acid (3‐HANA) was found to be present in the rat brain and peripheral organs. The highest concentrations were measured in the kidney (86 fmol/mg of tissue) and spleen (56 fmol/mg of tissue), whereas the adrenal gland, liver, heart, and several forebrain areas (hippocampus, striatum, parietal cortex, thalamus, amygdala/pyriform cortex, and frontal cortex) contained less 3‐HANA (between 15 and 22 fmol/mg of tissue). Slightly lower concentrations of 3‐HANA were found in the brainstem and the cerebellum. The metabolic disposition of 3‐HANA was examined in tissue slices which were incubated in Krebs‐Ringer buffer at 37°C in vitro. Incubation for up to 2 h did not affect 3‐HANA concentration in brain tissue. However, inhibition of 3‐HANA degradation by the specific 3‐hydroxyanthranilic acid oxygenase blocker 4‐chloro‐3‐hydroxyanthranilic acid (4‐C1‐3‐HANA; 10 μM) resulted in a rapid (within 2.5 min) doubling of 3‐HANA levels in slices from cerebral cortex. No further increases were observed after incubations of up to 120 min. Exposure of cortical slices to 3‐HANA's putative bioprecursors, 3‐hydroxykynurenine (3‐HK) and anthranilic acid (ANA), in the absence of 4‐C1‐3‐HANA resulted in rapid, transient increases in 3‐HANA production. Maximal 3‐HANA synthesis from ANA exceeded the maximal effect of 3‐HK by approximately 11‐fold. In the presence of 4‐C1‐3‐HANA, 1 mM3‐HK and 1 mMANA produced 9.0 ± 0.3 and 89.0 ± 9.3 (5 min) or 51.6 ± 7.9 and 187.5 ±11.2 (120 min) fmol of newly synthesized 3‐HANA/mg of brain tissue, respectively. In the brain, but not in the spleen, ANA proved to be a superior 3‐HANA precursor at lower concentrations as well. Time and dose relationships for the de novo production of 3‐HANA from ANA in brain slices were established in the presence of 10 μM4‐C1‐3‐HANA. Biosynthesis of 3‐HANA from ANA in the brain may be critically involved in the function or dysfunction of 3

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04553.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:Axonal transport of peptidylglycine α‐amidating monooxygenase (PAM) activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and reached a plateau between 48 and 72 h and then decreased. The flow rate was 100 mm/day, and the molecular mass of the active entity was 70 kDa, which was determined by gel nitration. In contrast, there was no evidence for significant retrograde axonal transport. Anterograde axonal transport of immunoreactive cholecystokinin, a carboxy‐terminal‐amidated putative neuropeptide, was also found. These results suggest that PAM is transported by a rapid axonal flow and may play a role as a processing enzyme during transport or in the terminals of rat sciatic

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04554.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:Commercially available and affinity‐purified bu‐tyrylcholinesterases isolated from human serum were examined for their esterasic activity and their ability to hydrolyze various neuropeptides, including neurotensin, substance P, and leucine‐enkephalin. The three pools that displayed the lowest esterasic activities were shown to hydrolyze neurotensin with the same HPLC degradative pattern. By contrast, noticeable qualitative and quantitative discrepancies were observed when hydrolyses of substance P and leucineenkephalin by these three butyrylcholinesterase pools were studied. The pool that exhibited the highest esterasic activity appeared to be homogeneously constituted by 90‐ and 180‐kDa protein bands by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis and was totally unable to hydrolyze these three neuropeptides. This suggested that the three other butyrylcholinesterase preparations could be contaminated by exogenous peptidases. This was confirmed by means of three distinct monoclonal antibodies directed toward human serum butyrylcholinesterase. The three IgG‐purified fractions precipitated the esterasic activity, whereas they failed to precipitate the neuropeptide‐hydrolyzing activities whatever the substrate examined. Altogether, these results demonstrate that peptidases associated with butyrylcholinesterase are contaminating enzymes that cannot be considered as intrinsic activities

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04555.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:Polyclonal and monoclonal antibodies were generated against a synthetic peptide (25 amino acid residues) corresponding to the amino acid sequence surrounding the active site serine ofTorpedo californicaacetylcholinesterase (AChE). Prior to immunization, the peptide was either coupled to bovine serum albumin or encapsulated into liposomes containing lipid A as an adjuvant. To determine whether this region of AChE is located on the surface of the enzyme and thus accessible for binding to antibodies, or located in a pocket and thus not accessible to antibodies, the immunoreactivity of the antibodies was determined using enzyme‐linked immunosorbent assay (ELISA), immunoprecipitation, Western blots, and competition ELISA. The polyclonal antibody and several of the monoclonal antibodies failed to react with eitherTorpedoor fetal bovine serum AChE in their native conformations, but showed significant cross‐reactivity with the denatured enzymes. Human serum butyrylcholinesterase, which has a high degree of amino acid sequence homology with these AChEs, failed to react with the same antibodies in either native form or denatured form. Chymotrypsin also failed to react with the monoclonal antibodies in either form. Eighteen octapeptides spanning the entire sequence of this region were synthesized on polyethylene pins, and epitopes of representative monoclonal antibodies were determined by ELISA. The reactivity of peptides suggests that a portion of the 25 mer peptide in AChE containing the active site serine is the primary epitope. It is not exposed on the surface of the enzyme and is most likely sequestered in a pocket‐like conformation in the native e

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04556.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:The normal developmental profile of preprotachykinin (PPT) gene expression was determined in rat striatum from embryonic day 20 to adulthood (>45 days of age). At embryonic day 20, the amount of striatal PPT mRNA was ∼8% of adult levels, and this specific mRNA approached adult levels by postnatal days 12–15. The absolute amount of PPT mRNA, determined by comparison with PPT mRNA synthesized in vitro, ranged from 0.2 pg/μg of total RNA at embryonic day 22 to 5–6 pg/μg of total RNA in adult striata. In addition, the proportion of the various PPT mRNAs derived from the same primary transcript by alternate splicing was determined in the developing animal. At those ages at which PPT mRNA levels were significantly less than those in the adult, there was a slight (10%) but statistically significant increase in the relative amount of γ‐PPT mRNA with respect to the amount of β‐PPT mRNA. Because these mRNA species encode different combinations of tachykinin peptides, these data suggest that the proportion of substance P versus various neurokinin A‐related peptides may be developmen

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04557.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:γ‐Aminobutyric acid (GABA) concentrations in human CSF are known to increase significantly after hydrolysis; however, the source of this increase has been unknown. Using either ion‐exchange or reverse‐phase chromatography coupled with on‐line alkaline hydrolysis, we have shown 2‐pyrrolidinone, the lactam of GABA, to be present in insufficient quantity to account for this increase. Subsequent experiments involving fraction collection of column eluents followed by acid hydrolysis and rechromatography demonstrated the presence of several previously undetected GABA‐containi

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04558.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:The monoamines dopamine, norepinephrine, epinephrine, and serotonin and their major metabolites 3,4‐dihydroxyphenylacetic acid, homovanillic acid, 3‐methoxy‐4‐hydroxyphenylethylene glycol, and 5‐hydroxyindoleacetic acid were measured in the CNS of the rat during development from fetal day 18 to young adult. The catecholamines, serotonin, and their major metabolites remained low during fetal life. Concentrations measured in total brain started to increase around birth till the end of the fourth week of life after which steady‐state levels were measured. Our results suggest that although monoamine systems are already morphologically well developed during late gestational life, they probably become a significant functional system only around birth and early pos

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04559.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:The major cholinergic innervation of the rat cerebral cortex arises from the nucleus basalis in the basal fore‐brain. Introduction of the excitotoxins kainate or ibotenate into the nucleus basalis by stereotaxic injecton results in degeneration of the cholinergic cells. We have investigated the effect of this excitotoxic action on ornithine decarboxylase (ODC) activity and cholinergic responsiveness in the cerebral cortex. A massive and rapid induction of ODC activity was seen in ipsilateral cortex after injection of excitotoxin. A maximal increase in ODC activity of 268 times the control value was seen in ipsilateral cerebral cortex 8 h after lesioning. Thereafter, ODC activity declined but remained significantly greater than control levels for 32 h. Pretreatment of animals with the irreversible ODC inhibitor difluoromethylornithine prevented the induction of ODC by kainate. Tissue content of the ODC product putrescine showed a marked increase in cerebral cortex ipsilateral to the lesion, increasing sevenfold at 24 h, the maximal concentration reached. After 24 h, the level of putrescine decreased but remained significantly elevated above control values for 5 days. Levels of the poly‐amines spermidine and spermine were unaffected by lesioning. Increases in ODC activity of much smaller magnitude were also seen in brain regions not directly innervated from the ipsilateral nucleus basalis. However, the response in ipsilateral cortex was found to be dependent on an intact projection from nucleus basalis to cortex. The induction of ODC was shown to be prevented by treatment of rats with MK‐801, a result indicating the involvement ofN‐Methyl‐d‐Aspartate (NMDA) receptors. The effect of MK‐801 was dose dependent, with the maximal effect being obtained at a dose of 1 mg/kg, and also critically dependent on the time of administration, with its effectiveness being significantly reduced if given as late as 4 h after lesioning. The anticonvulsant barbiturate pentobarbital produced an effect similar to that of MK‐801. The potentiation of cortical cholinergic responsiveness (assessed by measuring carbachol‐stimulated phosphoinositide turnover in cortical slices) was also prevented by MK‐801. Thus, both the induction of ODC and the enhanced postsynaptic responsiveness appear to be mediated by the involvement of NMDA receptors, in view of the

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04560.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:Bioassay analysis of extracts of the major neurose‐cretory structures of the American lobster have revealed several different agents with stimulatory effects on the cyclic GMP metabolism of various lobster tissues. The most potent of these is a peptide extracted from the sinus gland, a neurohemal organ found in the animal's eyestalk. This molecule, called peptide G1(for its effects on cyclic GMP metabolism), can increase the cyclic GMP content of every lobster tissue tested, sometimes by as much as 200‐fold. In this article, we describe the purification and some of the chemical properties of peptide G1. Purification was accomplished by sequential anion exchange and reverse‐phase HPLC. The purified peptide is a large, extremely hydrophobic molecule. Its apparent molecular mass on a reducing sodium dodecyl sulfate‐containing gel is 6.4 kDa, and its calculated molecular mass (based on an amino acid analysis of the purified material) is 8.2 kDa. Amino acid analysis reveals a high proportion of leucine and valine residues. The amino terminus of the molecule is not susceptible to Edman degradation, but sequencing studies were successfully carried out on tryptic fragments. Based on the estimated size of the molecule, these studies provide ∼60% of the total sequence. No homologies with any previously sequenced peptide were observed, but biochemical similarities to as yet unsequenced peptides found in extracts of sinus glands from other crustaceans (hyperglycemic hormone and moult‐inhibiting hormone) ar

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04561.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY