摘要:

AbstractA rat adrenal medullary recombinant clone has been isolated by cross‐hybridization with a tyrosine hydroxylase (TH) cDNA. The new clone has a mRNA size of 5.6 kb and also hybridizes to the 1.9‐kb tyrosine hydroxylase message. Southern blot analysis reveals several hybridizing bands in common between the TH cDNA and the adrenal medullary clone. These results demonstrate that the adrenal clone shares sequences in common with the TH gene and/or is closely linked to it in the genome. Hybrid‐selected mRNA translation products of the adrenal clone can be immunoprecipitated with dopamine beta hydroxylase antisera. This suggests that the adrenal medullary cDNA may code for another catecholamine pathway e

ISSN:0360-4012    

DOI:10.1002/jnr.490160103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractChronic cold stress and chemical sympathectomy are known to increase the synthesis and release of catecholamines in the adrenal medulla. Chromaffin cells adapt to altered functional requirements by increasing the synthesis of tyrosine hydroxylase (TH), the rate‐limiting enzyme in catecholamine biosynthesis. In this study, we investigated the molecular genetic mechanisms underlying these changes in enzyme activity. Estimates of TH mRNA levels were obtained by RNA dotblot analysis with a cloned TH cDNA hybridization probe. Exposure to cold produced a 4.3‐fold increase in the relative abundance of adrenomedullary TH mRNA. Increases in TH mRNA levels (90%) also were observed in the brainstem of cold‐stressed animals. The relative amount of TH synthesized in vitro in a rabbit reticulocyte cell‐free system, programmed with adrenal poly (A)+RNA, increased 4.3 times in cold‐stressed rats. Alteration in TH mRNA abundance appears to be specific, as we observed no significant difference in the levels of total RNA or poly(A)+RNA in this tissue. In addition, the relative abundance of adrenomedullary TH mRNA increased by 60% 4 days after systemic administration of the neurotoxin 6‐hydroxydopamine. This increase was transient and disappeared 2 weeks after the lesion. Changes in TH mRNA levels after cold stress or sympathectomy were eliminated by denervation of the adrenal gland. These results indicate that alterations in the relative abundance of TH mRNA mediate changes in TH activity induced by chronic stress or sympathectomy, and that these changes require an intact sympat

ISSN:0360-4012    

DOI:10.1002/jnr.490160104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractCholinesterases are serine esterases that rapidly hydrolyze the neurotransmitter acetylcholine. In humans, cholinesterases exhibit extensive polymorphism in terms of their substrate specificity, sensitivity to selective inhibitors, hydrophobicity, and cellular as well as subcellular localization. It is not yet known whether the various cholinesterase forms originate from different genes or are products of posttranscriptional and posttranslational processing. The extent to which these enzyme forms are homologous in their amino acid sequence is also not known. However, a consensus organophosphate‐binding hexapeptide sequence Phe‐Gly‐Glu‐Ser‐Ala‐Gly was found both in “true” acetylcholinesterase from the electric organ ofTorpedo[McPhee‐Quigley et al: J Biol Chem 260:12185–12189, 1985] and in “pseudocholinesterase” (butyrylcholinesterase) from human serum [Lockridge: “Cholinesterases—Fundamental and Applied Aspects.” New York: de Gruyter, pp 5‐12, 1984], suggesting that this region in the protein is conserved in all cholinesterases. Based on this common sequence, we prepared synthetic oligodeoxynucleotides and used them as labeled probes to screen a cDNA library from fetal human brain mRNA, cloned in lambda gt10 phages. A cDNA clone of 770 nucleotides in length was isolated. It contains an open reading frame terminating with the sequence Ser‐Val‐Thr‐Leu‐Phe‐Gly‐Glu‐Ser‐Ala‐Gly‐Ala‐Ala, which includes the consensus hexapeptide used for designing the DNA probe. Furthermore, the sequence of this 12‐amino acid peptide is identical to the sequence reported for the organophosphate binding site of human serum pseudocholinesterase [Lockridge: “Cholinesterases—Fundamental and Applied Aspects.” New York: de Gruyter, pp 5–12, 1984]. These findings confirm that t

ISSN:0360-4012    

DOI:10.1002/jnr.490160105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractcDNA libraries have been constructed in plasmid (pBR322) and bacteriophage (λgt10) vectors with poly (A +) RNA isolated from the nonfusing mouse muscle cell line BC3H‐1. The libraries were screened with a restriction fragment derived from a genomic clone coding for a human acetylcholine receptor γ subunit. Several clones were obtained whose cDNA inserts possessed nucleotide and deduced amino acid sequence homology with acetylcholine receptor γ subunits fromTorpedo californica, chick, calf, and human. One isolate, λBMG419, has 88 nucleotides of 5′‐untranslated sequence, an open reading frame of 1,557 nucleotides coding for the precursor to the mouse acetylcholine receptor γ subunit, and 144 nucleotides of 3′‐untranslated sequence. Alignment of the λBMG419‐deduced amino acid sequence with homologs from other species predicts a precursor peptide of 519 amino acids and a mature protein of 497 amino acids, with nonglycosylated molecular weights of 58,744 and 56,424 daltons, respectively. Comparison of the deduced amino acid sequence of the mouse γ subunit withTorpedo, chick, calf, and human sequences showed overall homologies of 54%, 67%, 90%, and 90%, respectively; however, significantly higher homologies were found in several putative functional domains. Radiolabeled λBMG419 has been used to identify homologous RNA species, one of approximately 2 kb and one of about 3.5 kb, in poly (A +) RNA prepared from BC3H‐1 cells and denervated mouse limb muscle. γ Subunit‐coding RNA species are considerably more abundant in denervated than in innervated muscle, suggesting that neural regulation of the abundance of theγ subunit is exerted through regulation

ISSN:0360-4012    

DOI:10.1002/jnr.490160106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractA detailed model for the acetylcholine binding site on the nicotinic acetylcholine receptor is proposed. It is derived from assumptions based on existing biochemical, structural, and pharmacological data, combined with molecular modeling and principles of protein evolution and architecture. Acetylcholine is proposed to fit into a pocket on one face of an antiparallel β‐pleated sheet formed by residues 128–142 on the α‐subunit. This sheet is flexible yet stable, in part because of a double cystine bridge at its end. Asp 138, Thr 133, and Gln 140 provide a ring of negative charges around the quaternary ammonium group of acetylcholine, Ile 131 and alkane segments of the other residues in the binding site provide hydrophobic interactions, and Gln 140 provides a hydrogen bond for acetylcholine's carbonyl group; Glu 129 would form part of the second anionic subsite for the bis‐quaternary ammonium compounds and curares. The model is compatible with the available evidence pertaining to the binding site and with structure‐activity relationship studies. It is precise and detailed, thereby making clear predictions, which are directly testable by affinity labeling and site‐directed mutagenesis. It should prove useful in the design of suc

ISSN:0360-4012    

DOI:10.1002/jnr.490160107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractThe dynamic state of the proenkephalin (PE) gene products during and after development of amygdaloid kindling was assayed by monitoring changes of the accumulation of PE mRNA and changes in proenkephalin‐related peptides. A parallel determination of PE mRNA and peptides from the same sample was conducted in this study.Electrical stimulation of the amygdala causes early increases in the PE mRNA content in that structure and in the hippocampus. Other areas related with the amygdaloid complex do not exhibit such an early increase, but this alteration occurs when the kindling process is fully established. Enkephalin content increases early in amygdala and hippocampus presumably owing to an increase in synthesis rate. Also, the enkephalin content of areas connected with amygdala and hippocampus such as the entorhinal cortex, the nucleus accumbens, and the frontal and occipital cortex exhibits an increase.A clear tendency towards normalization is observed after a recovery period of 2–3 months. Rekindling of the animals after this recovery period does not elicit a similar pattern of changes in the dynamic state of enkephalin system, even though the animals rekindle with just one single stimulation.The present data suggest that the enkephalinergic neurons participate in the development and spreading of kindling phenomena after amygdaloid stimulation, but they do not seem to play any role in mediating maintenance of the kindling st

ISSN:0360-4012    

DOI:10.1002/jnr.490160108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractIn situ cDNA:mRNA hybridization is a technique that has been developed for the visualization of cDNA:mRNA hybrids in individual cells. To use this technique to answer questions of regulation in heterogeneous populations of cells in the brain, it must be combined with other procedures allowing for the identification of functional subgroups of neurons. We report here a procedure by which in situ cDNA:mRNA hybridization may be combined with retrograde axonal tracing using the fluorescent tracer fast blue. Using this technique, it now becomes possible to measure mRNA regulation in functional subsets of cells defined by their axonal projections.

ISSN:0360-4012    

DOI:10.1002/jnr.490160109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractTechniques of in situ hybridization histochemistry, Northern blot hybridization, and immunocytochemistry were used to investigate the biosynthesis of glucagonlike immunoreactants (GLIs) in rat brain. Cells in the nucleus tractus solitarius of the medulla oblongata of adult rat brain hybridized to a synthetic oligonucleotide probe (GLP‐I oligomer) corresponding to nucleotide sequences in pancreatic proglucagon mRNA encoding glucagon‐like peptide I (GLP‐I), and stained with antisera specific for two antigenic determinants of pancreatic proglucagon, glucagon, and GLP‐I. These data suggest that there is de novo synthesis of proglucagon in cells of the nucleus tractus solitarius via expression of a proglucagon mRNA similar to that produced in pancreas. Previous studies have shown that cells in hypothalamus stain with GLP‐I antisera, but not with glucagon antisera. However, cells in the hypothalamus did not hybridize with the GLP‐I oligomer and may therefore produce a GLP‐I immunoreactant that is encoded by a mRNA different from the pancreatic proglucagon‐mRNA‐encoding glucagon and GLP‐I. Northern blot hybridizations with a cDNA probe encoding the entire pancreatic proglucagon sequence did not detect proglucagon/GLP‐I mRNAs in polyadenylated RNAs (Poly A RNA) from adult rat brainstem and hypothalamus, probably because of their low abundance. Poly A RNAs from fetal rat brain, however, contained two mRNAs that hybridized to the proglucagon cDNA probe. One mRNA of 1,300 bases is the same size as pancreatic proglucagon mRNA. The second mRNA of 1,500 bases may encode the GLP‐I immunoreactant detected in the hypothalamus of adult rat brain. The presence of neurons with glucagon and glucagon‐like peptides in the nucleus tractus solitarius suggests a role of these peptides in gustatory and/o

ISSN:0360-4012    

DOI:10.1002/jnr.490160110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractIn situ hybridization allows the detection and measurement of specific messenger RNAs in individual hypothalamic neurons, and has shown, among magnocellular neurons, not only which cells express the genes for oxytocin and vasopressin but also how they change with physiological stimulation. With this technique, neurons expressing a gene for luteinizing hormone releasing hormone‐like messenger RNA have been discovered in the preoptic area and diagonal bands of the rat forebrain. Seven days of estrogen treatment of ovariectomized female rats increases the LHRH‐like messenger RNA in this neuronal sys

ISSN:0360-4012    

DOI:10.1002/jnr.490160111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractIn situ hybridization histochemistry is a valuable technique for localizing specific messenger RNA (mRNA) and detecting changes in gene expression. Generally, the mRNA of interest has been detected by probes obtained from cloned DNA and labelled to high specific activity by nick translation. Such probes have a number of disadvantages which can be circumvented by the use of short synthetic oligonucleotides designed to be complementary to a known mRNA sequence. We report here that synthetic oligonucleotides complementary to part of the mRNA coding for rat arginine vasopressin (AVP) can be labelled to high specific activity with [125I], using either the primer extension method with the Klenow fragment of DNA polymerase I or the 3′‐tailing method with terminal deoxynucleotidyl transferase. Both AVP probes hybridized well to the magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei. A strong autoradiographic signal was present by 2 days, with grains largely confined to the perikaryon. These results compare favorably to those obtained with [32P]‐ or [3H]‐labelled probes. Given the ease of the 3′‐tailing method, [125I]‐labelled oligonucleotides appear to be especially useful probes for in situ hybridization h

ISSN:0360-4012    

DOI:10.1002/jnr.490160112

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY