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S Numa

Publications and source records attributed to S Numa.

At least 73 records · Page 4Linked to original sources
PubMed1984

Human leumorphin is a potent, kappa opioid receptor agonist.

The opioid activity and opioid receptor type specificity of synthetic human leumorphin were studied in vitro. Human leumorphin inhibited the contraction of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum and was similar in opioid potency to porcine leumorphin. This action of human leumorphin was antagonized less effectively by naloxone than by the opiate antagonist Mr 2266. Human leumorphin also inhibited the contraction of the rabbit vas deferens which has only the kappa-type opioid receptor. It is concluded that human leumorphin has potent opioid activity and acts as an agonist at the kappa-type opioid receptor, like porcine leumorphin and other peptides derived from preproenkephalin B.

Animals↗
PubMed1984

Leumorphin is a novel endogenous opioid peptide in man.

Porcine leumorphin, a putative opioid peptide corresponding to amino acid residues 228-256 of preproenkephalin B has been demonstrated to exist in the porcine neurointermediate pituitary. A recent study on the sequence analysis of genomic DNA of human preproenkephalin B has shown that human leumorphin differs in 3 amino acid residues from porcine leumorphin. In order to clarify whether leumorphin is an endogenous opioid peptide in man, we have studied its existence in the human brain using a radioimmunoassay and its opioid activity by a bioassay with the guinea-pig ileum myenteric plexus-longitudinal muscle preparation. High performance gel permeation chromatography and reverse-phase high performance liquid chromatography coupled with the radioimmunoassay for leumorphin have revealed that human leumorphin exists in water extracts of the human striatum. In the guinea-pig ileum assay, synthetic human leumorphin exhibited potent opioid activity, with the concentration of 3nM to give 50 per cent inhibition. These results indicate that leumorphin is a novel endogenous opioid peptide in man.

Adult↗
PubMed1984

Tissue distribution of messenger RNAs coding for opioid peptide precursors and related RNA.

All of the endogenous opioid peptides thus far identified are derived from three types of precursors, i.e. the corticotropin/beta-lipotropin precursor, preproenkephalin A and preproenkephalin B. Poly(A)-containing RNA from various bovine and porcine tissues has been subjected to blot hybridization analysis with the use of cDNA probes specific for the three opioid peptide precursors. Analysis with a corticotropin/beta-lipotropin precursor cDNA probe has revealed, in addition to the pituitary mRNA, a smaller hybridizable RNA species present in bovine extrapituitary tissues, such as the adrenal medulla, thyroid, thymus, duodenum and lung. The hypothalamus contains both these RNA species. DNA complementary to the smaller RNA species from the bovine adrenal medulla has been cloned. Analysis of the cloned cDNA, in conjunction with endonuclease S1 mapping of poly(A)-rich RNA from the adrenal medulla, has indicated that the smaller RNA species represents the 3'-terminal 712-729 nucleotides, excluding the poly(A) tail, of the pituitary corticotropin/beta-lipotropin precursor mRNA, having heterogeneous start sites. Analysis with a preproenkephalin A cDNA probe has shown the presence of hybridizable RNA in the bovine hypothalamus, duodenum and pituitary neurointermediate lobe in addition to the adrenal medulla. The hybridizable RNA species from all these tissues are indistinguishable in size. RNA hybridizable with a preproenkephalin B cDNA probe has been found in the porcine spinal cord and ileum besides the hypothalamus, and these RNA species exhibit an indistinguishable size. The results presented indicate that each opioid peptide precursor is synthesized in different tissues.

Animals↗
PubMed1984

Synthesis in vitro of precursor-type carnitine acetyltransferase with messenger RNA from Candida tropicalis.

Carnitine acetyltransferase was synthesized in vitro in the mRNA-dependent reticulocyte system with mRNA from alkane-grown or propionate-grown cells of Candida tropicalis. The protein synthesized in vitro was isolated by immunoprecipitation with antibody against peroxisomal or mitochondrial carnitine acetyltransferase and was compared with peroxisomal carnitine acetyltransferase (Mr of subunits, 64 000 and 57 000) and the mitochondrial enzyme (Mr of subunits, 64 000 and 52 000) of C. tropicalis by electrophoresis in the presence of sodium dodecyl sulfate. Nascent carnitine acetyltransferase prepared in vitro showed a hetero-oligomeric property, like the peroxisomal and mitochondrial enzymes isolated from C. tropicalis. The molecular weights of the subunits of nascent carnitine acetyltransferase were estimated to be 71 000 and 57 000, indicating the existence of the precursor form of the enzyme. By sucrose density gradient centrifugation of total mRNA, these two subunit proteins were shown to be synthesized with respective mRNAs of different sizes. The same precursor-type of carnitine acetyltransferase was obtained with the mRNAs from the alkane-grown cells and the propionate-grown cells. The results obtained suggest that a common precursor will be post-translationally modified to form the peroxisomal and mitochondrial enzymes.

Acetyltransferases↗
PubMed1983

Leumorphin is a novel endogenous opioid peptide derived from preproenkephalin B.

Using synthetic leumorphin, we obtained antisera for leumorphin and set up two radioimmunoassays (RIAs) with different specificities. Gel exclusion chromatography coupled with the two RIAs showed the existence of a considerable amount of leumorphin-like peptide in water extracts from porcine neuro-intermediate pituitaries. Reverse phase high performance liquid chromatography revealed that leumorphin-like peptide in the water extracts was indistinguishable from synthetic leumorphin. These results along with potent opioid activity of leumorphin indicate that leumorphin is a novel endogenous opioid peptide derived from preproenkephalin B.

Animals↗
PubMed1983

Complete nucleotide sequence of the human corticotropin-beta-lipotropin precursor gene.

The nucleotide sequence of an 8658-base-pair human genomic DNA segment containing the entire corticotropin-beta-lipotropin precursor gene has been determined, and some sequence features of the gene and its flanking regions have been analysed. The gene is composed of 7665 base pairs including two introns of 3708 and 2886 base pairs. Comparison of the 5'-flanking sequences of the human, bovine and mouse corticotropin-beta-lipotropin precursor genes reveals the presence of a highly conserved region, which contains sequences of 14-15 base pairs homologous with sequences located upstream of the mRNA start site of other glucocorticoid-regulated genes.

Adrenocorticotropic Hormone↗
PubMed1983

Sequence requirement for transcription in vitro of the human corticotropin/beta-lipotropin precursor gene.

Using HeLa whole cell extracts, we have demonstrated that transcription in vitro of the cloned human and bovine corticotropin/beta-lipotropin precursor genes is initiated accurately and efficiently. DNA sequences required for promoter function have been assessed by using a series of 5'-deletion mutants of a fusion gene that contains the 5'-flanking sequence and capping site of the human corticotropin/beta-lipotropin precursor gene and the structural sequence of the herpes simplex virus thymidine kinase gene. The results obtained have shown that the region between 22 base pairs and 35 base pairs upstream from the capping site is essential for the correct and efficient transcriptional initiation in vitro. Thus, the 'TATA box' present in this region seems to be the main promoter element for transcription of the human corticotropin/beta-lipotropin precursor gene in the HeLa cell-free system. We have also developed a transcription system in vitro from the corticotropin-producing mouse pituitary tumor cell line AtT-20 in culture. Deletion mapping of the fusion gene promoter has indicated that the 'TATA box' region is required for the accurate and efficient transcriptional initiation in this system as well. Characteristic of this system is that the deletion of the sequence lying between 53 base pairs and 59 base pairs upstream from the capping site increases the transcriptional efficiency. Because this effect is observed in the AtT-20 cell-free system, but hardly in the HeLa cell-free system, it seems reasonable to assume that the interaction of this upstream sequence with some factor(s) in the AtT-20 cell extract is responsible for the modulation of transcription of the human corticotropin/beta-lipotropin precursor gene.

Adrenocorticotropic Hormone↗
PubMed1983

A novel opioid peptide, leumorphin, acts as an agonist at the kappa opiate receptor.

The primary structure of the common precursor of porcine beta-neo-endorphin and dynorphin (preproenkephalin B) has shown the existence of a third leucine-enkephalin (leu-enkephalin) sequence with a C-terminal extension of 24 amino acids. This nonacosapeptide, named leumorphin, was approximately 70 times more potent than leu-enkephalin in inhibiting the contraction of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum. This action of leumorphin, like those of beta-neo-endorphin and dynorphin, was antagonized less effectively by naloxone than that of leu-enkephalin, but more effectively by Mr2266, an antagonist relatively specific for the kappa type opiate receptor. The inhibitory action of leumorphin or beta-neo-endorphin on the contraction of the guinea pig ileum muscle strip was reduced in a dose-dependent manner by pretreatment with dynorphin and vice versa. Leumorphin as well as beta-neo-endorphin and dynorphin inhibits the contraction of the rabbit vas deferens which is known to have only the kappa type opiate receptor. This action was also effectively antagonized by Mr2266. It is concluded that leumorphin has potent opioid activity and acts at the kappa receptor, like other opioid peptides derived from preproenkephalin B.

Animals↗
PubMed1983

Isolation and characterization of the mouse corticotropin-beta-lipotropin precursor gene and a related pseudogene.

Two mouse genomic DNA sequences homologous with human corticotropin-beta-lipotropin precursor gene sequences have been cloned. One of them represents the functional corticotropin-beta-lipotropin precursor gene, which exhibits a structural organization similar to those of its bovine and human counterparts. The other represents a pseudogene that corresponds to the functional mouse gene sequence encoding the carboxy-terminal 143 amino acid residues (including corticotropin and beta-lipotropin) and the 3'-untranslated region.

Animals↗
PubMed1983

Structural homology of Torpedo californica acetylcholine receptor subunits.

The nicotinic acetylcholine receptor (AChR) from the electroplax of the ray Torpedo californica is composed of five subunits present in a molar stoichiometry of alpha 2 beta gamma delta (refs 1-3) and contains both the binding site for the neurotransmitter and the cation gating unit (reviewed in refs 4-6). We have recently elucidated the complete primary structures of the alpha-, beta- and delta-subunit precursors of the T. californica AChR by cloning and sequencing cDNAs for these polypeptides. Here, we report the whole primary structure of the gamma-subunit precursor of the AChR deduced from the nucleotide sequence of the cloned cDNA. Comparison of the amino acid sequences of the four subunits reveals marked homology among them. The close resemblance among the hydrophilicity profiles and predicted secondary structures of all the subunits suggests that these polypeptides are oriented in a pseudosymmetric fashion across the membrane. Each subunit contains four putative transmembrane segments that may be involved in the ionic channel. The transmembrane topology of the subunit molecules has also been inferred.

Amino Acid Sequence↗
PubMed1983

Cloning and sequence analysis of cDNA for ovine corticotropin-releasing factor precursor.

Previously, Guillemin and Rosenberg and Saffran and Schally demonstrated the presence of hypothalamic factors that stimulated the secretion of adrenocorticotropic hormone (ACTH) by the pituitary gland. Recently, Vale et al. have isolated and sequenced an ovine hypothalamic peptide of 41 amino acids, which is believed to represent the major physiological corticotropin-releasing factor (CRF) (reviewed in refs 5, 6). Available data suggest that hypothalamic CRF enhances both the synthesis and secretion of ACTH and related peptides such as beta-endorphin and beta-lipotropin (beta-LPH) (reviewed in ref. 6), which are all derived from the common precursor, termed ACTH-beta-LPH precursor or preproopiomelanocortin (reviewed in ref. 7). Because CRF mediates the neural control of the pituitary-adrenocortical system, the characterization of its biosynthetic precursor and the gene encoding it is essential for understanding the molecular mechanism underlying the endocrine response to stress. We have now cloned DNA sequences complementary to the ovine hypothalamic mRNA encoding the CRF precursor (referred to hereafter as prepro-CRF). The nucleotide sequence of the cloned cDNA, reported here, has revealed the primary structure of prepro-CRF. The carboxyl end represents the CRF sequence preceded by the tetrapeptide, Arg-Lys-Arg-Arg, and followed by the dipeptide, Gly-Lys. Comparison of the amino acid sequence of prepro-CRF with those of the ACTH-beta-LPH precursor and the arginine vasopressin-neurophysin II precursor suggests that these precursor proteins may be evolutionarily related.

Amino Acid Sequence↗
PubMed1983

Primary structures of beta- and delta-subunit precursors of Torpedo californica acetylcholine receptor deduced from cDNA sequences.

The nicotinic acetylcholine receptor (AChR) from fish electric organ and mammalian skeletal muscle is the best characterized neurotransmitter receptor (reviewed in refs 1-3). The AChR from the electroplax of the ray Torpedo californica consists of five subunits present in a molar stoichiometry of alpha 2 beta gamma delta (refs 4-6); the apparent molecular weights of the alpha-, beta-, gamma- and delta-subunits are 40,000 (40K), 50K, 60K and 65K, respectively. Knowledge of the primary structures of these constituent polypeptides would facilitate the understanding of the molecular mechanism underlying the function of the neurotransmitter receptor. Recently, we have cloned cDNA for the alpha-subunit precursor of the T. californica AChR and have deduced the primary structure of this polypeptide from the nucleotide sequence of the cloned cDNA. Here we report the cloning and nucleotide analysis of cDNAs for the AChR beta- and delta-subunit precursors. The primary structures of the two polypeptides deduced from the cDNA sequences reveal conspicuous amino acid sequence homology among these and the alpha-subunits. The three subunits contain several highly conserved regions which may be essential for the receptor function or inter-subunit interaction.

Amino Acid Sequence↗
PubMed1983

Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene.

A human genomic DNA segment containing the gene for the corticotropin-releasing factor precursor has been isolated by screening a gene library with an ovine cDNA probe. The cloned DNA segment has been subjected to restriction endonuclease mapping and nucleotide sequence analysis. Comparison of the nucleotide sequence of the gene with that of the ovine cDNA indicates that an intron of 800 bp is inserted in the segment encoding the 5'-untranslated region of the mRNA. The segment corresponding to the protein-coding and the 3'-untranslated region of the mRNA is uninterrupted. The mRNA and amino acid sequences of the human corticotropin-releasing factor precursor have been deduced from the corresponding gene sequence. The deduced amino acid sequence of human corticotropin-releasing factor exhibits seven amino acid substitutions in comparison with the ovine counterpart.

Amino Acid Sequence↗
PubMed1983

Sequence requirement for transcription in vivo of the human preproenkephalin A gene.

The nucleotide sequence of the 5'-flanking region of the cloned human preproenkephalin A gene, extending to 949 bp upstream of the capping site, has been determined. The preproenkephalin A gene, when joined with an SV40 vector and introduced into COS monkey cells, is efficiently transcribed from its own promoter. To assess the DNA sequence required for promoter function, we have constructed a series of 5'-deletion mutants of a fusion gene that consists of the 949-bp 5'-flanking sequence and capping site of the preproenkephalin A gene and the structural sequence of the herpes simplex virus thymidine kinase gene. The deletions up to 757-172 bp upstream of the capping site exert essentially no effect on the expression of the fusion gene, whereas the deletions up to 145, 111, 81 and 67 bp upstream of the capping site result in a gradual decrease in the transcriptional efficiency. No detectable amount of the fusion gene transcript is produced with the mutants having deletions up to 67, 43 and 28 bp upstream of the capping site. These results indicate that a functional promoter of the preproenkephalin A gene lies between 67 and 171 bp upstream of the capping site. This promoter region corresponds to a highly GC-rich segment with short repeated sequences and palindromes.

Base Composition↗
PubMed1983

Comparison of the action of putative endogenous kappa-agonists, leumorphin and rimorphin in vitro.

Leumorphin and rimorphin (dynorphin B) were nearly equipotent in inhibiting the contraction of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum and the rabbit vas deferens. Leumorphin had long duration of action in vitro after wash-out, whereas the duration of action of rimorphin was relatively shorter. These action of leumorphin and rimophin were antagonized less effectively by naloxone than by Mr2266, an antagonist relatively specific for the kappa-receptor. Rimorphin as well as leumorphin inhibits the contraction of the rabbit vas deferens which contains solely kappa-receptors. It is concluded that leumorphin and rimorphin have equipotent opioid activity and act at the kappa-receptor, like other opioid peptides derived from preproenkephalin B.

Animals↗
PubMed1983

Synthesis of porcine leumorphin and some of its biological activities.

The carboxy-terminal nonacosapeptide sequence of porcine preproenkephalin B contains the sequence of Leu-enkephalin at its amino terminus. The endogenous existence of this peptide, leumorphin, has not yet been proved. Synthesis of leumorphin was carried out by a solid-phase technique and the purity and structure of the synthetic peptide were confirmed. Synthetic porcine leumorphin exhibited a dose-dependent opiate effect (ED50 4.70 X 10(-9) M) on electrically stimulated contraction of the guinea pig ileum preparation. The potency was about 100 times as high as that of Leu-enkephalin. Leumorphin was less potent than dynorphin(1-13) (ED50 0.38 X 10(-9) M) but it was more active than beta h-endorphin (ED50 18 X 10(-9) M). The opiate activity was only partially reversed by naloxone. Intracisternal injection of synthetic leumorphin caused significant analgesia in mice (ED50 7.31 nmol/mouse). The potency was lower than that of beta h-endorphin (ED50 0.60 nmol/mouse) but higher than that of dynorphin(1-13) (ED50 16.10 nmol/mouse). Intracisternally injected leumorphin did not produce such a violent behavioral effect as did dynorphin(1-13), and it exhibited a mild sedative effect. The data supports the concept that leumorphin is a new type of opioid peptide and that the synthetic preparation will be useful for further biological and immunological studies on this peptide.

Analgesia↗