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S Numa

Publications and source records attributed to S Numa.

At least 55 records · Page 3Linked to original sources
PubMed1987

Effects of substitution of putative transmembrane segments on nicotinic acetylcholine receptor function.

Mutants of the Torpedo nicotinic acetylcholine receptor in which each of the putative transmembrane segments of the alpha-subunit is replaced by the hydrophobic transmembrane segment of the vesicular stomatitis virus glycoprotein or of the human interleukin-2 receptor have been produced in Xenopus oocytes by cDNA manipulations. Functional analysis of these mutants shows that the hydrophobic segment M4 can be replaced by foreign transmembrane sequences without loss of channel activity. It is also suggested that the hydrophobic segments M1, M2 and M3 and the amphipathic segment MA are important for efficient expression of the acetylcholine receptor on the cell surface and that the specific amino acid sequence of segment M2 may be involved in channel activity.

Animals↗
PubMed1987

Functional properties of nicotinic acetylcholine receptor subunits expressed in various combinations.

The four kinds of subunits of the Torpedo californica nicotinic acetylcholine receptor have been produced in various combinations by injecting Xenopus oocytes with the corresponding subunit-specific mRNAs synthesized by transcription in vitro of the cloned cDNAs. Functional analysis suggests that association of the alpha-subunit with either the gamma- or the delta-subunit is a prerequisite for generating the conformation necessary for agonist binding. The acetylcholine receptor devoid of either the beta-, gamma- or delta-subunit exhibits weak channel activity.

Animals↗
PubMed1987

Functional expression of cloned cDNA encoding the alpha-subunit of adenylate cyclase-stimulating G-protein.

Cloned cDNA encoding the alpha-subunit of the adenylate cyclase-stimulating G-protein (Gs), carried by a simian virus 40 vector, has been introduced into the cyc- variant of S49 lymphoma cells by electroporation. In contrast to untransfected cys- cells, clones transformed with the cDNA exhibit an increase in intracellular cyclic AMP concentration in response to a beta-adrenergic agonist.

Adenylyl Cyclases↗
PubMed1987

Patch clamp characterization of sodium channels expressed from rat brain cDNA.

Sodium currents, INa, were recorded from Xenopus laevis oocytes which had been injected with mRNA synthesized by in vitro transcription of the rat brain sodium channel II cDNA (Noda et al. 1986 a, b). Patch pipettes were used to apply depolarizing voltage steps and to record macroscopic sodium currents of between 50 and 750 pA from cell-attached patches of the oocyte membrane. With a combination of whole-cell and patch clamp recording, the properties of the implanted sodium channels could be studied in detail. They were analyzed according to the model of Hodgkin and Huxley (1952 a) assuming three activation gates. The activation of the sodium currents is characterized by an equilibrium potential of -29 mV and an apparent gating charge of 8.7 e0. At -64 mV half of the sodium currents were inactivated. From single-channel current recordings, an elementary sodium channel conductance of 19 pS and an average open time of 0.43 ms were obtained at -32 mV membrane potential and 16 degrees C. The single-channel and activation properties of rat brain sodium channel II are therefore comparable to those found in peripheral nerve and skeletal muscle, but inactivation occurs at less negative potentials. This could be a specific property of the brain sodium channels and may underlie the maintained inward sodium currents reported in brain neurones (French and Gage 1985).

Animals↗
PubMed1987

Structure and function of sodium channel.

The primary structures of the Electrophorus electroplax sodium channel and two distinct sodium channel large polypeptides from rat brain have been deduced by cloning and sequencing the cDNAs. The sodium channel molecule contains four internal repeats with homologous amino acid sequences, which are oriented presumably in a pseudosymmetric fashion across the membrane, thus forming the channel. The mRNAs generated by transcription of the cloned cDNAs encoding the rat brain sodium channel large polypeptides, when injected into Xenopus oocytes, direct the formation of functional sodium channels. The transmembrane topology of the sodium channel molecule and the structure that may be involved in the voltage-dependent gating of the channel are discussed.

Amino Acid Sequence↗
PubMed1986

Primary structure of the beta-subunit of Torpedo californica (Na+ + K+)-ATPase deduced from the cDNA sequence.

DNA complementary to the Torpedo californica electroplax mRNA coding for the beta-subunit of (Na+ + K+)-ATPase has been cloned by screening a cDNA library with an oligodeoxyribonucleotide probe. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 305 amino acid residues (including the initiating methionine). The transmembrane topology and the potential N-glycosylation sites of this polypeptide are discussed.

Amino Acid Sequence↗
PubMed1986

Primary structure of the alpha-subunit of bovine adenylate cyclase-stimulating G-protein deduced from the cDNA sequence.

The primary structure of the alpha-subunit of the adenylate cyclase-stimulating G-protein (Gs) has been deduced from the nucleotide sequence of cloned DNA complementary to the bovine cerebral mRNA encoding the polypeptide. Comparison of the amino acid sequences of the alpha-subunits of Gs and transducin reveals that some of the highly conserved regions show sequence homology with elongation factor-Tu and ras p21 proteins and correspond to functional regions of guanine nucleotide-binding proteins.

Adenylyl Cyclases↗
PubMed1986

Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels.

Functional acetylcholine receptor (AChR) and sodium channels were expressed in the membrane of Xenopus laevis oocytes following injection with poly(A)+-mRNA extracted from denervated rat leg muscle. Whole-cell currents, activated by acetylcholine or by depolarizing voltage steps had properties comparable to those observed in rat muscle. Oocytes injected with specific mRNA, transcribed from cDNA templates and coding for the AChR of Torpedo electric organ, expressed functional AChR channels at a much higher density. Single-channel currents were recorded from the oocyte plasma membrane following removal of the follicle cell layer and the vitelline membrane from the oocyte. The follicle cell layer was removed enzymatically with collagenase. The vitelline membrane was removed either mechanically after briefly exposing the oocyte to a hypertonic solution, or by enzyme treatment with pronase. Stretch activated (s.a.) currents were observed in most recordings from cell-attached patches obtained with standard patch pipettes. S.a.-currents were evoked by negative or positive pressure (greater than or equal to 5 mbar) applied to the inside of the pipette, and were observed in both normal and mRNA injected oocytes indicating that they are endogenous to the oocyte membrane. The s.a.-channels are cation selective and their conductance is 28 pS in normal frog Ringer's solution (20 +/- 1 degree C). Their gating is voltage dependent, and their open probability increases toward more positive membrane potentials. The density of s.a.-channels is estimated to be 0.5-2 channels per micron 2 of oocyte plasma membrane. In cell-attached patches s.a.-currents are observed much less frequently when current measurement is restricted to smaller patches of 3-5 micron 2 area using thick-walled pipettes with narrow tips. In outside-out patches s.a.-currents occur much less frequently than in cell-attached or inside-out patches. AChR-channel and sodium channel currents were observed only in a minority of patches from oocytes injected with poly(A)+-mRNA from rat muscle. AChR-channel currents were seen in all patches of oocytes injected with specific mRNA coding for Torpedo AChR. In normal frog Ringer's solution (20 +/- 2 degrees C) the conductance of implanted rat muscle AChR-channels was 38 pS and that of sodium channels 20 pS. The conductance of implanted Torpedo AChR channels was 40 pS. The conductance of implanted channels was similar in cell-attached and in cell-free patches.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

A case of parietal lobe atrophy.

Bilateral parietal lobe atrophy was revealed by CT scan in a 48 year-old man, with a history of learning difficulties since childhood, encopretic up to the age of eight, and left-handedness. Nevertheless, he earned his living as a skilled potter, though unable to cope with the administration of the business.

Atrophy↗
PubMed1986

Molecular basis for the function of ionic channels.

The primary structures of nicotinic acetylcholine receptors and sodium channels from fish and mammals have been elucidated by cloning and sequencing the cDNAs. The structural basis for the operation of the acetylcholine receptor has been investigated by analysing the functional properties of mutant receptors produced by expression of the cDNAs altered by site-directed mutagenesis as well as those of hybrid receptors composed of subunits from different species. Structural features characteristic of the sodium channel are discussed in terms of its function. The evolution of the ionic channels is also discussed.

Animals↗
PubMed1985

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

DNA complementary to the rat hypothalamic mRNA coding for the corticotropin-releasing factor precursor (prepro-CRF) has been cloned by screening a cDNA library with a human genomic DNA probe. Nucleotide sequence analysis of the cloned cDNA has revealed that rat prepro-CRF consists of 187 amino acid residues including a putative signal peptide. The CRF and putative signal peptide regions are more highly conserved among rat, human and ovine prepro-CRF than is the cryptic portion.

Amino Acid Sequence↗
PubMed1985

Cloning and sequence analysis of human genomic DNA encoding gamma subunit precursor of muscle acetylcholine receptor.

Human genomic DNA encoding the gamma subunit precursor of the skeletal muscle acetylcholine receptor has been cloned by screening a gene library with a calf cDNA probe and has been subjected to nucleotide sequence analysis. Comparison of the nucleotide sequence of the cloned human genomic DNA with that of the calf cDNA has revealed that the protein-coding sequence of this gene is divided by 11 introns into 12 exons. Evidence is presented to suggest that the human muscle acetylcholine receptor gamma and delta subunit genes are juxtaposed. The primary structure of the gamma subunit precursor of the human muscle acetylcholine receptor has been deduced from the corresponding gene sequence. This polypeptide is composed of 517 amino acids including a hydrophobic prepeptide of 22 amino acids. The gamma subunit of the human muscle acetylcholine receptor, like the alpha subunit of the same receptor as well as the alpha, beta and gamma subunits of its calf counterpart, shares structural features common to all four subunits of the Torpedo electroplax receptor, such as the putative disulphide bridge corresponding to that in the alpha subunit proposed as being in close proximity to the acetylcholine binding site and the four putative, hydrophobic transmembrane segments M1-M4. Thus, the human gamma subunit molecule apparently exhibits the same transmembrane topology as proposed for the fish receptor subunits. The 12 exons seem to correspond to different structural and functional domains of the gamma subunit precursor molecule. Some exons and the protein regions encoded by them are more highly conserved between the mammalian and Torpedo sequences. The pattern of regional homology observed is consistent with the relatively high conservation of the region encompassing the putative disulphide bridge and of the region containing the putative transmembrane segments M1, M2 and M3.

Amino Acid Sequence↗
PubMed1985

Leumorphin in human brain.

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 and regional distribution in the human brain using a radioimmunoassay (RIA) for leumorphin. Reverse-phase HPLC and high performance gel permeation chromatography coupled with the RIA for leumorphin revealed that human leumorphin existed together with rimorphin (dynorphin B) in water extracts of the human brain. Leumorphin-like immunoreactivity (-LI) and dynorphin-LI distributed in parallel throughout the brain. These results indicate that leumorphin is a novel endogenous opioid peptide in man.

Animals↗
PubMed1985

Effects of adrenalectomy and dexamethasone administration on the level of prepro-corticotropin-releasing factor messenger ribonucleic acid (mRNA) in the hypothalamus and adrenocorticotropin/beta-lipotropin precursor mRNA in the pituitary in rats.

RNA blot hybridization analysis with cloned rat CRF precursor (prepro-CRF) cDNA as a probe showed that prepro-CRF mRNA existed in rat hypothalamic and extrahypothalamic brain tissue, whereas it was undetectable in the pituitary and adrenal. To study the effect of glucocorticoid on the level of prepro-CRF mRNA in the hypothalmus and that of ACTH/beta-lipotropin (beta LPH) precursor mRNA in the pituitary, effects of adrenalectomy and dexamethasone administration were studied in rats. Adrenalectomy markedly raised mRNA coding for ACTH/beta LPH precursor in the anterior pituitary, but not in the neurointermediate pituitary lobe. Hypothalamic pre-pro-CRF mRNA increased only to 152% of the control value, 7 days after adrenalectomy. The administration of dexamethasone (200 micrograms/day for 7 days) started immediately after adrenalectomy lowered the ACTH/beta LPH precursor mRNA level in the anterior pituitary to 19% of the intact control value, whereas the level of prepro-CRF mRNA in the hypothalamus decreased only to 102%. These results suggest that glucocorticoids exert their feedback effect at the level of gene expression on both hypothalamic CRF neurons and pituitary corticotropes. Although the possibility that CRF neurons insensitive to glucocorticoid in the hypothalamus might blunt the change in the prepro-CRF mRNA could not be ruled out, it is also possible that the effect of glucocorticoids on the pituitary is dominant.

Adrenalectomy↗
PubMed1985

A physiological study on acetylcholine receptor expressed in Xenopus oocytes from cloned cDNAs.

Nicotinic ACh receptor was expressed in Xenopus oocytes by injecting mRNAs produced from cloned cDNAs encoding the four subunits of ACh receptor of Torpedo californica. ACh responses recorded from oocytes 3 days after injection of the mRNAs were reversibly blocked by d-tubocurarine (1-2 microM), indicating that the newly synthesized receptor is of nicotinic type. The reversal potential of ACh response was found at around -1 - -5 mV. The reversal potential was not changed by removal of extracellular C1-, suggesting that the ionic channel of the newly expressed ACh receptor is permeable only to cations. Repetitive applications of ACh caused desensitization of the receptor. The rate of the desensitization was greater when the membrane potential was more negative. Subunit deletion studies showed that all four subunits are required for the formation of ACh receptors with normal ACh sensitivity. However, ACh receptors without delta subunit responded to ACh with low sensitivity. Studies on ACh receptor mutants with -subunits altered by site directed mutagenesis of the cDNA suggest that the anphipathic segment is involved in the channel function of the receptor as well as the four hydrophobic segments since partial deletion of amino acids in these segments essentially abolished ACh sensitivity with relatively little change in 125I-alpha-bungarotoxin binding activity.

Animals↗
PubMed1984

Primary structure of beta subunit precursor of calf muscle acetylcholine receptor deduced from cDNA sequence.

Clones harbouring cDNA sequences for the beta subunit precursor of the acetylcholine receptor from calf skeletal muscle have been isolated. Nucleotide sequence analysis of the cloned cDNA has disclosed the primary structure of this polypeptide, which consists of 505 amino acids including a hydrophobic prepeptide of 24 amino acids. The beta subunit of the calf muscle acetylcholine receptor, like the alpha and gamma subunits of the same receptor and the alpha subunit of its human counterpart, exhibits structural features common to all four subunits of the Torpedo electroplax receptor, apparently being oriented across the membrane in the same manner as proposed for the fish receptor subunits. The degree of sequence homology between the calf and Torpedo beta subunits (59%) is comparable to that between the gamma subunits (56%), but is lower than that between the alpha subunits of the two species (81%). Some regions of the beta subunit molecule, including the region corresponding to the putative acetylcholine binding area on the alpha subunit and the region encompassing the clustered putative transmembrane segments M1, M2 and M3, are relatively well conserved between the two species.

Amino Acid Sequence↗