Biomedical subjects

Y Furuichi

Publications and source records attributed to Y Furuichi.

At least 19 recordsLinked to original sources

PubMed1992

Identification of a domain of ETA receptor required for ligand binding.

Various chimeric ETA and ETB receptors were produced in CHO cells for the elucidation of a specific domain which influences the affinity of the receptor toward BQ-123, a selective ETA antagonist. Replacement of the first extracellular loop domain (B-loop) of the ETA receptor with the corresponding domain of the ETB receptor, reduced the inhibition by BQ-123 drastically, while the replacements of other extracellular domains of ETA did not. By contrast, the introduction of the B-loop of ETA in place of the corresponding domain of the ETB receptor endowed the ETB-based chimeric receptor with a sensitivity to BQ-123. These observations suggest that the B-loop domain of the ETA receptor is involved in ligand binding.

Amino Acid Sequence↗

PubMed1992

Truncation of N-terminal extracellular or C-terminal intracellular domains of human ETA receptor abrogated the binding activity to ET-1.

We have investigated the function of N-terminal and C-terminal domains of the human ETA receptor by expressing truncated mutants in COS-7 cells. Three kinds of ETA receptors truncated in the N-terminal extracellular or C-terminal intracellular domains were produced. Deletion of the entire extracellular N-terminal or intracellular C-terminal domain completely inactivated the ET-1 binding activity. However, the deletion of one half of the N-terminal extracellular domain of the ETA receptor, missing one of two N-linked glycosylation sites, maintained complete binding activity. Specific monoclonal antibodies detected all the truncated ETA receptors in the cell membrane fraction of transfected COS-7 cells. The size of the ETA receptor was heterogeneous due to differential glycosylation and distributed in 48K, 45K and 42K dalton bands in Western blot analysis. These results demonstrated that a part of the N-terminal domain in close proximity to the first transmembrane region is required for the ligand binding activity of the ETA receptor, and the C-terminal domain is perhaps necessary as an anchor for maintenance of the binding site.

Amino Acid Sequence↗

PubMed1992

Effects of nilvadipine on neuronal function in the ischemic cat brain.

The effect of nilvadipine, a dihydropyridine-type calcium entry blocker, on neuronal function during and following ischemia was investigated with a model of focal cerebral ischemia in cats and was compared with that of nicardipine. Drugs were given intravenously 30 minutes before occlusion of the left middle cerebral artery (MCA). Occlusion of the left MCA for 60 minutes was followed by reperfusion for 90 minutes. The amplitude of the somatosensory evoked potentials (SEPs), and the residual relative regional cortical blood flow in the left ectosylvian gyrus and the left posterior sigmoid gyrus, were higher or had a tendency toward higher values in the nilvadipine-treated (32 micrograms/kg) group than in other groups. After reperfusion, the amplitude of SEPs rapidly recovered in the nilvadipine-treated groups. When administered before MCA occlusion, nilvadipine improved neuronal function measured by SEPs both during and following the ischemic period. Thus, nilvadipine is effective against neuronal dysfunction in focal cerebral ischemia.

Animals↗

PubMed1992

A new method for constructing NotI linking and boundary libraries using a restriction trapper.

We have developed a novel method for constructing NotI linking and boundary libraries using a modified "solid-supported ligation primer" (restriction trapper). The restriction trapper could be used to purify the DNA fragments with a specific restriction enzyme cutting site(s) at their ends. The method uses a ligation and recutting reaction with double-stranded DNA ends of a hairpin-shaped oligolinker which is connected covalently to the surface of the latex beads. Selectivity is based on the specificity of the restriction enzyme for its recognition site, resulting in efficient purification. We applied this technique to the construction of high-quality NotI linking and NotI boundary libraries, which contain almost all the NotI sites of the genome and, in addition, are free of illegitimately ligated clones.

Base Sequence↗

PubMed1992

Biotin derivatives of endothelin: utilization for affinity purification of endothelin receptor.

Three different types of biotinylated endothelin 1 (ET-1) derivatives, [Cys1]-biotinylated ET-1, [Lys9]-biotinylated ET-1, and [Cys1][Lys9]-dibiotinylated ET-1, were obtained when the biotinylation reaction was carried out with sulfosuccinimidyl-6-(biotinamido)hexanoate in an aqueous solvent. The binding of [Lys9]-biotinylated ET-1 to the ET receptor was as efficient as that of natural ET-1, whereas the binding of either [Cys1]-biotinylated ET-1 or [Cys1][Lys9]-dibiotinylated ET-1 was significantly reduced. When ET-1 was reacted with succinimidyl-6-(biotinamido)hexanoate in an organic solvent, ET-1 was exclusively modified at lysine 9. The ET receptor was then isolated from human placenta by affinity chromatography with [Lys9]-biotinylated ET-1 and avidin-agarose. The purified ET receptor was active in ET binding and was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two polypeptides with apparent molecular masses of 45 and 35 kDa. The NH2-terminal amino acid sequence indicated that the two polypeptides were from an identical subtype of the ET receptor (ETB, the ligand-nonselective type). A signal peptide from Met1 to Gly26 was missing from the 45-kDa ETB, whereas 64 amino acids at the NH2 terminus were missing from the 35-kDa ETB due to proteolytic cleavage which occurred between Arg64 and Ser65. Indeed, incubation of purified ETB with endopeptidase Arg-C resulted in degradation of the 45-kDa ETB, giving rise to the 35-kDa species by a specific cleavage at Arg64. The 35-kDa ETB was active in binding to ET-1, indicating that the NH2-terminal 64-amino-acid residues are not essential for ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Different stability of ligand-receptor complex formed with two endothelin receptor species, ETA and ETB.

There are at least two types of endothelin receptors, ETA and ETB, present in various tissues. We found that although biotinylated ET-1 could bind to both ETA and ETB receptors, the stability of the formed ligand-receptor complexes was different. When the preformed complexes of receptor (solubilized from canine brain and lung membranes) and biotinylated ET-1 were subjected to avidin agarose column chromatography, most of the ETA activity was recovered in the pass-through fraction and the remainder was recovered in the 0.5 M KSCN eluate as ligand-free forms. On the other hand, the ETB activity bound firmly to the avidin agarose column was eluted with 1.5 M KSCN. The detection of the complex of 125I-ET-1 and its receptor by SDS-PAGE run at a low temperature was only possible with the ETB fractions and the complex of 125I-ET-1 and ETA was unstable during the separation. These results suggest that the conformation of the ligand binding sites of canine ETA and ETB as well as the stability of their ligand-receptor complexes to SDS are significantly different. Similar observations were also obtained for human ETA and ETB receptors.

Animals↗

PubMed1992

Characterization of endothelin receptors ETA and ETB expressed in COS cells.

The cloned cDNA genes for endothelin receptors ETA and ETB were expressed in COS cells, and the binding characteristics of the two receptors with three isopeptide ligands (ET-1, ET-2, and ET-3) were examined in detail. The results indicated that the stability of receptor-ET-1 complexes formed with ETA and ETB were significantly different from each other, while their affinities to ET-1 were similar. The preformed ETA-ET-1 complex readily dissociated upon SDS-PAGE, as did many of the other receptors so far studied, while the ETB-ET-1 complex survived SDS-PAGE when it was run at low temperature (approximately 4 degrees C). Clear differences in stability were also shown in comparative studies of acid treatment of the two types of complexes. Only the ETB-ET-1 complex was resistant to acid treatment (0.2 M acetic acid, 0.5 M NaCl), and its 50 kDa monomeric receptor-ligand complex remained intact. The ETB-ET-1 complex (50 kDa) formed at 4 degrees C on the surface of COS cells, however, was susceptible to limited proteolysis at 37 degrees C that reduced the molecular size of the complex to a distinct 35 kDa. No such size reduction was observed with the preformed ETA-ET-1 complex. The overall structure of two endothelin receptors, as deduced from the sequence of cloned cDNAs, is similar in many respects. However, the present findings demonstrate distinct differences in the biochemical nature of the two receptors, which suggest their distinct biological functions.

Base Sequence↗

PubMed1992

Patterns of de novo plaque formation in the human dentition.

The objective of the present clinical trial was to carefully assess the pattern of de novo plaque formation in the human dentition. 10 subjects aged 24-29 years were recruited for the study. None of the participants showed signs of destructive periodontitis. At the start of the preparatory period, they were given a thorough dental prophylaxis and oral hygiene instruction. For the next 2 weeks, the participants were carefully monitored and 1 x every 2 days examined with respect to plaque and gingivitis. At the re-examinations, they were also given professional tooth cleaning and instruction in proper plaque control measures. Towards the end of this 2-week period, the Day 0 examination was performed which revealed that the gingival tissues of all participants were in excellent clinical health. Following the Day 0 examination, all 10 subjects were exposed to professional tooth cleaning. During the next 14 days, they abolished all mechanical tooth cleaning efforts but were examined with respect to plaque on Days 1, 4, 7 and 14. The amount of plaque formed was examined using the criteria of the Plaque Index system (PlI). Each of 6 surfaces of each tooth in the dentition was given a score from 0 to 3. The results from the re-examinations demonstrated that in humans with clean teeth and normal gingiva, the abolishment of mechanical tooth cleaning rapidly resulted in de novo plaque formation. Most plaque, as assessed by the plaque index system, formed during the first 4 days of no tooth cleaning after which moderate additional amounts of plaque formed. It was observed that the mean PlI values for individuals, for groups of teeth and tooth surfaces, provide a proper overall estimation of plaque build up. The dynamics of plaque formation between examinations and in different parts of the dentition were more easily disclosed by the data describing % distributions of different score categories and the transition between scores from one examination to the next. The total amount of plaque formed on various tooth surfaces was best presented by so called "plaque pattern displays". The results also revealed that (i) the mandibular dentition harbored more plaque than the maxillary dentition, (ii) there was a difference in the mean PlI scores between the molar and the anterior tooth regions in the maxilla, but in the mandible such a difference could not be observed (except on Day 1), (iii) plaque accumulated most at the approximal surfaces and least at the palatal surfaces, and that (iv) differences in PlI scores between groups of teeth and tooth surfaces observed on Day 4 persisted through the Day 14 examination.

Adult↗

PubMed1992

A model for studying the effects of mouthrinses on de novo plaque formation.

The aim of the present study was to describe a 4-day no oral hygiene model to assess the pattern of de novo plaque formation and to use this model to appraise the potential of some mouthwash preparations to retard or inhibit plaque formation in the human dentition. 10 subjects were recruited for the trial. During a preparatory period, the participants were exposed to repeated professional plaque control and given oral hygiene instruction to eliminate signs of gingivitis. At the end of the preparatory period, each participant received a final professional tooth cleaning and was subsequently told to abstain from mechanical plaque control efforts for the next 4 days. They were asked to rinse twice daily for 60 s with 10 ml varying test solutions. On Day 4, the volunteers were exposed to a new clinical examination and the presence and amount of plaque were examined by the use of the plaque index system (P1I). The participants were subsequently given a professional tooth cleaning and asked to exercise proper self performed plaque control during the next 10 days. A new test period was then initiated. 6 different mouthwash preparations were tested in each subject namely, (1) placebo (a negative control rinse), (2) Veadent mouthrinse, (3) Listerine mouthrinse, (4) 0.06% triclosan + polyvinyl phosphonic acid (PVPA), (5) 0.06% triclosan + phenolic flavor and (6) 0.12% chlorhexidine digluconate (a positive control rinse). The results from the study revealed that the mean P1I values for individuals, groups of teeth and tooth surfaces provide an adequate but gross overall estimation of the potential of a given mouthrinse to retard/inhibit plaque build up. More detailed information on the effects of the test rinses could be obtained by data describing the % distribution of different P1I score categories; a high frequency of score 0 describes the potential of a mouthrinse to maintain tooth surfaces free from plaque while a low frequency of score 2/3 describes the ability of a treatment to retard/prevent gross plaque formation. The plaque pattern displays finally allowed assessment of the magnitude of plaque prevention, in comparison to the positive and negative controls, that could be achieved by a given compound in various parts and surfaces of the dentition. In this model, all test rinses (i) were significantly more effective than the placebo rinse in retarding de novo plaque build up and (ii) had a minor effects on plaque build up in the maxillary molars and at the approximal surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

PubMed1992

The effect of nilvadipine, a dihydropyridine type calcium channel blocker, on local cerebral blood flow in rats.

The effect of nilvadipine, a dihydropyridine type calcium channel blocker, on local cerebral blood flow (lCBF) was investigated using the autoradiographic iodo[14C]antipyrine technique in rats. In control rats, lCBF in cortical areas and in the superior colliculus and inferior colliculus was higher than that in the hippocampus, septal nucleus, globus pallidus and substantia nigra. Nilvadipine (32 micrograms/kg, i.v.) increased lCBF in all structures (significantly in 11 of 21 structures) in spite of a 33% reduction in mean arterial blood pressure. These results confirm that nilvadipine has the ability to increase cerebral blood flow.

Animals↗

PubMed1992

DNA polymerase alpha, beta, and gamma activities in human lymphocytes stimulated by Tora-mame (Phaseolus vulgaris) lectin.

We measured the levels of the DNA polymerases alpha, beta, and gamma in human peripheral lymphocyte cells stimulated with Tora-mame lectin (TM-lectin) and the induction patterns were compared to those with other plant lectins, i.e., phytohemagglutinin (PHA) and pokeweed mitogen (PWM). The maximum activity of DNA polymerase alpha in lymphocytes was achieved at the concentration of 10 micrograms/ml with TM lectin and the dose response curve of TM lectin showed a sharp peak in contrast to that of PWM. During prolonged stimulation for 10 days, the time course of DNA polymerase alpha induction was different among these three lectins. A peak of alpha-enzyme was correlated with maximal incorporation of [3H]thymidine and was observed on the fourth day with TM lectin, on the third day with PHA, and sixth day with PWM. DNA polymerase beta in lymphocytes was also activated by the addition of these proteins. Two different peaks were observed during a 10-day period with every lectin, and TM lectin was most potent stimulator among them. The activity of DNA polymerase gamma in lymphocytes was at a very low but detectable level which increased slightly in response to TM lectin treatment. Although some variability of gamma-enzyme activity was observed after the seventh day, the pattern in the course of 7 days was similar among the lectins.

Cells, Cultured↗

PubMed1991

Occurrence of multiple aberrantly spliced mRNAs upon a donor splice site mutation that causes familial lipoprotein lipase deficiency.

A donor splice site mutation was found in the lipoprotein lipase (LPL) gene of a patient with familial LPL deficiency. The mutation, a G----A substitution, occurred at the first nucleotide of intron 2. Northern blot analysis of total RNA from the patient showed strikingly low levels of LPL-specific mRNAs. Using the polymerase chain reaction, the LPL mRNA splicing was analyzed in detail. The results demonstrated that no normal splicing occurred at the authentic splice site; rather a cryptic splice site 18 bases upstream from the mutation site was preferentially utilized. Although the resulting alteration in mRNA was a minute in-frame 18-base deletion, the amount of the abnormal transcript was only 1/12 that of the normal. In addition to this major cryptic splice site, we also identified multiple minor sites which were utilized at extremely lower efficiencies. Unexpectedly, one of these minor sites was also used as an alternative splice site in the normal subject at a comparably low efficiency. The sequences of these minor cryptic sites possessed many of the characteristics common to those of other normal splice sites, indicating that even such minor sites should have also been selected according to the general rules for splice site selection. These results demonstrate that upon mutation, a broad spectrum of cryptic splice sites is activated in vivo at the sites' respective efficiencies.

Adult↗

PubMed1991

Molecular cloning and expression of the cDNA coding for a new member of the S100 protein family from porcine cardiac muscle.

We isolated a new calcium-binding protein from porcine cardiac muscle by calcium-dependent hydrophobic and dye-affinity chromatography. It showed an apparent molecular weight of 11,000 on SDS-PAGE. Amino acid sequence determination revealed that the protein contained two calcium-binding domains of the EF-hand motif. The cDNA gene coding for this protein was cloned from the porcine lung cDNA library. Sequence analysis of the cloned cDNA showed that the protein was composed of 99 amino acid residues and its molecular weight was estimated to be 11,179. Immunological and functional characterization showed that the recombinant S100C protein expressed in Escherichia coli was identical to the natural protein. Homologies to calpactin light chain, S100 alpha and beta protein were 41.1%, 40.9% and 37.5%, respectively. The protein was expressed at high levels in lung and kidney, and low levels in liver and brain. The tissue distribution was apparently different from those of the other S100 protein family. These results indicate that this protein represents a new member of the S100 protein family, and thus we refer to it as S100C protein.

Amino Acid Sequence↗

PubMed1991

Cloning and characterization of cDNA encoding human A-type endothelin receptor.

A cDNA coding for the human A-type endothelin receptor (ETA) was cloned from a human placenta cDNA library. The cDNA contained the entire coding sequence for the 427 amino acid protein with a relative Mr of 48,722. The deduced amino acid sequence of the human ETA was, respectively, 94% and 93% homologous with the sequence of bovine ETA and rat ETA, but was only 64% homologous with that of the human ETB receptor. Upon expression in COS-1 cells, the human ETA receptor showed binding activity to ETA, with the highest selectivity to ET-1. Northern blot analysis showed that the mRNA of human placenta ETA consists of one species 5 kilo-nucleotides in length, and the same analysis for the uterus, testis, heart and adrenal gland of Cynomolgus monkey showed that the cognate mRNAs are widely distributed.

Amino Acid Sequence↗

PubMed1991

Cadmium directly acts on endothelin receptor and inhibits endothelin binding activity.

The binding of endothelin (ET) to human placenta ET receptor was strongly inhibited by cadmium ions (Cd2+) (IC50 = 2 x 10(-5) M). Experiments with affinity cross-linking showed that the major 40 kDa receptor was inhibited to form a [125I]ET-1/receptor complex. The mode of inhibition was noncompetitive with respect to ET-1. The inhibitory effect of Cd2+ on solubilized ET receptor was partially reversed by the chelating agent, ethylenediaminetetraacetic acid (EDTA), whereas the effect was irreversible for the membrane-associated receptor. The rat aorta contractions by ET were prevented by pretreatment or addition of Cd2+.

Animals↗

PubMed1991

Detection of an endothelin-1-binding protein complex by low temperature SDS-PAGE.

We found that the complex of ET-1 and its binding protein was stable enough to be separated by SDS-PAGE when electrophoresis was run at a low temperature. Cross-linking was not necessary for the detection of [125I]-ET-1 and its binding protein complex by autoradiography. This simple method could be used in qualitative (estimation of apparent molecular weight of ET-1 binding protein) and quantitative (determination of relative content of ET-binding protein) analysis of the ET-binding protein complex. ET-binding protein complexes of various animal species and organs were investigated by this method.

Animals↗

PubMed1991

Heterogeneity of endothelin receptor.

The complex formed between endothelin (ET) and its binding protein was adequately stable to be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) at low temperature. Cross-linking was not necessary. This simple method was applied for both qualitative (determination of molecular weight of ET binding protein) and quantitative (determination of content of ET binding protein) analyses of ET-binding protein for various organs from different mammals. Molecular weights of the major ET binding proteins were around 35 and 55 kDa in several canine organs examined. The distribution of these protein species among organs was quite different. In the case of human placenta, the predominant one showed a molecular weight of 35 kDa. We obtained several monoclonal antibodies that could immunoprecipitate ET binding activity from the solubilized human placenta membrane fraction. One of the monoclonal antibodies recognized approximately 58-kDa protein, as determined by Western blot analysis, when the gel is run in the absence of beta-mercaptoethanol.

Animals↗

PubMed1991

Heterogeneous mutations in the human lipoprotein lipase gene in patients with familial lipoprotein lipase deficiency.

The DNA sequences were determined for the lipoprotein lipase (LPL) gene from five unrelated Japanese patients with familial LPL deficiency. The results demonstrated that all five patients are homozygotes for distinct point mutations dispersed throughout the LPL gene. Patient 1 has a G-to-A transition at the first nucleotide of intron 2, which abolishes normal splicing. Patient 2 has a nonsense mutation in exon 3 (Tyr61----Stop) and patient 3 in exon 8 (Trp382----Stop). The latter mutation emphasizes the importance of the carboxy-terminal portion of the enzyme in the expression of LPL activity. Missense mutations were identified in patient 4 (Asp204----Glu) and patient 5 (Arg243----His) in the strictly conserved amino acids. Expression study of both mutant genes in COS-1 cells produced inactive enzymes, establishing the functional significance of the two mis-sense mutations. In these patients, postheparin plasma LPL mass was either virtually absent (patients 1 and 2) or significantly decreased (patients 3-5). To detect these mutations more easily, we developed a rapid diagnostic test for each mutation. We also determined the DNA haplotypes for patients and confirmed the occurrence of multiple mutations on the chromosomes with an identical haplotype. These results demonstrate that familial LPL deficiency is a heterogeneous genetic disease caused by a wide variety of gene mutations.

Adolescent↗