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Biomedical subjects

Y Furuichi

Publications and source records attributed to Y Furuichi.

At least 163 records · Page 9Linked to original sources
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↗
PubMed1990

Purification of an endothelin receptor from human placenta.

We have identified an endothelin (ET) binding protein on the membranes of human placenta and purified it to homogeneity. It is a polypeptide with an apparent Mol. Wt. of 40,000 and is a major protein to be labeled by cross-linking with either 125I-ET-1, -2, or -3. Binding studies with Scatchard analysis indicated the presence of a single class, high-affinity binding site with Kds of 57 pM, 480 pM and 40 nM for 125I-labeled ET-1, ET-2 and ET-3, respectively. These results suggest that the 40K protein is a major ET receptor in placenta and, most likely, can bind differentially to ET-1, ET-2 and ET-3.

Affinity Labels↗
PubMed1990

Nutritional characteristics of a neoglycoprotein, casein modified covalently by glucose.

Glucose was combined covalently with the epsilon-amino groups of lysyl residues of bovine casein in the presence of sodium cyanoborohydride as a reducing reagent by reductive alkylation, forming stable secondary amine linkages. Solubility characteristics and nutritional values of the neoglycoprotein were examined. The degree of modification (%) of the glucosylated casein was 82.5. Solubility of the modified casein was increased by the attachment of glucose. The modification did not disturb the digestion of casein by pepsin or trypsin. Rat feeding experiments using 10% protein diets demonstrated that the protein efficiency ratio (PER) of the modified casein was 0.35 +/- 0.33 compared with 2.99 +/- 0.29 for the unmodified casein. When the modified casein was supplemented with L-lysine to equal the level of total lysine of unmodified casein, the PER value was increased to 2.21 +/- 0.29. Nitrogen balance experiments showed that the modified casein was digested completely. On the other hand, biological value and net protein utilization of the modified protein were shown to be considerably lower than those of the unmodified casein.

Journal Article↗
PubMed1989

Gene polymorphism identified by PvuII in familial lipoprotein lipase deficiency.

We previously demonstrated that the PvuII polymorphism is a useful marker to analyze the genetic defects in familial lipoprotein lipase (LPL) deficiency. In this study, we have mapped this polymorphic site and cloned the gene fragments containing this site from a patient and a normal subject. Comparative sequence analysis revealed that a C-T transition occurred in the gene of the patient at the PvuII site in the intron 6. Interestingly, the sequence near the PvuII site showed a significant homology to the consensus sequence of the 3' splice site. In addition, the insertional event into the human LPL gene, which was recently reported for a population of Caucasian patients, was not observed for eight unrelated Japanese patients, suggesting that genetic defects underlying familial LPL deficiency should be heterogeneous among races.

Base Sequence↗
PubMed1989

Biological activities of synthesized 20K and 22K hGH in Nb2 bioassay and IM-9 radioreceptor assay.

We investigated the bioactivities of the recombinant DNA-derived methionyl 20K hGH (20K-Met-hGH) and methionyl hGH (22K-Met-hGH). The growth-promoting activities in Nb2 cells of 20K-Met-hGH and 22K-Met-hGH were 10.7% and 93.5% of pituitary hGH (P-hGH), respectively. In the IM-9 lymphocyte assay, the binding activities of 20K-Met-hGH and 22K-Met-hGH to hGH receptor were 29.0% and 87.1% of P-hGH, respectively. Our data demonstrate that 20K-Met-hGH may have weaker biological potency than P-hGH.

Animals↗
PubMed1988

Molecular cloning and characterization of cytoplasmic polyhedrosis virus polyhedrin and a viable deletion mutant gene.

The double-stranded RNA genome of Bombyx mori cytoplasmic polyhedrosis virus (CPV) was converted to double-stranded DNA and cloned into plasmid pBR322. The complete nucleotide sequence of cloned genome segment 10, which encodes virus polyhedrin polypeptide, was determined. The CPV polyhedrin gene consists of 942 based pairs and possesses a long open reading frame that codes for a polypeptide of 248 amino acids (molecular weight, 28,500), consistent with an apparent molecular weight of 28,000 previously determined for purified polyhedrin. No sequence homology was found between CPV polyhedrin and polyhedrins from several nuclear polyhedrosis viruses. In addition to the polyhedrin gene, we completed the sequence analysis of a small deletion mutant gene derived from the polyhedrin gene. This mutant gene consists of two subset domains of the polyhedrin gene, i.e., the 5'-terminal 121 base pairs and the 3'-terminal 200 base pairs. An in vitro transcription demonstrated that the small mutant gene is transcribed by virion-associated RNA polymerases. These data confirm the importance of CPV terminal sequences in virus genome replication.

Amino Acid Sequence↗
PubMed1988

A rapid and efficient purification of poly(A)-mRNA by oligo(dT)30-Latex.

Latex particles were covalently linked to the 5'-proximal region of oligo(dT)30. The resultant oligo(dT)30-Latex was tested for its hybridizability to poly(A) containing mRNA. Several advantages were noted as compared to the conventional oligo(dT)30-cellulose column chromatography; (1) a highly efficient (approximately 95%) hybridization occurs in a short reaction period (10min), (2) more than 95% of poly(A) mRNA can be recovered from oligo(dT)30-Latex by a simple heating followed by brief centrifugation, (3) multiple samples can be handled simultaneously and moreover, (4) the poly(A)-mRNA on the oligo(dT)30-Latex can be directly transcribed by AMV reverse transcriptase to form the cDNA. These properties of oligo(dT)30-Latex promise an excellent reagent for nucleic acid technology.

Chromatography, Affinity↗
PubMed1987

Synthesis in Escherichia coli of the HTLV-I trans-acting protein p40x.

The pX gene of human T-cell leukemia virus type I (HTLV-I) encodes a protein that activates the expression of viral genes in trans. Plasmid constructs designed to express the pX gene under the control of either the temperature-inducible lambda PL promoter or the trp promoter were used to transform several Escherichia coli strains, including murein-lipoprotein and Ion mutant strains. Upon induction it was possible to detect the synthesis of a new polypeptide of approximately 40 kDa which reacted specifically with serum from an ATL patient.

Cloning, Molecular↗