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Biomedical subjects

C Miyamoto

Publications and source records attributed to C Miyamoto.

At least 55 records · Page 3Linked to original sources
PubMed1993

Application of oligo(dT)30-latex for rapid purification of poly(A)+ mRNA and for hybrid subtraction with the in situ reverse transcribed cDNA.

The carboxyl groups on the surface of latex beads were linked to amino moiety of cytidine residue of oligo(dC)10(dT)30. The resultant latex beads-(dC)10(dT)30 showed a very stable suspension and yet is precipitable to a small pellet by centrifugation. These properties merits the oligomer-linked beads to be applied for experiments in which poly(A)+ mRNAs are involved. An efficient (> 95%) hybridization to poly(A)+ mRNA occurred in a short reaction period (10 min), and more than 95% of bound mRNAs were recovered from the beads by heating (65 degrees C, 5 min) followed by centrifugation. Interestingly, the poly(A)+ mRNAs could be transcribed to cDNAs in situ by reverse transcriptase, with the covalently linked oligo(dT)30 as primers. These properties allowed the oligo(dT)30-latex to prepare the cDNA covalently bound to latex which was used for mRNA hybrid subtraction. In a model experiment with the mixture of vaccinia virus and HeLa mRNAs, about 200-fold enrichment of vaccinia mRNA species was obtained after four cycles of hybrid subtraction with HeLa cDNA-latex.

DNA↗
PubMed1993

Molecular characterization of the 5'-flanking region of human genomic ETA gene.

A clone containing the promoter, the exon 1 and exon 2 of human genomic ETA gene was isolated and sequenced. The transcription initiation site and TATA box were identified at 528 bp upstream of the initiation codon and 24 bp upstream of the transcription initiation site, respectively. The 534 bp fragment containing 5'-flanking sequence and most of the exon 1 of the human ETA gene showed a promoter activity corresponding to 55% of SV40 early promoter activity when placed upstream of the luciferase gene and transfected into CHO cells. Thus, the TATA box which consists of the TAAAAA sequence is functional for transcription of the human ETA gene. The coding region of the human ETA receptor, therefore, starts from exon 2 which contained the first and the second transmembrane regions. The human ETA gene was demonstrated to be a single pair of copy by Southern blot analysis, and it was localized in chromosome 4 by analysis with a panel of human-rodent somatic cell hybrid lines.

Amino Acid Sequence↗
PubMed1993

Clinical experience with interstitial thermoradiotherapy for localized implantable pelvic tumors.

Twenty-six patients (20 females, 6 males) with localized tumors of the pelvis, including 3 primary advanced (PRIM), 7 persistent (PERS), 10 recurrent (REC), and 6 metastatic (MET) tumors, were treated with a combination of low-dose rate (LDR) iridium 192 interstitial radiotherapy (IRT), interstitial 915 MHz microwave hyperthermia (IHT), and external beam radiotherapy (RT). Histological diagnoses were squamous cell carcinoma in 13 (50%), adenocarcinoma in 12 (46%) and soft tissue sarcoma in 1 (4%) lesion. Tumor sites were cervix in 8 (31%), colorectum in 6 (23%), vagina in 4 (15%), anus in 3 (12%), ovary in 2 (8%), and other sites in 3 (12%) lesions. IHT was administered immediately before iridium 192 was placed and after its removal for 45-60 minutes at 41-44 degrees C. On December 31, 1991 median follow-up was 25 months (mean: 23 months; range: 5-65 months). At 3 months follow-up (FU), complete remission (CR) occurred in 17 (65%), partial remission (PR) in 7 (27%), and no change or progressive disease (NC/PD), in 2 (8%) lesions. At 12 months FU, in 16 of 21 patients (76%) local control (LC) was achieved, with 1 (5%) patient exhibiting a slow tumor regression. After combined IRT-IHT locoregional relapse or tumor regrowth occurred in 8/26 (31%): 5 (19%) outside and 3 (12%), inside the previously treated volume; relapses occurred within 8-30 (mean: 18) months of follow-up. Factors influencing initial (3 months FU) and long-term tumor response (12 months FU) included tumor class, tumor volume, total radiation dose, and thermal parameters with "good quality of heating" (TQ 41 degrees C > or = 75%) and high minimum tumor temperature (Tmin(av) > or = 41 degrees C). Treatment toxicity was acceptable: whereas 8 (31%) patients experienced acute side effects, which subsided within weeks, 7 (27%) developed long-term complications. Thermal damage was associated with IHT treatments exceeding maximum average temperatures of > or = 44 degrees C and maximum peak temperatures of > or = 45 degrees C.

Actuarial Analysis↗
PubMed1993

Functional domains of human endothelin receptor.

The ligand binding site to the ETA receptor was investigated by substitution of each 5-amino acid sequence located in the second extracellular (B) region of the ETA receptor with the cognate sequences of the beta 2-adrenergic receptor. A 5-amino acid sequence (140-KLLAG-144) in the B-loop region was implicated as the most important element required for ligand binding. In addition, both the third and the fourth extracellular regions (C- and D-loops), including the flanking transmembrane regions, were found to play an important role in ligand selection. As for the biological significance of the intracellular regions of the ETA receptor, we have found that the C-terminal 8-amino acid residues located in close proximity to the seventh transmembrane region and the C-terminal 16-amino acid residues in the third intracellular loop are important for the binding of ET-1. Therefore, the intracellular third loop and C-terminal domains seem to contribute to the three-dimensional structure of the ligand binding site located in the extracellular domains. The same lines of experiment showed that the ETA receptor requires > 13 amino acid residues at the proximal cytoplasmic tail and 10 amino acid residues in the C-terminal region of the third intracellular loop to induce an ET-1-dependent increase in [Ca2+]i. Both regions are possibly involved in the interaction with G-protein.

Amino Acid Sequence↗
PubMed1993

Seroepidemiological survey of chlamydial infections in light horses in Japan.

To investigate the overall prevalence of chlamydial infections in light (i.e. non-draught) horses in Japan, 599 sera obtained from 12 localities in 1991 were tested for complement fixation antibodies. The mean antibody positive rates of the all sera were 15.2% (91/599) and the regional positive rates were higher in Honshu (19.1%, 48/251) and Kyushu (20.0%, 20/100) than in Hokkaido (9.3%, 23/248). In Honshu, the highest rate (56.0%, 28/50) was observed in Utsunomiya. Analysis of the positive rate in different age groups showed that the 2-5 years age-group had the highest prevalence of chlamydial infections. This indicates that chlamydial infection is prevalent in light horses in Japan.

Animals↗
PubMed1993

Identification of specific intracellular domains of the human ETA receptor required for ligand binding and signal transduction.

We have investigated the function of the C-terminal and the third intracellular domains of the ETA receptor by expressing truncated and mutated ETA receptors in COS-7 and CHO cells. All the C-terminal truncated ETA receptors were produced at a similar expression level and were detected in the cell membrane using indirect immunostaining. The sizes of the truncated ETA receptors were decreased in proportion to the molecular mass of the truncated amino acid sequence. When the ligand binding activities were determined for various truncated ETA receptors, it was found that more than eight amino acid residues at the proximal cytoplasmic tail of the ETA receptor were required for ET-1 binding. In addition, the deletion of 16 C-terminal amino acid residues from the third intracellular loop severely decreased the ligand binding activity. It seems that deletion of these cytoplasmic domains of the ETA receptor influences the three-dimensional structure of the ligand binding site located in the extracellular domains. The ETA receptor required more than 13 amino acid residues in the proximity of C-terminal cytoplasmic tail and 10 amino acid residues in the C-terminal region of the third intracellular loop to induce the ET-1 dependent increase in intracellular calcium concentration. Both regions are possibly coupled with G-protein to transmit the ET-1 signal.

Amino Acid Sequence↗
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

Malignant astrocytomas treated with iodine-125 labeled monoclonal antibody 425 against epidermal growth factor receptor: a phase II trial.

Twenty-five patients with primary presentation of malignant astrocytoma, astrocytoma with anaplastic foci, and glioblastoma multiforme were treated with surgical resection and definitive radiation therapy followed by intravenous or intra-arterial administration of Iodine-125 labeled monoclonal antibody-425, which binds specifically to human epidermal growth factor receptor. The patients presented with primary untreated disease, positive contrast enhanced computed tomography scans of the brain, and compatible clinical symptoms. In this Phase II clinical trial, the patients had surgical debulking or biopsy followed by definitively administered external beam radiation therapy and one or multiple doses (35 to 90 mCi per infusion) of radiolabeled antibody. The total cumulative doses ranged from 40 to 224 mCi. The administrations of the radiolabeled antibody were performed in most cases 4-6 weeks following completion of the primary surgery and radiation therapy. Ten patients had astrocytoma with anaplastic foci and 15 had glioblastoma multiforme. No significant life-threatening toxicities were observed during this trial. At 1 year 60% of the patients with astrocytoma with anaplastic foci or glioblastoma multiforme are alive. The median survival for both groups was 15.6 months.

Actuarial Analysis↗
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

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

Photosensitivity in atopic dermatitis: demonstration of abnormal response to UVB.

It is a well-known fact among clinicians that sunlight may exacerbate atopic dermatitis (AD), but little is known beyond that. In a preliminary study investigating this phenomenon, 19 patients with AD were selected for phototests. All of them had a normal minimal erythema dose (MED). However, 3 patients (15.7%) demonstrated abnormal cutaneous responses 24-72 h after provocation with ultraviolet light B (UVB). None of the patients had a positive response to pure ultraviolet light A (UVA) irradiation of up to 9 J/cm2. The photobiological results of this study confirm the existence of photosensitivity in AD and indicate that UVB wavelengths are responsible for it.

Adolescent↗
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

Allergic photocontact dermatitis due to suprofen. Photopatch testing and cross-reaction study.

We report 5 cases of photocontact dermatitis due to suprofen, a nonsteroidal anti-inflammatory drug introduced to the Japanese market in 1989, and available as a 1% ointment. The patients developed pruritic eczematous lesions after applying the ointment for from 2 weeks to 3 months. All 5 patients reacted positively to photopatch testing with ultraviolet A (UVA) and suprofen down to 0.1-0.01% pet., and 3 patients showed positive reactions with ultraviolet B (UVB) and suprofen down to 1.0-0.1%. Moreover, all patients showed a cross-reaction with tiaprofenic acid, which has a very similar chemical structure to suprofen. However, there was no cross-reaction between suprofen and ketoprofen. Prescribers should be aware of the existence of photocontact sensitivity due to these drugs.

Adolescent↗
PubMed1990

A new hematopoietic cell line, KMT-2, having human interleukin-3 receptors.

A novel human cell line, KMT-2, from umbilical cord blood cells was established based on the selection of cultures in the presence of recombinant human interleukin-3 (IL-3) and the sorting of cells with anti-My 10 antibody. Morphologic and cytochemical studies (peroxidase negative, Sudan-black negative, chloroacetate esterase negative, PAS positive, nonspecific esterase positive) and phenotyping (HLA-DR, My7 = CD13, My9 = CD33, My10 = CD34, MCS-2, LeuM1 positive, glycophorin A negative, and P2 negative) suggest that the KMT-2 cells are myelomonocytic cells, probably of immature progenitor origin. Besides IL-3, granulocyte-macrophage colony-stimulating factor supported the growth of the KMT-2 cells, but IL-1 alpha, IL-2, IL-4, IL-5, and erythropoietin did not. IL-6 showed only slight activity. Binding studies with 125I-labeled recombinant human (rh) IL-3 indicated that IL-3 bound to a single class of high affinity receptors (approximately 4,000 receptors/cell) on KMT-2 cells with a kd of approximately 200 pmol/L. The chemical cross-linking assay demonstrated that radiolabeled hIL-3 bound three molecules with molecular masses of 170, 130, and 70 Kd. Present data suggest that the newly established human cell line will be a valuable tool for the biologic assay of hIL-3, and a model for biochemical studies of IL-3 receptors.

Blood Cells↗
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↗