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Biomedical subjects

W Sato

Publications and source records attributed to W Sato.

At least 91 records · Page 5Linked to original sources
PubMed1990

Practical utility of an antibody specific to a synthetic peptide corresponding to the N-terminal amino acid sequence of human urine DNAse I for genetic analysis of human DNase I isozymes.

An antibody specific to a synthetic peptide corresponding to the N-terminal 27 amino acid residues of human urine DNase I (anti-DNase I peptide) was obtained. The antibody did not inhibit the activity of the enzyme, but reacted well with the enzyme upon immunoblotting following electrophoresis. The urine DNase I isozyme patterns detected using this antibody were almost identical to those produced with an antibody specific to purified DNase I. Therefore, the anti-DNase I peptide antibody should prove to be valuable for genetic analysis of human DNase I isozymes.

Amino Acid Sequence↗
PubMed1990

Human serum deoxyribonuclease I (DNase I) polymorphism: pattern similarities among isozymes from serum, urine, kidney, liver, and pancreas.

We have devised a zymogram method with high sensitivity and resolution for investigating molecular heterogeneity and genetic polymorphism of deoxyribonuclease I. A combination technique of polyacrylamide-gel isoelectric-focusing electrophoresis and the newly developed zymogram method have led to the discovery of genetic polymorphism of human serum DNase I. Family studies showed that the three common phenotypes--DNASE1 1, DNASE1 1-2, and DNASE1 2--and the other five relatively rare phenotypes--DNASE1 1-3, DNASE1 2-3, DNASE1 2-4, and DNASE1 3-4--represent homozygosity or heterozygosity for four autosomal codominant alleles, DNASE1 *1, DNASE1 *2, DNASE1 *3, and DNASE1 *4. The frequencies of DNASE1 *1, DNASE1 *2, DNASE1 *3, and DNASE1 *4 calculated in a Japanese population were .5517, .4358, .0104, and .0021, respectively. Moreover, it was found that urine and extracts of kidney, liver, and pancreas, as well as serum, can be used for DNase I phenotyping.

Deoxyribonuclease I↗
PubMed1989

Specific amplification of deleted mitochondrial DNA from a myopathic patient and analysis of deleted region with S1 nuclease.

Heteroplasmy of the normal-sized and the deleted mitochondrial genome has been observed in mitochondrial myopathy. The deleted region of the genome in the skeletal muscle of a patient was analyzed both by the conventional Southern blot method and by the novel method of employing the combination of polymerase chain reaction and S1 nuclease digestion. The results obtained by these methods were compared. Southern hybridization using various mitochondrial DNA fragments localized the deletion from at least position 9020 to 14,955, but regions of uncertainty of 1 kb remained on both ends of the deletion. Using the polymerase chain reaction, a fragment from the deleted genome was specifically amplified by choosing a pair of primers surrounding the deletion, and two fragments adjacent to the starting and end of the deletion were amplified from the normal-sized genome. S1 nuclease analysis of the heteroduplexes formed among these fragments demonstrated that the deletion extended from positions 8650 +/- 50 to 15,660 +/- 60. This method does not require radioisotopes and, moreover, can determine the deleted region within 5 h, in contrast to the 2 days required by the conventional Southern blot analysis. These results indicate that the novel method is faster and more accurate than the conventional method for the determination of the deleted region of genome.

Adult↗
PubMed1989

Direct sequencing of deleted mitochondrial DNA in myopathic patients.

To investigate the mechanism of mitochondrial DNA deletion in human diseases, we amplified the deleted mitochondrial DNA of five patients with mitochondrial myopathy by using the polymerase chain reaction, and directly sequenced the crossover regions of the deleted mitochondrial DNA without cloning. In Patient 1, a 7-bp directly repeated sequence of 5'-ATCCCCA-3' was found at the boundaries of deleted segment spanning 7,039 bp between the ATPase 6 and the cytochrome b genes. In Patients 2, 3, and 4, a 13-bp sequence of 5'-ACCTCCCTCACCA-3' was found in the boundaries of deleted segment spanning 4,977 bp between the ATPase 8 and the ND5 genes. In Patient 5, a 3-bp sequence of 5'-CCT-3' was found in the boundaries of deleted segment spanning 3,717 bp between the ATPase 6 and the ND5 genes. Similar directly repeated sequences may contribute to mitochondrial DNA deletions in human degenerative diseases.

Adolescent↗
PubMed1989

Multiple populations of deleted mitochondrial DNA detected by a novel gene amplification method.

A gene amplification method for detecting small populations of deleted mitochondrial DNA was used in analysis of skeletal muscle from a patient with ocular myopathy. Multiple populations of differently deleted mtDNA were detected in the patient muscle. The presence of deleted mtDNAs was further confirmed by comparison of the shift in the sizes of the amplified fragments with the shift in the positions of the primers used for the amplification, (the primer shift PCR method). Other methods, namely Southern blotting, enzymic activity measurement, and Western blotting, were inefficient at detecting the mitochondrial abnormality. These findings suggest that the primer shift PCR method could be valuable for accurate diagnosis of ocular myopathy associated with mtDNA deletion.

Blotting, Western↗
PubMed1989

Catecholamines increase nerve growth factor mRNA content in both mouse astroglial cells and fibroblast cells.

Previous studies have shown that catecholamines increase the nerve growth factor (NGF) content in medium conditioned by mouse L-M fibroblast cells and mouse astroglial cells. In this study, the NGF mRNA levels in these cells were measured by Northern blot analysis. In astroglial cells treated with epinephrine (EN), the cellular NGF mRNA level increased prior to accumulation of NGF in the culture medium. 3-Hydroxytyramine (DA) and norepinephrine (NE) also increased the cellular NGF mRNA content. An increased level of NGF mRNA elicited by EN was also observed in mouse L-M cells. These results indicate that catecholamines enhance NGF synthesis of L-M fibroblast cells and astroglial cells by increasing the cellular content of NGF mRNA. The present results also indicate that the effects of catecholamines are not mediated by adrenergic receptors.

Albuterol↗
PubMed1988

Maternal inheritance of deleted mitochondrial DNA in a family with mitochondrial myopathy.

Skeletal muscles from a mother and her daughter both with chronic progressive ophthalmoplegia were analyzed. Histological and biochemical analyses of their muscle samples showed typical features of this type of mitochondrial myopathy. Southern blot analysis revealed that, in both patients, there were two species of mitochondrial DNA (mtDNA): normal one and partially deleted one. The sizes of the deletion were different; the mutant mtDNAs from the mother and the daughter had about 2.5- and 5-kilobase deletions, respectively. The two mutant mtDNAs shared a common deleted region of 1.2-kilobase. However, both the start and the end of deletion were different between them, implying a novel mode of inheritance. This is the first report that the mutant mtDNA is responsible for the maternal inheritance of a human disease.

Adolescent↗
PubMed1988

Purification and characterization of a human urine ribonuclease (RNAase 1) showing genetic polymorphism.

A ribonuclease (RNAase) was isolated and purified from the urine of a 45-year-old man by column chromatographies on DEAE-Sepharose CL-6B, cellulose phosphate and CM-cellulose followed by gel filtrations on Bio-Gel P-100 and Sephadex G-75, and finally to a homogeneous state by SDS-polyacrylamide gel electrophoresis. The enzyme was designated RNAase 1. It was possible to detect RNAase 1 isozymes in urine and serum without difficulty using isoelectric focusing electrophoresis followed by immunoblotting with a rabbit antibody specific to RNAase 1. The existence of genetic polymorphism of RNAase 1 was detected in human serum utilizing this technique (Yasuda, T. et al. (1988) Am. J. Hum. Genet., in press). RNAase 1 in serum and urine seemed to exist in multiple forms with regard to molecular weight and pI value. Genetically polymorphic RNAase 1 was a glycoprotein, containing three mannose, one fucose, four glucosamine and no sialic acid residues per molecule, with a molecular weight of 16,000 and 17,500 determined by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively. The enzyme was most active at pH 7.0 on yeast RNA substrate and inhibited remarkably by Cu2+, Hg2+ and Zn2+. It also showed definite substrate preference for poly(C) and poly(U), but much less activity against poly(A) and poly(G). Thus, the enzyme is a pyrimidine-specific RNAase.

Amino Acids↗
PubMed1988

Genetic polymorphism of human serum ribonuclease I (RNase I).

One of the human urinary ribonucleases (RNases) was isolated and purified to homogeneity (SDS-PAGE) by means of a series of column chromatographies. The enzyme, designated RNase 1, is a glycoprotein with a molecular weight of approximately 16,000. Rabbit antibody to the purified RNase 1 reacted with human urine and sera, as well as with the purified RNase 1. The genetic polymorphism of serum RNase 1 was studied by polyacrylamide gel isoelectric focusing (IEF-PAGE) in a pH range of 5-8, followed by immunoblotting with antisera specific for RNase 1. Two common phenotypes, RNASE1 1 and RNASE1 1-2, were easily recognized. The homogeneous phenotype, RNASE1 1, consisted of four major bands with different pI values, and the heterogeneous phenotype, RNASE1 1-2, was presumed to represent a mixture of each of the homogeneous phenotypes 1 and 2; however, the other homogeneous phenotype, RNASE1 2, was not detected in our samples. Family studies are in agreement with an autosomal codominant transmission of the two alleles. Population studies indicate that the frequencies of the RNASE 1 and RNASE1 2 alleles are .988 and .012, respectively.

Adult↗
PubMed1987

New detection method for uropepsinogen (PGA) using isoelectric focusing and immunoblotting techniques.

Uropepsinogen (PGA) was isolated and purified from human urine using a column chromatography series. The purified PGA was injected into a rabbit and a PGA-specific antibody was obtained. PGA isozymogen in human urine could be detected reproducibly by immunoblotting using this antibody after isoelectric focusing electrophoresis (IEF) on polyacrylamide gels. This technique may prove to be useful in the genetic study of PGA polymorphism.

Child↗
PubMed1985

Identification and partial characterization of the cytoplasmic androgen receptor in bovine ovarian capsule.

[3H]Dihydrotestosterone (DHT) binding to a specific protein in the cytosol of bovine ovarian capsule was studied in vitro. The specific androgen-binding protein in the cytosol was analyzed by chromatographic and ultracentrifugal techniques. From Scatchard analysis, the dissociation constant was 7.4 nM and the number of binding sites was 58.8 fmol/mg protein. Testosterone and 17 alpha-methyltrienolone (R1881) compete for [3H]DHT binding. In the presence of sodium molybdate and at low salt concentrations, the steroid-protein complex sediments as a 9S form, while in the presence of high salt, it sediments at 3.5S. In the absence of molybdate or in the presence of high salt, the 9S form dissociates in a temperature-dependent manner into smaller units. These properties are consistent with the presence of a typical androgen receptor in the bovine ovarian capsule.

Animals↗
PubMed1985

Characterization of androgen-binding protein in the human ovarian capsule and its changes by polycystic ovarian disease.

In sucrose density gradient analysis, the cytosol of human ovarian capsules was found to contain two (3H)dihydrotestosterone (DHT)-binding macromolecules with sedimentation constants of 9S and 4.6S, respectively. In the tissue, however, more than 95% of the total (3H)DHT binding to the cytosol was found to be due to the 4.6S macromolecule. This molecule was further characterized and identified as a tissue-specific androgen-binding protein (ABP). The dissociation constant and number of binding sites of ABP were 2.60 nM and 231 fmol/mg protein, respectively. ABP was further characterized by affinity chromatography on concanavalin A-Sepharose and was found to separate into two parts: one showed no interaction with the resin and was eluted in the void volume (peak I) and the other bound to the resin and was eluted by alpha-methyl-D-glucoside (peak II). The apparent amount of cytoplasmic ABP in capsules with polycystic ovarian disease was significantly lower than normal. This decrease in ABP was mainly due to the characteristic disappearance of peak II.

Adult↗
PubMed1985

Rapid purification of nucleosome assembly protein (AP-I) and production of monoclonal antibodies against it.

A nucleosome assembly protein (AP-I) was purified approximately 50% from the cytosol of HeLa S3 cells by three purification steps. Using this protein fraction as an antigen, we established three stable hybridomas that secrete monoclonal antibodies specific for AP-I by the conventional method of cell fusion. Immunoblotting of the HeLa S3 cytosol, proved AP-I exists as a 58-kDa peptide in vivo, not as the 53-kDa peptide previously identified as active in nucleosome assembly (Ishimi, Y., et al., Eur. J. Biochem., 142, 431-439, 1984). An immunocytochemical study using the monoclonal antibody with the highest specificity against AP-I pin pointed the intranuclear localization of AP-I in HeLa S3 cells.

Animals↗
PubMed1985

Diagnosis of submucosal tumors by injecting a water soluble contrast medium: diagnosis of extra-gastric tumors and gastric varices.

Extra-gastric compression caused tumescent lesions that are difficult to differentiate from gastric submucosal tumors, and gastric varices similar in appearance to circumscribed tumors were sometimes experienced clinically. Up to now, the differential diagnosis of these lesions has been done by the palpation through x-ray examination or the tactile test under endoscopic examination. Even by the recent use of the CT scan, the differential diagnosis still remains unsatisfactory except in a few specific cases. Under these present circumstances, submucosography is recommended for routine screening test for outpatients. Our method is simple, safe and time-saving. Recently, it has become easy to diagnose hemangiomas as well as varices in the stomach by application of submucosography. Accordingly, in the cases of vascular tumors or tumescent lesions caused by extra-gastric compression, the risk of incidental major bleeding or perforation can be prevented by using the submucosography.

Adult↗
PubMed1984

Purification and initial characterization of a protein which facilitates assembly of nucleosome-like structure from mammalian cells.

A protein, which facilitates assembly of a nucleosome-like structure in vitro, was previously partially purified from mouse FM3A cells [Ishimi, Y. et al. (1983) J. Biochem. (Tokyo) 94, 735-744]. The protein has been purified to approximately 80% from FM3A cells by using histone-Sepharose column chromatography. It sedimented at 4.6 S and had a molecular mass of 53kDa. A preincubation of core histones with the 53-kDa peptide before DNA addition was necessary for the nucleosome assembly. The 53-kDa peptide bound to core histones and formed a 12-S complex. This complex contained stoichiometrical amounts of the 53-kDa peptide and core histones, and the core histones in this complex were composed of equal amounts of H2A, H2B, H3 and H4 histones. The nucleosomes were assembled by adding pBR322 DNA to the 12-S complex. When mononucleosome DNA and core histones were mixed in the presence of the 53-kDa peptide, formation of a 10.5-S complex was observed. The complex contained DNA and core histones in equal amounts, while no 53-kDa peptide was detected in the complex. From above results it is suggested that the 53-kDa peptide facilitates nucleosome assembly by mediating formation of histone octamer and transferring it to DNA. Rat antibody against the 53-kDa peptide did not bind to nucleoplasmin from Xenopus eggs. The relationship between the 53-kDa peptide and nucleoplasmin is discussed.

Animals↗
PubMed1984

Serum group I pepsinogen (PG I) levels and their changes in the healing process of the ulcer in patients with and without unoperated recurrent ulcer.

We measured serum group I pepsinogen (PG I) levels in subjects with endoscopically normal gastric and duodenal mucosa and patients with peptic ulcer. The release mechanism of PG I into blood stream was also investigated. The mean (+/- S.E.) serum PG I level in 136 subjects with endoscopically normal mucosa was 61 +/- 2 ng/ml and the normal range was calculated to be 30-109 ng/ml from the frequency distribution. In the patients with unoperated recurrent duodenal ulcer, the serum PG I levels remained high with the healing process of the ulcer. On the other hand, in the patients with non-recurrent duodenal ulcer and those with recurrent or non-recurrent gastric ulcer, the serum PG I levels decreased with the healing process of the ulcer gradually and significantly from the value in the active stage. These findings suggest that duodenal ulcer patients with high levels of serum PG I throughout the healing process of the ulcer tend to have the recurrence. Therefore, the serial measurements of serum PG I with the healing process will be helpful for the prediction of ulcer recurrence. Administration of atropine caused a significant decrease in serum PG I in the patients with duodenal ulcer, which suggests the vagal control of PG I release in duodenal ulcer patients.

Adolescent↗