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K Umetsu

Publications and source records attributed to K Umetsu.

At least 37 records · Page 2Linked to original sources
PubMed1999

Determination of the HUMTH01 alleles by the APLP method.

We present a simple and rapid technique for determination of alleles at the HUMTH01 locus. The amplified product length polymorphism (APLP) method using two primers different in length, permits the differentiation between allele 9.3 and other alleles. The primers were designed to have an allele-specific nucleotide at the 3' terminal and 11 non-complementary nucleotides were added to the 5' terminal of one of the primers for the allele 9.3. The amplified fragment sizes for the alleles 9.3 and 10 were 80 bp and 70 bp, respectively. This method has proved to be very useful for forensic applications.

Base Sequence↗
PubMed1998

Simultaneous genotyping of alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2) loci by amplified product length polymorphism (APLP) analysis.

Genotyping of the alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2) loci is important in alcohol studies. We describe a method for simultaneous genotyping of ADH2 and ALDH2 based on amplified product length polymorphism (APLP) analysis. Two polymerase chain reaction (PCR) fragments for ADH2 (57 bp, 53 bp) and two for ALDH2 (78 bp, 73 bp) are simultaneously amplified. Nine banding patterns reflecting the genotypes of the ADH2 and ALDH2 loci are clearly and unambiguously distinguished. The APLP method seems to be particularly suited for large-scale population studies of ADH2 and ALDH2 loci because it is simple and rapid.

Alcohol Dehydrogenase↗
PubMed1998

Neonatal hyperbilirubinemia and mutation of the bilirubin uridine diphosphate-glucuronosyltransferase gene: a common missense mutation among Japanese, Koreans and Chinese.

We analyzed the bilirubin uridine diphosphate-glucuronosyltransferase (B-UGT) gene in 42 Japanese newborns with hyperbilirubinemia and determined that 21 infants were heterozygous while 3 was homozygous for Gly71Arg. Allele frequency of Gly71Arg was 0.32 in newborns with hyperbilirubinemia, which was significantly higher than 0.13 in healthy Japanese controls. This mutant allele is also prevalent among Korean and Chinese healthy controls with a frequency of 0.23 in both populations. However, this mutation was not detected in 50 healthy German controls. These data suggest that the high frequency of the Gly71Arg mutation of the B-UGT gene is associated with high incidence of neonatal hyperbilirubinemia in Japanese, Korean and Chinese populations.

Alleles↗
PubMed1998

Simultaneous evaluations of contractility and energy metabolism of stunned myocardium using 31P magnetic resonance spectroscopy.

A canine model of postischemic myocardial dysfunction (15 min ischemia, 60 min reperfusion) was used to evaluate the relationship between energy metabolism and myocardial contractile function by on-line measurements of ECG, left ventricular pressure, coronary blood flow and regional segment shortening (%SS) with the continuous acquisition of 31PMR spectra. Two groups emerged from these studies; the first (n = 7) in which regional myocardial %SS remained significantly depressed after 60 min of reperfusion (stunned) and the second (n = 5) in which regional %SS returned to control levels after 60 min of reperfusion (non-stunned). Both groups exhibited rapid, similar decreases in %SS and parallel rapid decreases in the phosphocreatine to inorganic phosphate (PCr/Pi) ratio with the onset of ischemia. The PCr/ATP ratio exceeded control levels in the stunned group immediately upon reperfusion and remained significantly above control after 60 min of reperfusion. Measurements of tissue myocardial creatine kinase (CK) revealed a significant decrease in total tissue CK activity in stunned myocardium compared to control. A significant inverse relationship (r = -0.904, p < 0.003) was found between myocardial tissue CK specific activity and the PCr/ATP ratios. We postulate that the elevated PCr/ATP ratio caused by the impairment of energy transfer to the contractile apparatus constitutes a contractile dysfunction in the postischemic heart.

Animals↗
PubMed1998

Simultaneous determination of STR polymorphism and a new nucleotide substitution in its flanking region at the CD4 locus.

In the course of the investigation of a pentanucleotide repeat polymorphism at the human CD4 locus, a C-A transversion was found at the position corresponding to the 3' end of the original forward primer presented by Edwards et al. (1). In the present study, the simultaneous determination of the new sequence polymorphism and the pentanucleotide repeat polymorphism at the CD4 locus was attempted. To achieve this purpose, we adopted amplified product length polymorphism (APLP) analysis and designed some new allele-specific forward primers tagged with non-complementary nucleotides differing in length. A total of 646 DNA samples from peripheral blood of Japanese, Chinese and German populations were investigated. Although the C-A transversion was restricted to CD4*5, a new subtype allele with A and 5 repeats, designated CD4*5A, was observed at polymorphic frequencies in the three populations. The simultaneous genotyping by APLP analysis resulted in dramatically increased heterozygosity and discriminating power of the human CD4 locus.

Base Sequence↗
PubMed1998

A transferrin D variant (DHR5) identical with DSaga by polyacrylamide gel isoelectric focusing was found in a disputed paternity case.

A transferrin (TF) variant was found in the child in a paternity case by polyacrylamide gel isoelectric focusing (PAGIF). Further analysis of serum TF disclosed that this rare TF variant was inherited from the alleged father. This TF variant was identified as TF DHR5, being identical with a variant TF DSaga, in a direct comparison with several known TF D variants by PAGIF. The isoelectric points (pIs) of holo-type (Fe(II) binding type) of TF DHR5 (= DSaga) and TF C were 5.6 and 5.4, respectively. After sialidase treatment, TF DHR5 (= DSaga) showed a single band migrating at a more basic position than TF C, thus suggesting that the pI difference between the two was due to amino acid substitution.

Acrylic Resins↗
PubMed1997

Nucleotide substitution in the 5' flanking region of D1S80 locus.

We have analyzed a mutation responsible for a Hinf I polymorphism of the D1S80 system by Taq dye deoxy terminator cycle sequencing. A G-T transversion at the 58th nucleotide from the 5' end of the forward primer was identified. This mutation has been firmly established by the restriction enzymes. The type G was digested with Hinf I but not with Tsp 509 I. Conversely, the type T was digested with Tsp 509 I but not with Hinj I. We applied this technique to analyze D1S80 system in German and Japanese populations. In the Japanese population, the type T frequencies were much lower than those in the German population, and the type T was almost exclusively observed in the allele 24.

Base Sequence↗
PubMed1997

Structure of the gene encoding human alpha 2-HS glycoprotein (AHSG).

Alpha 2-HS glycoprotein (AHSG) is a human plasma glycoprotein and fetuin is the homologue in the calf. In this report, we present the structure and organization of the AHSG gene. Introns and the 5' and 3'-flanking regions were obtained by polymerase chain reaction (PCR) and the inverted PCR, respectively, from genomic DNA using AHSG cDNA-specific oligonucleotide primers. The sequence of the PCR products shows that the coding region spans approximately 8.2 kb and is composed of seven exons interrupted by six introns. The exon-intron splice junctions agree with the consensus sequence, and the positions interrupted by introns are precisely identical to those of the rat insulin receptor tyrosine kinase inhibitor (fetuin) gene. The 5'-promoter region contains several characteristic sequences such as an A + T-rich sequence of TAAATAA, C/EBP-binding site, and hepatocyte nuclear factor-5 (HNF-5) and serum response factor (SRF) sites.

Animals↗
PubMed1997

Simple and rapid determination of the acetaldehyde dehydrogenase (ALDH2) genotypes by nonradioactive single-strand conformation polymorphism analysis.

The genotyping of mitochondrial acetaldehyde dehydrogenase (ALDH2) is very important in alcohol studies. We describe an ALDH2 genotyping method based on nonradioactive single-strand conformation polymorphism (SSCP) analysis on mini-gels following amplification with a mutated primer set. The three ALDH2 genotypes were clearly and unambiguously distinguished. This method was applied to the ALDH2 genotyping of 129 unrelated Japanese. The allele frequency of ALDH2*2 was calculated to be 0.271, which was consistent with the previous data. The method proved to be simple, rapid and reliable, and dispensed with isotopic reagent and expensive restriction enzymes and equipment. The SSCP method described here is valuable in routine ALDH2 genotyping.

Aldehyde Oxidoreductases↗
PubMed1997

ITIH1*Q0iwate, a null allele of inter-alpha-trypsin inhibitor H1 caused by deletion/frameshift mutation.

The molecular characterization of the first example of null allele in the inter-alpha-trypsin inhibitor H1 (ITIH1) system, ITIH1*Q0iwate, encountered as apparent inverse homozygosity of ITIH1 phenotypes between mother and child in a paternity case, is described. Single-strand conformation polymorphism analysis and subsequent sequencing showed that deletion of a single nucleotide in the codon for Lys87 results in a frameshift causing a terminator codon downstream of the deletion. This leads to premature termination of ITIH1 protein translation at amino acid 128, resulting in a truncated protein.

Alleles↗
PubMed1997

Molecular evidence for human alpha 2-HS glycoprotein (AHSG) polymorphism.

Alpha 2-HS glycoprotein (AHSG) is a human plasma glycoprotein that exhibits genetic polymorphism on isoelectric focusing (IEF). To identify the origin of two common alleles, AHSG*1 and *2, we examined nucleotide exchanges in the gene. AHSG cDNA was obtained by RT-PCR from poly(A) RNA of seven liver tissue samples and subcloned into a plasmid vector. After sequencing, we found six single nucleotide differences in comparison with the originally reported sequence. In particular, the nucleotide substitutions of C to T at amino acid position 230 and C to G at position 238 were common among the samples exhibiting phenotype 2-1 or 2. Since these substitutions might give rise to a NlaIII site and a SacI site, respectively, for the potential AHSG*2, we analyzed these substitutions by PCR-RFLP using genomic DNA of 68 individuals. The result was consistent with the IEF analysis of the corresponding serum, indicating that AHSG*1 was characterized by ACG (Thr) at position 230 in exon 6 and ACC (Thr) at position 238 in exon 7, and that AHSG*2 was characterized by ATG (Met) at position 230 and AGC (Ser) at position 238.

Amino Acid Sequence↗
PubMed1997

Amplified product length polymorphism (APLP): a novel strategy for genotyping the ABO blood group.

We present a simple rapid reproducible polymerase chain reaction based technique, termed amplified product length polymorphism (APLP), as a new strategy for primer design for ABO genotyping. The method involves the use of primers differing in length and permits the identification of the major ABO genotypes (A1, A2, B, OA, OG, and O2) according to the molecular size of the allele-specific amplified products. Ten different primers designed to analyze the variations in nucleotide positions 261, 297, 796, 802, and 1059-1061 of cDNA are mixed in one reaction, and the amplified products are resolved on a polyacrylamide gel. Of eight PCR fragments (132 bp, 120 bp, 108 bp, 98 bp, 88 bp, 80 bp, 72 bp, and 64 bp), two to five are amplified in the reaction according to ABO genotypes. The new technique has been successfully applied to the genotyping of 221 peripheral blood samples from Japanese and Germans whose ABO phenotypes had previously been determined; a novel A allele (AG) was found in Japanese individuals.

ABO Blood-Group System↗
PubMed1997

Human orosomucoid polymorphism: molecular basis of the three common ORM1 alleles, ORM1*F1, ORM1*F2, and ORM1*S.

The human orosomucoid (ORM) is controlled by two closely linked loci, ORM1 and ORM2, and two tandem genes, AGP1 and AGP2, encoding the proteins produced by the two loci, have been cloned. In this study the molecular basis of ORM1 polymorphism was investigated. For the detection of mutations the products of the six exons of each gene, amplified by the polymerase chain reaction (PCR), were screened by single-strand conformation polymorphism analysis. Subsequently, the exons with an altered migration pattern were gene-specifically amplified by nested PCR. Sequencing of the gene-specific PCR products showed that the three common ORM1 alleles result from A-->G transitions at the codons for amino acid positions 20 in exon 1 and 156 in exon 5 of the AGP1 gene: ORM1*F1 was characterized by CAG (Gln) and GTG (Val), ORM1*F2, by CAG (Gln) and ATG (Met), and ORM1*S, by CGG (Arg) and GTG (Val). The phylogenesis of the genes encoding these three ORM1 alleles is discussed.

Alleles↗
PubMed1997

The effects of dopamine, dobutamine and amrinone on mitochondrial function in cardiogenic shock.

The impairment of mitochondrial in non-infarcted myocardium under cardiogenic shock complicated by acute myocardial infarction was studied. We induced acute myocardial infarction in dogs by ligating the circumflex branch of the left coronary artery (LCX). On basis of left ventricular systolic pressure (LVPs) after 60 minutes, we divided the dogs into two groups: a group in which LVPs fell to below 70% of the pre-LCX ligation level, and a Control group in which LVPs remained more than 90%. The former group was further divided into four subgroups, depending on infusion of dopamine, dobutamine, amrinone or saline after 90 minutes. Mitochondria were prepared and mitochondrial respiratory activity determined. In the Saline group, hemodynamics became reduced to less than 70% of the preligation level after 120 minutes, however, in the Dopamine and Dobutamine groups, hemodynamics became restored to the preligation level. In the Amrinone group, LVPs decreased slightly, while cardiac output, LV Max. dp/dt and myocardial blood flow increased. In the Saline group, mitochondria in the non-infarcted myocardium functioned at a lower level of activity than that of the Control group. However, in the Dopamine, Dobutamine, and Amrinone groups, the mitochondria functioned at a higher level. Electron microscopy revealed mitochondrial damage in the Saline group only. The results indicate that an energy production disorder in the non-infarcted myocardium may have pathogenetic implications in cardiogenic shock associated with acute myocardial infarction, while dopamine, dobutamine, and amrinone improve mitochondrial function, and ultimately improve cardiac function.

Adenosine Triphosphatases↗
PubMed1997

[Three cases of recombinated gene in the ABO blood group system].

We found the AG allele in three cases (two cases are AGOA type and one case is A1AG type) out of unrelated 150 Japanese by amplified product length polymorphism technique for genotyping of ABO blood group system. The AG alleles amplified with allele-specific PCR method, were analyzed from exon 6 to 7 by direct sequencing. The AG allele was divided into suballees, AG1 and AG2. The AG1 and AG2 were revealed to have the same sequence as B allele in exon 6. In exon 7, however, the AG1 allele had the same sequence as A1 allele and the AG2 allele had hybrid sequence of B and OG allele.

ABO Blood-Group System↗
PubMed1997

[A simple technique for the genotyping of TH01 locus].

It is difficult to differentiate between type 9.3 and 10 at the TH01 locus. Therefore, we designed three primers including allele-specific primer for the type 9.3. Our primer sets gave shorter fragments ranging from 62 to 81 bp than those in other reports, allowing each allelic band to be resolved by 10% non-denaturing polyacrylamide gel electrophoresis and stained with SYBR Green I. TH01 typing was successfully applied to human remains such as sera, whole bloods and blood stains made about 80 years ago without extracting DNA from them. The simple and fast methods for TH01 typing as demonstrated herein will be useful in the field of forensic practice.

Base Sequence↗
PubMed1996

Mitochondrial respiratory impairment in streptozotocin-induced diabetic rat heart.

The purpose of this study was to examine mitochondrial respiratory impairment in the diabetic heart. Diabetes mellitus was induced in male Wistar rats by intravenous injection of streptozotocin (STZ) for 2 to 16 weeks (Group D). In some of the diabetic rats, insulin was injected for 2 or 3 weeks prior to sacrifice (Group I). Fasting blood glucose was markedly elevated to greater than 300 mg/dl in Group D and was similar to normal glucose levels in Group I. At 2 weeks after STZ injection, state 3 was only 59.1% of that in the control. Complex I and complex V activities were also significantly reduced to 43.4% and 71.7% of those in the control, respectively. These reductions recovered with insulin treatment. This phenomenon persisted for 16 weeks. Morphological studies revealed swelling of the mitochondria and an increase in lipid droplets in diabetic cardiomyocytes, and these were also improved with insulin treatment. We conclude that in the diabetic heart, disturbance of energy production in cardiac mitochondria is generated by the impairment of oxidative phosphorylation due to depression of complex I and complex V. These changes may contribute the cardiac dysfunction that is a complication of diabetes mellitus.

Animals↗