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Y Takarada

Publications and source records attributed to Y Takarada.

At least 37 records · Page 2Linked to original sources
PubMed1994

Development of enzyme-labeled oligonucleotide probe for detection of mecA gene in methicillin-resistant Staphylococcus aureus.

A DNA hybridization method with an enzyme-labeled oligonucleotide probe (mecA-ELONP) was developed to detect the methicillin-resistant gene (mecA) in methicillin-resistant Staphylococcus aureus. For rapid identification, bacterial colonies were transferred from agar plates directly onto nylon membranes. Lysis of cells, denaturation of DNA, and hybridization were performed on the membranes. These procedures required only 3 h for completion. The results obtained by this test closely corresponded with those obtained by determining the MICs of oxacillin against S. aureus. The results of the mecA-ELONP also correlated well with those of a commercially available PCR test. Thus, mecA-ELONP proved to be a reliable and convenient method for the rapid identification of methicillin-resistant S. aureus, which could be useful in clinical microbiology laboratories.

Bacterial Proteins↗
PubMed1994

[Genetic diagnosis of phenylketonuria. III. Mutations of phenylalanine hydroxylase gene in Orientals].

Phenylketonuria (PKU) is an autosomal recessive disorder caused by lesions in the phenylalanine hydroxylase (PAH) gene. The recent studies on PAH mutations show the genetic drift of PKU alleles among some Oriental populations. Therefore, we searched for PKU mutations among Japanese, Chinese and Taiwanese. Direct sequencing was conducted on DNA fragments amplified by the polymerase chain reaction, using solid-phase technology involving the biotin-streptavidin system. Two new mutations (R241C and G247V) and two of the known mutant alleles (Y204C and R243Q) were found in two Taiwanese and two Chinese PKU patients, and three known mutations (R111X, Y204C and R413P) were recognized in three Japanese; two new mutations were identified in exon 7 of the PAH gene at codon 241 and codon 247, where the single base changes from C to T and from G to T substituted cysteine for arginine and valine for glycine, respectively. Further all the PAH mutations detected are common in Oriental populations as they have been thus far unreported among Caucasians. From these data as well as the clinical phenotype of the patients, we suggest that the R241C and G247V substitutions may interfere with proper enzyme function, although we have not yet performed functional studies. More detailed studies would be needed to clarify the regional distribution of mutant chromosomes in Oriental populations and other unidentified mutations.

Alleles↗
PubMed1994

[Genetic diagnosis of phenylketonuria. IV. Mutations of phenylalanine hydroxylase gene in Caucasian and Gypsy populations in Czech and Slovakia Republics].

Direct sequencing was conducted on the regions of the exon 7 and 12 in the phenylalanine hydroxylase (PAH) gene amplified by the polymerase chain reaction, using solid-phase technology involving the biotin streptavidin system. A novel mutation and seven previously known mutations were identified in the PAH genes among 15 Caucasians and 10 Gypsies in the Czech and Slovakia republics, affected with classical phenylketonuria (PKU). Two of these substitutions (R243X and G272X) resulted in the generation of a premature stop codon, and a single base transition of G to A at codon 261 resulted in the substitution of Arg for Gln (R261Q). These three mutations together accounted for 16.7% of PKU alleles among 15 Caucasians. The R252W mutation was detected in these two groups: two Caucasians were compound heterozygous for the P281L or R408W mutations (6.7% of all mutant alleles). However, all 10 Gypsy PKU patients were homozygous for the R252W transition. The R408W mutation accounted for 43% of PKU alleles in 15 Caucasian PKU patients. A novel heterozygous C-to-G transversion at the third base of codon 263 in the exon 7, resulting in the substitution of Phe for Leu (F263L), was detected in a Caucasian PKU patient (3.3% of all mutant alleles). In this study, we revealed a novel PKU mutation of the F263L in Caucasian populations and a high-risk PKU mutation of the R252W in Gypsy populations.

Base Sequence↗
PubMed1994

[Detection of methicillin-resistant Staphylococcus aureus using PCR and non-radioactive DNA probes: IV. Mutational sequences in the region upstream of the mec A gene in clinical staphylococcal strains].

Expression of mec A is regulated by two regulatory genes (mecR1 and mecI); the intact regulatory genes exist in the N315 strain of methicillin-sensitive S. aureus, whereas the repressor gene (mecI) is deleted in the MR108 strain of methicillin-resistant S. aureus (MRSA). However, our previous findings that clinical strains of N315 type were methicillin-resistant suggest possible mutations within the operator region, by which the inhibitory effect of MecI is released to induce the constitutive expression of mec A. Therefore, we analyzed nucleotide sequences in the region upstream of the mec A gene and in the regulatory genes, by using PCR-direct sequencing and ASO probes. Analysis of selected clinical strains by ASO probes revealed that 74% of MRSA and 36% of MRSE (methicillin-resistant S. epidermidis) lacked the mecI gene whereas 26% of MRSA and 64% of MRSE possessed the intact regulatory genes. Furthermore, PCR-direct sequencing identified four different mutations in the operator nucleotide sequence and one mutation in Shine-Dalgarno sequence. Additionally, these mutations were shown to occur in the strains of N315 type with higher probability rather than in those of MR108 type. The data suggest that one or more of these mutational sequences at least may have some influence on the expression of methicillin-resistance in clinical staphylococcal strains.

Base Sequence↗
PubMed1994

[Detection of Campylobacter species by using polymerase chain reaction and nonradioactive DNA probes. III. DNA probe for identification of C. laridis].

The development of a rapid and specific DNA probe assay for identification of Campylobacter species, including C. jejuni, C. coli, C. laridis, C. fetus, and C. hyointestinalis is important in determining the precise diagnosis of Campylobacter infections. Sequence data of our previous studies for a 240-base DNA fragment was used to select primers and probes conjugated to alkaline phosphatase, complementary to a portion of DNA between primers. However, a 21-base probe (CL (1)) tested here for detection of C. laridis was cross-reactive with PCR-amplified fragments of C. jejuni, C. coli and C. hyointestinalis, although it was not reactive with C. fetus and C. fetus subsp. fetus. To solve this problem, further modifications of the probe were therefore made to improve the specificity for those particular species. A second 21-base probe with a single base-substitution (CL (2)) and a third 20-base probe (CL(3)) were ineffective for identification of C. laridis, too. A fourth 20-base probe with a single base substitution (CL(4)) was a significant improvement over the results obtained by other three probes specifically to detect C. laridis, Thus, the alkaline phosphatase-labeled probe method developed so far is an interesting alternative without access to radioisotopes for clinical laboratories for identification of Campylobacter species, including C. jejuni/coli/hyointestinalis, C. laridis, and C. fetus/fetus subsp. fetus.

Base Sequence↗
PubMed1994

[Detection of methicillin-resistant Staphylococcus aureus using PCR and non-radioactive DNA probes: III. Mutations of the fem A gene in clinical strains of Staphylococcus aureus].

Methicillin resistance in S. aureus is primarily due to the presence of the mec A gene. However, in addition to mec, the phenotypic expression of methicillin resistance requires the presence of an additional gene(s), fem A which is chromosomally encoded. Previous studies suggest an increase in the biochemical function of fem A gene products due to base substitutions in the region upstream of the fem A gene and in its coding frame. The partial nucleotide sequences of fem A regions in reference and clinical strains of S.aureus were therefore analyzed by PCR-direct solid-phase sequencing and suitable DNA probes. Amplified target DNAs of 251, 330 and 271 bp were resolved on ethidium bromide-stained gels and hybridized with DNA probes conjugated to alkaline phosphatase. In ATCC 12600 strain, a palindromic sequence was conserved in the region upstream of fem A. However, it was destroyed by the occurrence of mutations in other reference, and clinical strains tested regardless of whether they are methicillin-susceptible or resistant. Furthermore, in the coding frame of fem A, two missense mutations were present in MSSA and MRSA without any regularity. These findings suggest that mutations in the fem A region may not be a single factor essential for regulation of methicillin resistance, although fem A probably functions cooperatively with mec A.

Base Sequence↗
PubMed1993

Development of an enzyme-labeled oligonucleotide probe for the cholera toxin gene.

An alkaline phosphatase-conjugated 30-mer oligonucleotide probe was developed to detect the cholera toxin gene (ctx) in Vibrio cholerae O1. For rapid identification, V. cholerae O1 was grown on selective agar (thiosulfate-citrate-bile salts agar) or in alkaline peptone water and organisms were transferred directly to nylon membranes. Lysis of cells, denaturation of DNA, neutralization, and hybridization were carried out on the membrane. These procedures required only 3 h for completion. The results of the hybridization test with 88 clinical and 20 environmental isolates agreed almost exactly with the results of the immunological tests (anti-cholera toxin antibody-sensitized latex agglutination tests). The specificity of the probe was also tested with strains of enterotoxigenic Escherichia coli, V. cholerae non-O1, and Vibrio mimicus.

Alkaline Phosphatase↗
PubMed1992

[Genetic diagnosis of phenylketonuria: identification of the mutations of phenylalanine hydroxylase gene by PCR direct sequencing].

To investigate the mutations of the phenylalanine hydroxylase (PAH) gene in Orientals, direct sequencing was conducted on DNA fragments amplified by the polymerase chain reaction, using solid phase technology involving the biotin-streptavidin system. Four mutations possibly associated with phenylketonuria (PKU) were identified in a Chinese and four Japanese patients. A novel Arg158 (CGG)-to-Trp158 (TGG) mutation was identified in exon 5 of the PAH gene in a Chinese PKU patient. The second change was due to a G-to-A transversion at the last base of intron 4. The third change was a compound heterozygote; one mutation was a G-to-A transversion at the last base of intron 4. The other was a G-to-C transversion at the second base of codon 413, which resulted in a substitution of Arg(CGC) by Pro(CCC) in exon 12. The last change was due to a Tyr204(TAT) -to-cys204(TGT) mutation in exon 6 of the PAH gene in two Japanese. This preliminary study revealed a novel PKU mutation and considerable genetic heterogeneity in the PAH gene among Orientals.

Asian People↗
PubMed1992

[Detection of Campylobacter species by using polymerase chain reaction and nonradioactive DNA probes. II. PCR direct sequencing of the Campylobacter DNA].

We have developed a sensitive DNA hybridization assay for the detection and identification of Campylobacter species which are recognized as important pathogens of acute diarrheal disease in humans. This technique utilizes DNA probes complementary to nucleotide sequences present in 16S ribosomal RNA (rRNA) of C. jejuni, C. coli, C. laridis, C. fetus, C. fetus subsp. fetus, C. fetus subsp. venerealis and C. hyointestinalis, and polymerase chain reaction. The partial sequence of DNAs encoding the Campylobacter rRNA was first analyzed by direct solid phase sequencing in order to select suitable DNA probes. Amplified target DNA of 240 base pairs could be resolved on ethidium bromide-stained gels, and hybridized with DNA probes conjugated to alkaline phosphatase. In identification experiments, one of the 10 probes tested here gave a positive hybridization reaction with C. jejuni, C. coli and C. hyointestinalis but not with other Campylobacter species. The other was specifically reactive with C. fetus, C. fetus subsp. fetus and C. fetus subsp. venerealis. When applied to stool specimens, a good correlation was found between the results obtained by the present assay and by biochemical tests. These findings suggest that the nonradioactive probe assay can be used as the practical criterion for differentiating Campylobacter species.

Base Sequence↗
PubMed1992

[Detection of Campylobacter species by using polymerase chain reaction and nonradioactive labeled DNA probe].

We have detected Campylobacter species which are now recognized as major pathogens of acute diarrheal disease in humans using polymerase chain reaction (PCR) and a nonradioactive labeled DNA probe. Diagnosis of Campylobacter enteritis without doing culture from stool samples is of great benefit in the laboratory. Two oligonucleotide primers (20 mer) complementary to a unique sequence of the DNA encoding ribosomal RNA (rRNA) of Campylobacter jejuni for PCR were synthesized by solid-phase phosphoamidite method. Amplified target DNA of 275 base pairs could be resolved on ethidium bromide-stained gels, and hybridized with an oligodeoxynucleotide probe (28 mer) conjugated to alkaline phosphatase. In identification experiments, it was shown that the nonradioactive probe was hybridized to clinical strains of C. jejuni (104), C. coli (5), C. laridis (5), C. hyointestinalis (1) and C. fetus subsp. fetus (1) with an accuracy of 99-100%, while it was not for Helicobacter pylori. Further, there was no evidence of amplification in strains of K. pneumoniae, S. marcescens and E. coli. Using direct detection to stool specimens, this method could be performed in C. jejuni in 39 of 43 culture-positive specimens (91%), and in 19 of 141 culture-negative specimens (13.5%), respectively. The results of this comparative study suggested that the DNA probe assay became a rapid and reliable technique to confirm culture of Campylobacter species.

Animals↗
PubMed1992

Serum erythropoietin concentrations and iron status in patients on chronic hemodialysis.

We studied the relationship between serum erythropoietin (EPO) concentration and iron status in 67 patients undergoing chronic hemodialysis. Serum concentrations of EPO were measured by RIA with recombinant human EPO. The geometric mean of the serum EPO concentration was 10.9 int. units/L (mean +/- SD range = 7.8 - 15.3 int. units/L) in hemodialysis patients, considerably lower than that in normal subjects (12.9 int. units/L). We found no significant correlation between concentrations of serum EPO and hemoglobin in hemodialysis patients, but found a significant negative correlation between serum concentrations of EPO and iron in hemodialysis patients. Moreover, we also found a significant positive correlation between the EPO concentration and the unsaturated iron-binding capacity (UIBC) in serum, and a significant negative correlation between the serum concentrations of EPO and ferritin in hemodialysis patients. Several patients who had relatively high EPO concentrations for hemodialysis patients also had low iron concentrations, high UIBC values, and low ferritin concentrations. These findings suggest that iron was utilized even at these EPO concentrations, which were very low for the degree of anemia observed in the hemodialysis patients.

Adult↗
PubMed1990

[An early cancer in adenoma of the papilla of Vater].

A 61-year-old male was admitted to our hospital with a complaint of epigastric discomfort and back pain. Chronic pancreatitis was suspected after an abdominal US study. Therefore, an ERCP study was performed. Macroscopically, the papilla of Vater showed an irregular shaped swelling. An ERCP X-ray revealed no abnormal findings, but biopsied specimens from the papilla showed a well differentiated adenocarcinoma in the adenoma, so that a pancreatoduodenectomy was performed. The lesion was 15 x 9 x 6 mm in size, and a histological examination revealed that a minor portion of the tumor was a carcinoma while the major portion was an adenoma, and the invasion as being d0 panc0 n0.

Adenocarcinoma↗
PubMed1986

[A case of secondary linitis plastica of the colon developing 14 years after gastrectomy in advanced carcinoma].

A 59-year-old woman was admitted to our hospital complaining of weight loss. Fourteen years earlier, she had undergone gastrectomy for gastric carcinoma of the Borrmann III type on the lesser curvature of the body. Biopsy specimens of the lesion revealed poorly differentiated adenocarcinoma. On the fifth hospital day of the admission under study, she complained of vaginal bleeding, and a diagnosis of endometrial carcinoma was made by a gynecologist. Transverse colectomy and total hysterectomy were done. The lesion was localized only in the transverse colon, was histologically poorly differentiated adenocarcinoma similar to the specimen of gastric carcinoma resected 14 years earlier and involved mainly the serosa. Therefore, secondary linitis plastica of the transverse colon was diagnosed.

Adenocarcinoma, Papillary↗