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Biomedical subjects

Y Kosugi

Publications and source records attributed to Y Kosugi.

At least 19 recordsLinked to original sources
PubMed1999

Thermostable flap endonuclease from the archaeon, Pyrococcus horikoshii, cleaves the replication fork-like structure endo/exonucleolytically.

The flap endonuclease gene homologue from the hyperthermophilic archaeon, Pyrococcus horikoshii, was overexpressed in Escherichia coli and purified. The results of gel filtration indicated that this protein was a 41-kDa monomer. P. horikoshii flap endonuclease (phFEN) cleaves replication fork-like substrates (RF) and 5' double-strand flap structures (DF) using both flap endonuclease and 5'-3'-exonuclease activities. The mammalian flap endonuclease (mFEN) is a single-strand flap-specific endonuclease (Harrington, J. J., and Lieber, M. R. (1994) EMBO J. 13, 1235-1246), but the action patterns of phFEN appear to be quite different from those of mFEN at this point. The DF-specific flap endonuclease and 5'-exonuclease activities have not yet been reported. Therefore, this is the first report of the specific endo/exonuclease activities of phFEN. The DF-specific 5'-exonuclease activity degraded the downstream primer of 3' single-flap structure and was 15 times higher than the activities against nicked substrates without 3' flap strand. DF-specific flap endonuclease cleaved the 5' double-flap strand in DF and the lagging strand in RF at the junction portion. Because the RF appears to be the intermediate structure, due to the arrest of the replication fork, the double strand breaks after the arrests of the replication forks are probably caused by phFEN.

Amino Acid Sequence↗
PubMed1999

Thermostable aminopeptidase from Pyrococcus horikoshii.

From the genome sequence data of the thermophilic archaeon Pyrococcus horikoshii, an open reading frame was found which encodes a protein (332 amino acids) homologous with an endoglucanase from Clostridium thermocellum (42% identity), deblocking aminopeptidase from Pyrococcus furiosus (42% identity) and an aminopeptidase from Aeromonas proteolytica (18% identity). This gene was cloned and expressed in Escherichia coli, and the characteristics of the expressed protein were examined. Although endoglucanase activity was not detected, this protein was found to have aminopeptidase activity to cleave the N-terminal amino acid from a variety of substrates including both N-blocked and non-blocked peptides. The enzyme was stable at 90 degrees C, with the optimum temperature over 90 degrees C. The metal ion bound to this enzyme was calcium, but it was not essential for the aminopeptidase activity. Instead, this enzyme required the cobalt ion for activity. This enzyme is expected to be useful for the removal of N(alpha)-acylated residues in short peptide sequence analysis at high temperatures.

Aminopeptidases↗
PubMed1999

Increased heterogeneity of chromosome 17 aneuploidy in endometriosis.

OBJECTIVE: Endometriosis is a complex gynecologic disorder that may display features similar to malignancy, including aggressive growth and localized invasion of the myometrium or spread to various organs outside the uterus. Molecular studies of cancer have demonstrated that genomic instability involving chromosome 17 plays a role in the development and progression of various tumor types. These involve gain and/or loss, deletions, and mutations of candidate tumor suppressor genes (eg, BRCA1 and p53 ) on chromosome 17. STUDY DESIGN: We used a 2-color fluorescence in situ hybridization method for analysis of endometriotic and normal archival tissue. Centromere-specific and locus-specific p53 probes localized to chromosome 17 were selected to study 8 patients with late-stage (severe) endometriosis. Single cells localized to endometriotic lesions or normal endometrial glands were analyzed and identified as normal or abnormal on the basis of the distribution of fluorescence in situ hybridization signals. RESULTS: Overall, chromosome 17 aneuploidy was significantly greater (P <.05) in the endometriosis specimens (mean of 65%) than in normal endometrial cells (mean of 25%). No significant difference (P =.1071) in the distribution of fluorescence in situ hybridization signals was observed among the 5 normal endometrial specimens. However, significant differences (P <. 0001) were observed between the 8 endometriosis tissue specimens. CONCLUSION: We found increased heterogeneity of chromosome 17 aneuploidy in endometriosis. These findings support a multistep pathway involving somatic genetic alterations in the development and progression of this common disease.

Aneuploidy↗
PubMed1999

Counteracting moment device for reduction of earthquake-induced excursions of multi-level buildings.

A vibration-control mechanism for beams and columns was presented in our previous report in which the earthquake force was transformed into a vibration-control force by using a gear train mechanism. In our previous report, however, only the principle of transforming the earthquake force into the control force was presented; the discussion for real structures and the design method were not presented. The present article provides a theoretical analysis of the column which is used in multi-layered buildings. Experimental tests were carried out for a model of multi-layered buildings in the frequency range of a principal earthquake wave. Theoretical results are compared to the experimental data. The optimal design of the control mechanism, which is of importance in the column design, is presented. Numerical calculations are carried out for the optimal design. It is shown that vibrations of the column involving the mechanism are suppressed remarkably. The optimal design method and the analytical results are applicable to the design of the column.

Architecture↗
PubMed1998

Agonist stimulation of B1 and B2 kinin receptors causes activation of the MAP kinase signaling pathway, resulting in the translocation of AP-1 in HEK 293 cells.

In response to bradykinin, phosphorylated MAP kinases (ERK-1 and ERK-2) were abundantly increased in HEK 293 cells, which overexpress the rat B2 kinin receptor. In a similar way des-Arg9-bradykinin stimulation of B1 kinin receptor-overexpressing HEK 293 cells caused activation of the same species of MAP kinase. Furthermore, nuclear translocation of transcription factor AP-1 was also found in the cells after stimulation with either agonist. PD98059, a MAP kinase kinase (MEK-1) inhibitor, blocked the agonist-induced AP-1 translocation as well as the phosphorylation of the MAP kinases. This communication provides the first evidence for both B1 and B2 kinin receptors mediating the MAP kinase signaling pathway to activate AP-1.

Animals↗
PubMed1998

Stabilities of N-acetyl-beta-D-glucosaminidase (NAG) isoenzymes in urine: advantage of NAG isoenzyme B measurement in clinical applications.

N-Acetyl-beta-D-glucosaminidase (NAG) is a widely used urinary enzyme for the assessment of renal diseases. We studied the stabilities of NAG isoenzymes in urine at 37 degrees C by enzyme assay and ELISA using a model simulating in vivo conditions. The stabilities were found to be affected by the pH. Under mild acidic condition (about pH 6), there was no significant loss of enzymatic activity of NAG isoenzyme A, enzymatic activity of NAG isoenzyme B and immunological activity of NAG isoenzyme B even after 8 h incubation. In contrast, under alkaline condition (about pH 8), the enzymatic activity of NAG isoenzyme A was rapidly lost, whereas both enzymatic and immunological activities of NAG isoenzyme B were maintained at more than 80% of their initial values. Also, we found that the ratios of endogenous NAG isoenzyme B to total NAG were elevated in alkaline urine samples. These results indicate that NAG isoenzyme A, which is a major isoenzyme in normal urine (pH 5-7), seems to be inactivated in alkaline urine. Our results suggest that for alkaline urine, NAG isoenzyme B should be measured to avoid misinterpretation of total NAG enzymatic activities.

Acetylglucosaminidase↗
PubMed1997

Inhibition by 1-aminocyclobutane-1-carboxylate of the activity of 1-aminocyclopropane-1-carboxylate oxidase obtained from senescing petals of carnation (Dianthus caryophyllus L.) flowers.

We partially purified 1-aminocyclopropane-1-carboxylate (ACC) oxidase from senescing petals of carnation (Dianthus caryophyllus L. cv. Nora) flowers and investigated its general characteristics, and, in particular, the inhibition of its activity by ACC analogs. The enzyme had an optimum pH at 7-7.5 and required Fe2+, ascorbate and NaHCO3 for its maximal activity. The Km for ACC was calculated as 111-125 microM in the presence of NaHCO3. Its M(r) was estimated to be 35 and 36 kDa by gel-filtration chromatography on HPLC and SDS-PAGE, respectively, indicating that the enzyme exists in a monomeric form. These properties were in agreement with those reported previously with ACC oxidases from different plant tissues including senescing carnation petals. Among six ACC analogs tested, 1-aminocyclobutane-1-carboxylate (ACBC) inhibited most severely the activity of ACC oxidase from carnation petals. ACBC acted as a competitive inhibitor with the Ki of 20-30 microM. The comparison between the Km for ACC and the Ki for ACBC indicated that ACBC had an affinity which was ca. 5-fold higher than that of ACC. Whereas ACC inactivated carnation ACC oxidase in a time-dependent manner during incubation, ACBC did not cause the inactivation of the enzyme. Preliminary experiments showed that ACBC and its N-substituted derivatives delayed the onset of senescence in cut carnation flowers.

Amino Acid Oxidoreductases↗
PubMed1997

New sandwich ELISA for human urinary N-acetyl-beta-D-glucosaminidase isoenzyme B as a useful clinical test.

We have developed a new ELISA for quantifying N-acetyl-beta-D-glucosaminidase (NAG) isoenzyme B in human urine after raising monoclonal antibodies against the isoenzyme from human placenta. Though the obtained antibodies reacted not only to isoenzyme B but also to A, we could detect isoenzyme B selectively by a two-step sandwich ELISA with a pair of selected antibodies at low pH in the first reaction. The detected limit was 0.5 microgram/L for a sample volume of 25 microL. Within-run CVs ranged from 2.5% to 5.4% and between-run CVs ranged from 6.2% to 9.1%. Recoveries of NAG isoenzyme B added to each of three urine samples ranged from 91% to 114%. The dilution curves of urine samples showed good linearity. The cross-reactivity of NAG isoenzyme A was practically negligible (2-3%). The mean value for NAG isoenzyme B in spot urines from healthy adults was 2.9 micrograms/g creatinine. This ELISA method is rapid and precise enough for routine determination of NAG isoenzyme B in human urine.

Acetylglucosaminidase↗
PubMed1997

Neural network-based PET image reconstruction.

In PET image analysis, conventional deconvolution alone will not give sufficient information for a precise study of a localized brain function. In the deconvolution process, which is a type of inverse problem, it is important to confine the solution space by incorporating a priori knowledge such as the tissue distribution given by MR images as well as smoothness in the blood flow distribution profile. An MR-embedded neural-network model is described to reduce the partial volume effect in the restoration of blood flow profiles from PET images.

Brain↗
PubMed1996

[Usefulness of newly developed subcutaneous one point lifting method in gynecological laparoscopic operation].

We first introduced the abdominal wall lifting method in gynecological laparoscopic operations in 1994. Now we have developed a new one-point lifting technique (Tokyo Medical College method) that enables gynecological laparoscopic operations to be performed more safely, easily and economically. 1. The TMC method provides a sufficiently spacious operative field in the same way as artificial pneumoperitoneum and can overcome the negative influence of obesity on the operative field. 2. Because the abdominal wall is lifted up by means of a single stainless steel wire passed under the skin in this method, the limitations of previous methods are completely overcome. Furthermore setting up for this method is very easy and can be completed in a short time. 3. Because the aperture for the Surgigrip made in the abdominal wall is short and has a large internal diameter, we can manipulate operative instruments freely. Especially suturing and ligation can be carried out simply and accurately. 4. We were able to continue irrigation and suction without deterioration of the operative field and to easily remove resected tissue from the abdominal cavity. 5. The frequency of intracorporeal suture and ligation in this method increased significantly compared with the pneumoperitoneum method in cases of ovarian diseases. As a result, most operative procedures in ovarian diseases became cystectomies, preserving the healthy part of the ovary. This also reduced operative cost by avoiding expensive autosuture equipment. 6. There were no complications such as subcutaneous bleeding with this method. There was no significant difference between this method and pneumoperitoneum in terms of postoperative pain. The TMC method is a new laparoscopic operative technique that allows procedures to be performed under conditions close to laparotomy procedures and solves most of the problems of the pneumoperitoneum method.

Abdominal Muscles↗
PubMed1996

[Analysis of the mechanism of trophoblast infiltration].

A cytotrophoblast (CT) infiltrates into the stroma, forming an extravillous trophoblast (EVT) in the placenta early in gestation and the phenomenon is strictly controlled, differing from the infiltration of cancer cells. The expression of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9), which deeply involve infiltrative metastasis of cancer, and the reactivity to transforming growth factor beta 1 (TGF beta 1), which controls the expression of these MMPs and inhibits the growth of epithelial cells, were investigated in CT derived from villi at normal gestational week 6 (early CT) and CT derived from villi at normal gestational week 37 (full-term CT), and also the choriocarcinoma cell line BeWo (BeWo). The ability of normal epithelial cells and BeWo cells to proliferate and infiltrate were evaluated in vitro by northern blotting, gelatin zymography, and invasion assay. It was revealed that early CT had a higher capacity for infiltration than full-term CT as well as BeWo. MMP2 and MMP9 appeared in the early CT, whereas only MMP9 was observed in the full-term CT. MMP2 and MMP9 were more abundantly observed in the early CT and the full-term CT rather than in BeWo. In uterine stroma-derived cells, membrane type matrix metalloproteinase (MT-MMP), which activates MMP2, was observed. These results indicated that the motility of normal villous cells was higher in the early CT than in the full-term CT. The expression of MMP2 in the early CT, which was not observed in the full-term CT, was thought to be related to this difference in motility. As for the responsiveness to TGF beta 1, which is a growth inhibiting factor for epithelial cells, the villous carcinoma cell line was insensitive to the growth inhibiting effect of TGF beta 1, but the early CT was sensitive to this effect. When TGF beta 1 was added, MMP2 and MMP9 increased in the early CT. This response was also seen in BeWo. That is, it was assured that the growth capacity was not inhibited in BeWo, but was certainly inhibited in the early CT. The overall results of these evaluations indicated that the development to EVT by infiltration of the early CT was associated with the increase in the mobility of cells caused by MMP2 and the increase in amounts of MMP2 and MMP9 caused by TGF beta 1, and the predominant inhibitory effect of TGF beta 1 on the growth of normal epithelial cells could explain why normal epithelial cells do not grow as cancer cells do.

Animals↗
PubMed1993

Changes in the parameters of oxygen metabolism in a clinical course recovering from potassium cyanide.

Poisoning with potassium cyanide is usually fatal because of the inhibition of cytochrome oxidase. The parameters of oxygen metabolism in a patient with cyanide poisoning who was admitted in a coma with seizures was monitored. The administration of amyl nitrite and sodium thiosulfate led to a rapid improvement: the parameters reflecting oxygen metabolism improved and the plasma level of cyanide decreased. The patient revived 1 1/2 hours after treatment. The arterial ketone body ratio (AKBR), which is the ratio of acetoacetate to beta-hydroxybutyrate in arterial blood and which reflects the redox state in liver mitochondria, improved dramatically following treatment. Because the AKBR changes in relation to electron transport in liver mitochondria, it seems to be a logical parameter for evaluating the effect of potassium cyanide poisoning on electron transport. The AKBR also reflects the efficacy of treatment for cyanide poisoning.

3-Hydroxybutyric Acid↗
PubMed1992

A biocompatible needle-type glucose sensor based on platinum-electroplated carbon electrode.

A biocompatible needle-type glucose sensor with a 3-electrode configuration was constructed. A platinum-electroplated carbon stick was used as the working electrode, Ag/AgCl as the reference electrode, and a disposable hypodermic needle made of stainless steel as the counter electrode. A Nafion membrane, an immobilized glucose oxidase (GOD) membrane, and a biocompatible membrane with diffusion-limiting effect were coated successively onto the working electrode. The sensor showed a rapid response (< 120 s in batch operation), good reproducibility (RE < 3%), good stability (over 36 h in control serum), a wide dynamic range (5-600 mg/dL glucose), and superior biocompatibility. It was used to determine glucose in serum. The data obtained from the sensor showed good agreement with that from a clinical autoanalyzer (R > 0.95).

Biocompatible Materials↗