Biomedical subjects

K Hirano

Publications and source records attributed to K Hirano.

At least 271 records · Page 15Linked to original sources

PubMed1997

Purification and characterization of prostate specific antigen from human urine.

Prostate specific antigen (uPSA) was purified to homogeneity from human urine using SuperQ-Toyopearl, Sulfate-Cellulofine, Phenyl-Toyopearl, CM-Sepharose, anti-urokinase IgG Sepharose and Sephadex G-100. The purified uPSA gave a major band at 32.9 kDa on SDS-PAGE under the reduced condition. However, it shows multiple bands on native PAGE. Substrate specificity of purified uPSA is identical with that of PSA from human seminal plasma and uPSA shows the kallikrein and chymotrypsin-like activities. On the analysis of N-terminal amino acid, two amino acid residues at N-terminal position of uPSA were detected and other amino acid sequence of uPSA was identical with that of sPSA. In addition, we isolated the multiple components of uPSA using anion-exchange chromatography. They were almost the same in amino acid composition and N-terminal amino acid sequences and showed differences in lectin-blotting pattern.

Amino Acid Sequence↗

PubMed1997

The tumor-derived fetal-intestinal alkaline phosphatase cDNA is identical in sequence to the adult intestinal alkaline phosphatase isozyme gene.

The alkaline phosphatase (AP) of Caco-2 cells, a cell line derived from a human adenocarcinoma of the colon, is quite similar to fetal intestinal AP in its enzymatic properties. The nucleotide sequence of a cDNA encoding AP produced in Caco-2 cells was examined. The sequence was identical to one of the three sequences of adult intestinal AP reported previously. We further investigated the entire nucleotide sequence of cDNA of intestinal-type AP produced in cancer cell lines such as HuH-7 cells, FL-amnion cells, and HuG-1 cells. The sequence of these cell APs was identical to that of Caco-2 cell AP. These results indicate that cancer cells producing intestinal-type AP have the same nucleotide sequence as that of adult intestinal AP, and suggest that the differences in electrophoretic mobilities of these cell APs compared with adult intestinal AP may be due to post-translational modifications.

Adult↗

PubMed1997

Operation of a Two-Crystal X-ray Interferometer at the Photon Factory.

A two-crystal X-ray interferometer for phase-contrast X-ray imaging is reported. Mechanical stability of less than 0.1 nm is required to operate the two-crystal interferometer. The feasibility of using such an interferometer at the Photon Factory using synchrotron radiation has been investigated. Interference fringes of 70% visibility were observed with 0.092 nm X-rays. This result indicates that the two-crystal X-ray interferometer can be applied to phase-contrast X-ray imaging.

Journal Article↗

PubMed1997

A possible mechanism for hyperthermic radiosensitization mediated through hyperthermic lability of Ku subunits in DNA-dependent protein kinase.

DNA-dependent protein kinase (DNA-PK), composed of p470 catalytic subunit and p85/p70 heterodimer of Ku autoantigen, is considered a critical enzyme in DNA double-strand break repair. We purified DNA-PK from human leukaemic MOLT-4 cells by successive column chromatography and separated into p470 and Ku subunits by ultracentrifugation in glycerol gradient. We studied hyperthermic stability of DNA-PK holoenzyme and its separated subunits to test a possible role of DNA-PK in hyperthermic radiosensitization. DNA-PK was found to lose its activity rapidly at hyperthermic 44 degrees C, and further, Ku subunits instead of p470 catalytic subunits were found to be sensitive to hyperthermia. These results indicate a possibility that hyperthermic radiosensitization is mediated through the heat lability of Ku subunits of DNA-PK, impairing repair of radiation-induced double-strand break of DNA.

DNA Damage↗

PubMed1997

Cloning and characterization of a protein phosphatase type 1-binding subunit from smooth muscle similar to the glycogen-binding subunit of liver.

A yeast two-hybrid screen of a chicken gizzard cDNA library detected the interaction of the catalytic subunit of protein phosphatase type 1 with a novel subunit. Subsequent characterization established similarity (58%) to the rat liver glycogen-binding subunit. Northern analyses showed expression in a wide range of tissues.

Amino Acid Sequence↗

PubMed1997

Interactions of the subunits of smooth muscle myosin phosphatase.

Myosin phosphatase from smooth muscle consists of a catalytic subunit (PP1c) and two non-catalytic subunits, M130 and M20. Interactions among PP1c, M20, and various mutants of M130 were investigated. Using the yeast two-hybrid system, PP1c was shown to bind to the NH2-terminal sequence of M130, 1-511. Other interactions were detected, i.e. PP1c to PP1c, M20 to the COOH-terminal fragment of M130, and dimerization of the COOH-terminal fragment of M130. Mutants of M130 were constructed to localize the PP1c and light chain binding regions. Results from the two-hybrid system indicated two binding sites for PP1c on M130: one site in the NH2-terminal 38 residues and a weaker site(s) in the ankyrin repeats region. Inhibition of PP1c activity with phosphorylase a by the M130 mutants also was consistent with the assignment of these two sites. Overlay assays showed binding of phosphorylated light chain to the ankyrin repeats, probably in the COOH-terminal repeats. Activation of PP1c with phosphorylated light chain required binding sites for PP1c and substrate, plus an additional sequence COOH-terminal to the ankyrin repeats. Thus, activation of phosphatase and binding of PP1c and substrate are properties of the NH2-terminal one-third of M130.

Enzyme Activation↗

PubMed1997

Preservation of the larynx after resection of a carcinoma of the posterior wall of the hypopharynx: versatility of a free flap patch graft.

BACKGROUND: Preservation of the larynx after resection of a pharyngeal tumor remains a challenging problem for the head and neck surgeon. METHODS: Nine patients with T1 or T2 carcinoma of the posterior wall of the hypopharynx (UICC 1987), who were treated surgically between 1984 and 1994, were studied. All patients underwent surgical resection of the tumor with laryngeal preservation and immediate reconstruction with free flap transfer. A free forearm flap was transferred in four patients and a free jejunal patch graft, in five patients. RESULTS: There was one flap loss, due to venous thrombosis. Successful larynx preservation was achieved in the remaining eight patients (89%). Although there was one local control failure, three patients remained free of disease for more than 5 years. CONCLUSIONS: Laryngeal preservation surgery using a free flap patch graft has proven very beneficial in selected cases with a carcinoma of the posterior wall of the hypopharynx.

Aged↗

PubMed1997

Interferon alpha induces disorder of lipid metabolism by lowering postheparin lipases and cholesteryl ester transfer protein activities in patients with chronic hepatitis C.

The effect of recombinant interferon alpha 2a (rIFN-alpha2a) on serum lipoprotein metabolism was assessed in 39 patients with chronic viral hepatitis C. rIFN-alpha2a was administered intramuscularly at a dose of 9 x 10(6) U/d for 2 weeks and then for 3 times a week over 6 months. The serum cholesterol concentration significantly decreased one week after rIFN-alpha2a administration. Approximately 67% of this decrease was attributed to the reduction of high-density lipoprotein (HDL)-cholesterol; a decrease in HDL2-cholesterol was more evident. By contrast, serum triglyceride levels, largely derived from very-low density lipoprotein (VLDL), significantly increased following rIFN-alpha2a treatment. Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) activities in the postheparin plasma were reduced by 75.7% and by 79.4%, respectively, and decreases in plasma cholesteryl ester transfer protein (CETP) activity and its protein mass were also observed. However, prothrombin time was ameliorated by rIFN-alpha2a, suggesting that the decrease in LPL, HTGL, and CETP activities may not be due to a reduction in protein synthesis by the liver. Simple correlation analysis demonstrated that the changes in LPL activity before and after 2 weeks of treatment with rIFN-alpha2a showed a significant negative correlation with changes in serum triglyceride and VLDL-triglyceride and a positive correlation with changes in HDL-cholesterol and HDL2-cholesterol. These results suggest a major contribution of reduced LPL activity with regard to the lipoprotein disorders. In conclusion, rIFN-alpha2a treatment on patients with chronic hepatitis C causes marked changes in serum lipoprotein metabolism associated with decreases in LPL, HTGL, and CETP activities.

Adult↗

PubMed1997

Polymorphism of extracellular superoxide dismutase (EC-SOD) gene: relation to the mutation responsible for high EC-SOD level in serum.

Extracellular superoxide dismutase (EC-SOD) with amino acid substitution R213G generated by the nucleotide substitution 760C-->G in the heparin binding domain is responsible for the high EC-SOD level in serum. We identified the two DNA polymorphic sites in the coding region of EC-SOD gene related to the 760C-->G and determined the allele frequencies. The polymorphism were A and G at nucleotide position (nt.) 241 and C and T at nt. 280 near the N-terminal. The haplotype frequencies in Japanese were 241A280C: 0.45, 241G280T: 0.37, and 241G280C: 0.18. The haplotype of 241A280T did not exist. The mutation 760C-->G must occur on the allele having the haplotype of 241G280T.

Extracellular Space↗

PubMed1997

Intestinal alkaline phosphatase isoforms in rabbit tissues differ in glycosylation patterns.

OBJECTIVES: In patients with advanced liver cirrhosis or chronic nephritis, an intestinal alkaline phosphatase (IAP)-like enzyme is ectopically expressed in the liver or kidney. In this study, we used rabbit organs as a human pathological model, because the rabbit liver or kidney expresses an IAP-like enzyme as the predominant isozyme, unlike humans. METHODS: IAP and the IAP-like enzyme were purified from rabbit intestine and kidney, respectively, by immunoaffinity chromatography using a monoclonal antihuman IAP antibody. Some properties of the IAP and IAP-like enzyme expressed in rabbit organs are compared. RESULTS: Some of their catalytic and physicochemical properties differed. In particular, the net charge, molecular mass, and hydrophobicity of IAP from rabbit intestine was slightly different from the IAP-like enzyme from rabbit kidney. There was a difference in the sugar chain structure between the two enzymes according to the results of lectin affinity chromatography, and part of the peptide maps differed slightly. However, there was no difference in the peptide maps after treatment with endo-N-acetylglucosaminidase F. CONCLUSIONS: The primary structures of IAP and the IAP-like enzyme are basically similar, except for the glycosylation process of the respective AP isozymes.

Alkaline Phosphatase↗

PubMed1997

Increased serum remnant lipoproteins in patients with apolipoprotein E7 (apo E Suita).

Apolipoprotein (apo) E7 was originally identified by Yamamura et al. in subjects with atherosclerotic cardiovascular diseases (J. Clin. Invest. 1984;74:1229). However, the lipoprotein abnormalities associated with apo E7 phenotype have not been elucidated. In the current study, to clarify the physiological roles of apo E7, lipoprotein abnormalities were studied in 12 apo E7 heterozygotes. A total of seven subjects were hyperlipidemic and five subjects were normolipidemic. The apo E phenotype was apo E7/3 in 11 subjects and apo E7/4 in one subject. Polymerase chain reaction revealed that all of the subjects with apo E7 phenotype had the same mutation as that of apo E(Suita) as reported previously (J. Biochem. 1989;105:249). All the hyperlipidemic subjects were over 40 years of age and two of them also had and severe coronary heart disease. Ultracentrifugal analysis revealed that the cholesterol level both in very low density lipoprotein and in intermediate density lipoprotein (IDL) was substantially higher in hyperlipidemic apo E7 heterozygotes, compared with control subjects and that the IDL cholesterol was also increased even in normolipidemic apo E7 heterozygotes. Polyacrylamide gel electrophoresis of lipoproteins showed a midband, which implies the increase of remnant lipoproteins, in 11 subjects out of 12, irrespective of the presence or absence of hyperlipoproteinemia. In two cases, a broad beta pattern was observed similar to that seen in type III hyperlipoproteinemia. Dietary therapy was dramatically effective for the treatment of hyperlipidemia in patients with apo E7. These findings confirm that apo E is crucial for remnant lipoprotein metabolism and that apo E7 is related to the increase in serum remnant lipoproteins, which leads to hyperlipoproteinemia in association with obesity, aging and impaired glucose metabolism.

Adult↗

PubMed1997

Detection of maternofetal transfusion by placental alkaline phosphatase levels.

We investigated the volume of maternofetal transfusion by measuring placental alkaline phosphatase (PLAP) as an indicator of placental passage. PLAP levels were measured in 135 pairs of maternal-neonatal sera. Estimated volumes of maternofetal transfusion were calculated with the following formula: Volume of transfusion (in milliliters) = 85 ml/kg x BW (in kilograms) x (PLAPAB-PLAP1MO)/ PLAPMA, where BW is the birth weight, PLAPAB is the PLAP level in neonatal serum at birth, PLAP1MO is the PLAP level in neonatal serum at 1 month of age, and PLAPMA is the PLAP level in maternal serum at delivery. The mean PLAPMA, PLAPAB, and PLAP1MO levels were 276.95 +/- 159.74 IU/L, 3.83 +/- 2.17 IU/L, and 0.25 +/- 0.22 IU/L, respectively. The mean volume of maternofetal transfusion was estimated to be 3.33 +/- 1.68 ml. The mean estimated volume of maternofetal transfusion per kilogram of birth weight was significantly lower in cases of scheduled cesarean delivery (0.74 +/- 0.35 ml/kg) than in cases of vaginal delivery (1.18 +/- 0.54 ml/kg; p < 0.001) and emergency cesarean delivery (1.73 +/- 1.00 ml/kg; p < 0.01). In scheduled cesarean delivery a significant positive correlation between gestational age and the estimated volumes of transfusion per kilogram of birth weight was observed. In cases of vaginal delivery, the estimated volume of transfusion per kilogram of birth weight was significantly lower in the group with short labor (< 5 hours: 0.92 +/- 0.32 ml/kg) than in the group with prolonged labor (> or = 5 hours: 1.29 +/- 0.61 ml/kg; p < 0.001). PLAP was considered to be useful for estimating the volume of maternofetal transfusion. The transfer volume appeared to relate to uterine contractions and to histologic changes in the placenta with aging.

Adult↗

PubMed1997

Mutation in pncA is a major mechanism of pyrazinamide resistance in Mycobacterium tuberculosis.

OBJECTIVE: To characterize the correlation of the mutations in the pncA gene encoding pyrazinamidase (PZase) of Mycobacterium tuberculosis to a loss of PZase activity and development of pyrazinamide (PZA) resistance. DESIGN: The association of PZase activity, minimum inhibitory concentrations (MICs), and mutations in the pncA gene of M. tuberculosis isolated in mostly Asian countries was investigated. RESULTS: One hundred thirty-five out of 168 isolates were PZase positive, and 33 were negative. The MICs of PZA at pH 6.0 were over 400 micrograms/ml for all 33 PZase-negative isolates, while those of PZase-positive isolates were equal to or less than 200 micrograms/ml. Among 33 PZase-negative isolates sequenced, 32 (97%) had mutations within the pncA gene. A mutation was seen in various regions throughout the pncA gene. It was surprising that all three strains of in vitro selected PZA resistant mutants were PZase-positive and showed no change in the pncA gene. These results indicate that additional mechanisms may be involved in PZA resistance. No mutations were observed in all of 135 PZase-positive M. tuberculosis isolates tested, indicating that mutations in the pncA gene could be involved in the loss of PZase activity. CONCLUSIONS: Sequencing analysis of the pncA gene should provide rapid diagnosis of PZA resistant clinical isolates of M. tuberculosis.

Amidohydrolases↗

PubMed1997

High-density lipoproteins from probucol-treated patients have increased capacity to promote cholesterol efflux from mouse peritoneal macrophages loaded with acetylated low-density lipoproteins.

To elucidate the anti-atherogenic effect of probucol, high-density lipoprotein (HDL) was isolated from probucol-treated patients (n = 14) and compared with that from control subjects (n = 12). The HDL obtained from probucol-treated patients was low in cholesteryl ester (CE) in comparison with that from control subjects (21.3 +/- 3.9 mol per cent vs. 27.6 +/- 3.2 mol% of total lipids. P < 0.001), and the peak diameters of patients' HDL were significantly smaller than those of control subjects on 4-30% non-denaturing polyacrylamide gradient gel electrophoresis (10.6 +/- 0.6 nm vs. 12.1 +/- 0.4 nm, P < 0.001). These data may be explained by the increased cholesteryl ester transfer protein (CETP) activities of probucol-treated patients (129 +/- 12% of control subjects, P < 0.001). The in vitro ability of HDL to remove CE from lipid-laden macrophages induced by incubation with acetylated low-density lipoprotein (Ac-LDL) was studied. The small and CE-poor HDL obtained from probucol-treated patients had a greater capacity to promote CE efflux from macrophages than did control HDL (59.8 +/- 6.9% vs. 44.2 +/- 5.4%, P < 0.01). Furthermore, the ability of HDL to promote cholesterol efflux correlated negatively with the CE content and particle diameter of HDL (r = -0.561 and r = -0.583 respectively; P < 0.01). When the inhibitory effect of HDL on the incorporation of [14C]-oleate into cellular cholesteryl ester was compared, the HDL from patients and control subjects inhibited CE formation to a similar extent. The enhanced ability of probucol-treated patients' HDL may, therefore, be involved in the acceleration of hydrolysis of the CE pool in macrophages. Taken together, we conclude that CETP plays a crucial role in making HDL more active in its anti-atherogenic function by reducing CE and making HDL smaller, and that probucol may enhance reverse cholesterol transport by activating CE transfer in vivo. The current study demonstrated, for the first time, that HDL modified by enhanced CETP activity in vivo is potentially anti-atherogenic.

Adult↗

PubMed1997

Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts.

Myosin II light chains (MLC20) are phosphorylated by a Ca2+/calmodulin-activated kinase and dephosphorylated by a phosphatase that has been purified as a trimer containing the delta isoform of type 1 catalytic subunit (PP1C delta), a myosin-binding 130-kDa subunit (M130) and a 20-kDa subunit. The distribution of M130 and PP1C as well as myosin II was examined in smooth muscle cells and fibroblasts by immunofluorescence microscopy and immunoblotting after differential extraction. Myosin and M130 colocalized with actin stress fibers in permeabilized cells. However, in nonpermeabilized cells the staining for myosin and M130 was different, with myosin mostly at the periphery of the cell and the M130 appearing diffusely throughout the cytoplasm. Accordingly, most M130 was recovered in a soluble fraction during permeabilization of cells, but the conditions used affected the solubility of both M130 and myosin. The PP1C alpha isoform colocalized with M130 and also was in the nucleus, whereas the PP1C delta isoform was localized prominently in the nucleus and in focal adhesions. In migrating cells, M130 concentrated in the tailing edge and was depleted from the leading half of the cell, where double staining showed myosin II was present. Because the tailing edge of migrating cells is known to contain phosphorylated myosin, inhibition of myosin LC20 phosphatase, probably by phosphorylation of the M130 subunit, may be required for cell migration.

Amino Acid Sequence↗

PubMed1997

Properties of phospholipase A1/transacylase in the white muscle of bonito Euthynnus pelamis (Linnaeus).

The properties of phospholipase A1 (PLA1) obtained from the white muscle of bonito, Euthynnus pelamis (Linnaeus), were examined. The PLA1 activity had a pH optimum from 6.5 to 7.0 for phosphatidylcholine (PC), and calcium ion was not required. The optimum temperature was from 20 to 30 degrees C. When a fatty alcohol was used as an acceptor, a wax ester was produced by transferring a fatty acid at the sn-1 position of the donor's PC. The maximum production of lysophosphatidylcholine was shifted by 0.5 pH units to the acidic side and the pH optimum of wax ester synthesis was from 6.0 to 6.5. The synthesis was independent of calcium ion and Coenzyme A. The transacylation was also observed when 1-lyso-2-acyl-sn-glycero-3-phosphocholine was used as an acceptor. Fatty acid at the sn-1 position of the donor PC was transferred to the unoccupied hydroxy group of the acceptor at the sn-1 position. When 2,3-dipalmitoyl-sn-glycero-1-phosphocholine was used as the acyl donor, a similar amount of palmitic acid was transferred as in the case of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. However, 1-acyl-2-lyso-sn -glycero-3-phosphocholine, a positional isomer, was a poor acceptor. These results indicate that the transacylation by the PLA1 from bonito muscle is not stereospecific, but is position-specific both for the acyl donor and acceptor.

Acyltransferases↗

PubMed1997

Molecular genetics of plasma cholesteryl ester transfer protein.

Plasma cholesteryl ester transfer protein facilitates the transfer of cholesteryl ester from HDL to apolipoprotein B-containing lipoproteins, and is a key protein in the reverse cholesterol transport system. The importance of plasma cholesteryl ester transfer protein in lipoprotein metabolism was highlighted by the discovery of deficient individuals with a marked hyper-HDL-cholesterolemia. Cholesteryl ester transfer protein deficiency causes various abnormalities in the concentration, composition, and functions of both HDL and LDL. Its significance in atherosclerosis is still controversial. However, in-vitro evidence shows large cholesteryl ester-rich HDL particles in cholesteryl ester transfer protein deficiency are defective in cholesterol efflux. Recent epidemiological studies have demonstrated an increased incidence of coronary atherosclerosis in deficient patients. The current review will also focus on the molecular genetics of the protein.

Animals↗

PubMed1997

Effects of endothelin 1 on phosphate transport in brush border membrane vesicles.

Endothelin is the most potent vasoconstrictive agent released from endothelial and many other types of cells. It has been reported that endothelin has physiological effects on the rat kidney, especially on the renal proximal tubules. We investigated the role of endothelin 1 in the renal brush border membrane. The V(max) of P(i) uptake by brush border membrane vesicles (BBMV) in the endothelin-1-treated group was significantly greater than that in the control group. There were no significant changes in apparent K(m) values between the two groups. To define the direct effect of endothelin 1 on P(i) transport of BBMV, BBMV from normal rats were incubated with endothelin 1 in vitro. The P(i) uptake by BBMV incubated with endothelin 1 did not differ from that by BBMV incubated with a salt solution as a control. These results suggested the probability of the presence of endothelin 1 receptor in the proximal tubules. Administration of the endothelin 1 might increase the P(i) reabsorption by BBMV according to changes in the capacity of the transporter.

Animals↗