Search PubMed⌕ Search

Biomedical subjects

T Higaki

Publications and source records attributed to T Higaki.

At least 19 recordsLinked to original sources
PubMed2001

Clonal chromosomal aberrations accompanied by strong telomerase activity in immortalization of human B-lymphoblastoid cell lines transformed by Epstein-Barr virus.

Human B-lymphoblastoid cell lines transformed by Epstein-Barr (EBV-LCLs) are considered to be immortalized, although most of them show a normal diploid karyotype. Recently, we and others have shown that only part of EBV-LCLs is immortalized by developing strong telomerase activity that stabilizes the telomeres. In this study, we investigated the change in karyotypes during immortalization. All the eight immortalized cell lines developed clonal chromosomal aberrations accompanied by the development of strong telomerase activity. Interestingly, abnormal chromosomes were not shared among the immortalized cell lines. These results strongly suggest that chromosomal rearrangements and induction of strong telomerase activity are two events that take place in parallel in the process of immortalization of EBV-LCLs, and indicate that EBV-LCLs are clearly divided into two distinct groups, pre-immortal cell lines mostly with a normal diploid karyotype and post-immortal cell lines with a clonally abnormal karyotype.

B-Lymphocytes↗
PubMed2001

Effect of cicletanine on the nitric oxide pathway in human umbilical vein endothelial cells.

The purpose of this study was to evaluate the effects of cicletanine, a slightly diuretic antihypertensive drug, on human vascular endothelial cells with regard to nitric oxide, intracellular calcium concentration ([Ca2+]i), cyclic nucleotide, inositol 1,4,5-trisphosphate (IP3), and prostacyclin generation. Primary cultured human umbilical vein endothelial cells were used in this study. [Ca2+]i was measured by fura-2/AM. Cyclic adenosine monophosphate (AMP), cyclic guanosine monophosphate (GMP), IP3, and prostacyclin were measured by radioimmunoassay. Nitric oxide was measured by the Griess method. Cicletanine had no effect on [Ca2+]i. Cicletanine (10(-6)-10(-4) M) increased cyclic GMP but decreased prostacyclin generation. Cicletanine had no stimulating effect on cyclic AMP or IP3 generation. IP3 increased 45Ca release from storage sites. Cicletanine decreased prostacyclin generation via increase in cyclic GMP. Cicletanine had no stimulating effect on nitrogen oxides for 2 h after incubation but increased it after 3-24 h. Pretreatment with L-N(G)-monomethyl-arginine (L-NMMA) prevented this increase. The inhibitory effect of L-NMMA was prevented by pretreatment with L-arginine. These results indicate that nitric oxide and cyclic GMP may contribute to the antihypertensive action of cicletanine.

Antihypertensive Agents↗
PubMed2000

Effects of bradykinin on prostaglandin I(2) synthesis in human vascular endothelial cells.

The effects of bradykinin on the regulatory mechanisms of prostacyclin synthesis in endothelial cells were investigated in association with intracellular Ca(2+) kinetics, cytosolic phospholipase A(2) (cPLA(2)) activity, and mRNA expression of cPLA(2) and prostaglandin H synthase (PGHS) isoforms. Bradykinin enhanced prostacyclin release from endothelial cells time-dependently, but pretreatment with EGTA H-7 or HOE 140 inhibited bradykinin-induced prostacyclin release. Bradykinin increased both the influx of extracellular Ca(2+) and Ca(2+) release from the intracellular Ca(2+) storage sites. These reactions occurred within 5 minutes after bradykinin stimulation. Within 15 minutes, bradykinin activated cPLA(2) to 1.3-fold the control level. The constitutive expressions of mRNA of cPLA(2), PGHS-1, and PGHS-2 was 87, 562, and 47 amol/microg RNA, respectively. With the stimulation of bradykinin, cPLA(2) mRNA increased to 746 amol/microg RNA in 15 minutes, PGHS-1 mRNA increased to 10 608 amol/microg RNA, and PGHS-2 mRNA increased to 22 400 amol/microg RNA in 180 minutes. Pretreatment with cycloheximide superinduced cPLA(2) and PGHS-2 mRNA expression but almost completely inhibited PGHS-1. Pretreatment with EGTA had effects similar to pretreatment with cycloheximide in the case of cPLA(2) and PGHS-1 but did not affect PGHS-2. These findings suggest that the elevation of cPLA(2) activity caused by the increase of intracellular Ca(2+) concentration is important in the early phase of bradykinin-induced prostacyclin synthesis and that the mechanisms regulating cPLA(2) are different from those regulating PGHS isoforms in endothelial cells.

Bradykinin↗
PubMed1999

A role of protein kinase C in the regulation of cytosolic phospholipase A(2) in bradykinin-induced PGI(2) synthesis by human vascular endothelial cells.

The purpose of this study was to elucidate the mechanism by which bradykinin (BK) enhances prostacyclin (PGI(2)) production in human umbilical vein endothelial cells (HUVEC). BK-induced enhancement of PGI(2) synthesis was observed in a dose- and time-dependent manner, and it also increased [Ca(2+)](i) followed by enhancement of cytosolic phospholipase A(2) (cPLA(2)) activity. The PKC inhibitors GF109203X and H7 attenuated the BK-induced increase in [Ca(2+)](i) and inhibited the BK-induced PGI(2) synthesis. Phorbol 12-myristate 13-acetate increased cPLA(2) activity and PGI(2) synthesis but failed to alter [Ca(2+)](i). BK increased cPLA(2) mRNA eightfold by 15 min, and this increase was inhibited by pretreatment with the PKC inhibitors. In response to cycloheximide pretreatment, cPLA(2) mRNA was superinduced. These results suggest that BK stimulates PGI(2) synthesis in HUVEC by activation of cPLA(2) by dual mechanisms: an elevation of [Ca(2+)](i) and a PKC-dependent pathway. Moreover, changes in calcium kinetics and expression of cPLA(2) mRNA may underlie the BK-induced PGI(2) enhancement in these cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1999

Effect of probucol on intracellular pH and proliferation of human vascular endothelial cells.

We investigated the effect of probucol on the intracellular pH ([pH]i) and proliferation of human umbilical vein endothelial cells (HUVEC), as well as their production of prostacyclin (PGI2). The addition of probucol produced a biphasic shift in [pH]i, with a brief initial acidification followed by a rapid alkaline shift. After pretreatment with EGTA, the initial decrease in [pH]i was abolished, and the subsequent increase was inhibited. After pretreatment with amiloride, only the increase of [pH]i was abolished. These results suggest that the probucol-induced increase of [pH]i was mainly dependent on Na+/H+ exchange and partly on extracellular Ca2+. In contrast, the addition of LDL produced a decrease of [pH]i. Under Ca2+-free condition, [pH]i was further decreased by LDL. In cells pretreated with amiloride, however, [pH]i was not further decreased by LDL. It was found that probucol promoted cell proliferation, and LDL inhibited cell proliferation. Addition of probucol also enhanced prostacyclin generation by HUVEC. This enhancement of PGI2 generation resulted from increased release of Ca2+ from the storage sites, due not only to increased production of inositol 1,4,5-triphosphate (IP3) but also to the increase of [pH]i. These findings may help to explain the antiatherosclerotic action of probucol.

Amiloride↗
PubMed1999

The mechanism of thrombin-induced prostacyclin synthesis in human endothelial cells with reference to the gene transcription of prostacyclin-related enzymes and Ca2+ kinetics.

This study was designed to evaluate the effect of thrombin on prostacyclin (PGI2) production in cultured human vascular endothelial cells in association with intracellular Ca2+ and with the gene expression of prostaglandin H2 synthase (PGHS) and phospholipase A2 (PLA2) using competitive polymerase chain reaction. Thrombin enhanced the PGI2 synthesis dependent with time. Additionally, thrombin increased the intracellular Ca2+, which stimulates PLA2, resulting in arachidonic acid cleavage from membrane phospholipids and its subsequent conversion into PGI2 through the PGHS pathway. The elevation of intracellular Ca2+ was a result of Ca2+ influx and Ca2+ release from its intracellular storage sites. In this study, PGHS-1 mRNA was constitutively expressed, whereas PGHS-2 mRNA was not. With the stimulation of thrombin, cytosolic PLA2 (cPLA2) mRNA increased 9-fold at 15 min, PGHS-1 mRNA increased 3.4-fold at 180 min, and PGHS-2 mRNA increased 38-fold at 60 min. These results suggest that the elevation of intracellular Ca2+ and the expression of cPLA2, PGHS-1, and PGHS-2 mRNA cause PGI2 generation.

Calcium↗
PubMed1999

Interleukin-1alpha stimulated prostacyclin release by increasing gene transcription of prostaglandin H synthase and phospholipase A2 in human vascular endothelial cells.

This study was conducted to evaluate the effects of interleukin-1alpha (IL-1alpha) on prostacyclin (PGI2) production in cultured human vascular endothelial cells in association with intracellular Ca2+, inositol 1,4,5-trisphosphate (IP3), and with prostaglandin H synthase (PGHS) and phospholipase A2 (PLA2) gene expression by using the competitive polymerase chain reaction (PCR) method. IL-1alpha did not increase PGI2 production for 15 min, but induced an increase of about three-fold relative to that in controls at 60 and 180 min. IL- 1alpha had no effect on intracellular Ca2+ levels throughout the experimental period. In this study, consistent with previous reports, PGHS-1 messenger RNA (mRNA) was constitutively expressed, whereas PLA2 mRNA was not. After stimulation with IL-1alpha, PLA2 mRNA level showed an eightfold increase within 15 min, and PGHS-2 mRNA level increased by 76-fold within 180 min. PGHS-1 mRNA level was increased 1.6-fold at 180 min. These results suggest the existence of regulatory mechanisms of IL-1alpha-induced PGI2 production, which involve PGHS and PLA2 gene transcription.

Base Sequence↗
PubMed1999

A quantitative comparison of induction and challenge concentrations inducing a 50% positive response in three skin sensitization tests; the guinea pig maximization test, adjuvant and patch test and Buehler test.

The sensitivities of three skin sensitization tests such as the Guinea pig maximization test (GPMT), Adjuvant and patch test (APT) and Buehler test (BT), were quantitatively compared with reference to induction and challenge concentrations. Four chemical which had different physico-chemical properties (octanol-water partition coefficient (logP) and reactivity with NH2-group) were used in order to clarify the effect of the physico-chemical properties of chemicals on the sensitivity of the different methods. The induction concentrations inducing a 50% positive response (IC50) demonstrated extreme variation with the three methods. For example, the BT/GPMT ratio of IC50 values for 2,4-dinitrochlorobenzene was 33, whereas that for maleic anhydride was 300,000. The results were thought to be caused by difference properties such as the logP and reactivity of chemicals. This correlation was confirmed by using 2-dodecen-1-yl succinic anhydride, which had the same reactivity but higher logP than that of maleic anhydride. On the other hand, the challenge concentrations inducing 50% positive responses (CC50) were less affected by the methods and the BT/GPMT ratios for CC50 values were all within a 10-fold range. These results suggest that the sensitivity might be strongly different with reference to induction concentration, but not challenge concentration among the three methods.

Adjuvants, Immunologic↗
PubMed1998

Comparison of sex differences in guinea-pig maximization test for detection of skin-sensitizing potential using OECD recommended positive control sensitizers.

Both male and female guinea pigs are widely used in tests to detect the skin-sensitizing potential of new chemicals. The aim of the present study was to investigate the influence of the animal gender on sensitization rates, with the guinea-pig maximization test (GPMT) using OECD recommended positive control sensitizers, and the differences of the density of Langerhans cells in male and female guinea pigs. The sensitization rates of males and females challenged with HCA, MBT or benzocaine gave almost the same results. There were no statistical differences between the mean responses of males and females. These results were confirmed by rechallenge with the OECD recommended positive controls. Furthermore, the density of Langerhans cells in abdominal epidermis in males (1229 +/- 45 cells/mm2) was the same as that in females (1242 +/- 89 cells/mm2). These results indicate that there is no significant influence of guinea-pig gender in the assessment of skin-sensitizing potential using the GPMT.

Acrolein↗
PubMed1996

Effects of high-density lipoproteins on intracellular pH and proliferation of human vascular endothelial cells.

We investigated the effects of high-density lipoprotein (HDL) on the intracellular pH ([pH]i), and on the proliferation of human vascular endothelial cells (HUVEC), as well as on their production of prostacyclin (PGI2). The [pH]i was slightly acidified when extracellular Ca2+ was chelated with EGTA. Pretreatment of HUVEC with amiloride, the Na+/H+ exchange inhibitor, caused the [pH]i to become strongly acidic. The addition of HDL produced a biphasic shift in [pH]i, with a brief initial acidification followed by a rapid alkaline shift. The initial decrease in [pH]i was abolished in the cells pretreated with EGTA, and subsequent alkalinization was inhibited. The alkalinization of [pH]i disappeared in the cells pretreated with amiloride. These results suggest that [pH]i depends mainly on Na+/H+ exchange and partially on the extracellular Ca2+ of the HUVEC either in the resting unstimulated state or during HDL stimulation. In contrast, the addition of LDL produced an acidification of [pH]i, which was increased by LDL in the Ca(2+)-free condition. In the cells pretreated with amiloride, [pH]i was not further acidified by LDL. As a result, HDL promoted the proliferation of cells, an action that was inhibited by pretreatment with EGTA. However LDL inhibited cell proliferation, an action unaffected by EGTA pretreatment. The addition of HDL also enhanced the generation of prostacyclin in endothelial cells, the enhancement of PGI2 generation resulted from an increase in the release of Ca2+ from storage sites, due not only to an increased production of inositol 1,4,5-trisphosphate (IP3), but also to the alkalinization of [pH]i. These effects may be involved in the mechanism of HDL's anti-atherosclerotic action.

Amiloride↗
PubMed1994

NMR studies of 1H resonances in the 10-18-ppm range for cytosolic aspartate aminotransferase.

Continuing a previous investigation (Kintanar, A., Metzler, C. M., Metzler, D. E., and Scott, R. D. (1991) J. Biol. Chem. 266, 17222-17229), we have recorded 1H NMR spectra at 500 MHz in the 10-18-ppm range for the 93-kDa porcine cytosolic aspartate aminotransferase and for four specific mutant forms of the enzyme in which histidine 68 has been replaced by lysine or histidine 143, 189, or 193 has been replaced by glutamine. We have correlated resonances for apoenzyme, pyridoxamine and pyridoxal phosphate forms, and dicarboxylate complexes and have assigned imidazole NH resonances of active site histidines. The chemical shifts of several resonances undergo pH-dependent changes around the pKa of the Schiff base proton at the active site. Other resonances shift upon binding of dicarboxylates or other ligands. Phosphate or carboxylate ions, which can also occupy the site of the substrate's alpha-carboxylate, cause rapid exchange of the Schiff base proton. Although most resonances in the 10-18-ppm range disappear rapidly in D2O, a few are retained for months in the presence of the dicarboxylate inhibitor glutarate. We demonstrate that changes in chemical shifts and in exchange rates are sensitive indicators of electronic interactions of the enzyme with ligands and of conformational change. Nuclear Overhauser effects from NH protons have allowed us to identify resonances of CH protons of the imidazole rings of histidines 143, 189, and 193. Observed and predicted chemical shifts have been compared. We conclude that the net charge on this histidine cluster is zero but that some negative charge from the aspartate 222 carboxylate is donated inductively into the histidine 143 ring. Studies of the related enzyme from Escherichia coli are provided in an accompanying paper (Metzler, D. E., Metzler, C. M., Scott, R. D., Mollova, E. T., Kagamiyama, H., Yano, T., Kuramitsu, S., Hayashi, H., Hirotsu, K., and Miyahara, I. (1994) J. Biol. Chem. 269, 28027-28033). Our approach should be applicable to the study of active sites of a broad range of relatively large proteins.

Amino Acid Sequence↗
PubMed1994

Functional role of the amino-terminal mobile segment in catalysis by porcine cytosolic aspartate aminotransferase. Critical importance of Val17 and Phe18 for productive binding of substrates.

A notable feature of porcine cytosolic aspartate aminotransferase is the closure of the active site cleft by a mobile amino-terminal segment (residues 15-40) upon binding substrate. The functional roles of Val17 and Phe18, residues that are part of the mobile loop, have been studied in the site-directed mutants in which the size and hydrophobic nature of these residues have been changed. Absorption, circular dichroism spectra, susceptibility to protease 401, and thermal stability did not differ appreciably between wild type and mutant enzymes. In the overall transamination between aspartate and 2-oxoglutarate, V17A represented a typical Km mutant while V17I retained the substrate binding affinity fairly well. In contrast, replacement of Phe18 by Ala resulted in a large decrease in both catalytic rate and binding affinity for substrates. F18W, F18Y, and F18H showed a moderate decrease in kcat and a considerable increase in Km values. Single-turnover reactions with four individual substrates yielded analogous results to those obtained for the overall reaction and, in addition, revealed that k/Kd values of mutants F18A and F18H were over 10 times lower for C5 substrates (glutamate and 2-oxoglutarate) than those for C4 substrates (aspartate and oxalacetate). All mutant enzymes showed variously increased Kd values for substrate analogs such as 2-methylaspartate, succinate, and glutarate. 1H NMR observations of F18H, in which His18 served as a built-in probe, were in accord with the behavior that would be expected from the conformational transition. We conclude that, although Val17 and Phe18 may not be essential for catalysis, the presence of a bulky residue of appropriate size at each position is critical for productive binding of substrate.

Animals↗
PubMed1994

[The significance of portal infusion chemotherapy for prevention of recurrence in residual liver after hepatectomy for metastases from colorectal cancer].

We performed portal infusion chemotherapy using a reservoir for prevention of recurrence in residual liver after hepatectomy for metastases from colo-rectal cancer. To study the usefulness of portal infusion chemotherapy, the period from hepatectomy to recurrence in residual liver was investigated by three treatment groups for H2 cases; (a) a group of systemic chemotherapy, (b) a group of arterial infusion chemotherapy and (c) a portal infusion chemotherapy group. Treatment in (a) group was for 204 +/- 98.2 days (n = 6), in (b) group for 343.0 +/- 238.3 days (n = 8) and in (c) group for 961.0 +/- 1,172.4 days (n = 5). There was no statistically significant difference in the three groups, but (c) group had a better result in recurrence in residual liver. As for prevention of recurrence in residual liver after re-hepatectomy, there was no significant difference in the three groups, but (c) group had the longest survival.

Aged↗
PubMed1994

[Evaluation of arterial infusion chemotherapy for advanced gallbladder cancer using implantable port].

Seven patients with advanced gallbladder cancer were treated by arterial infusion chemotherapy. Five patients had unresectable tumor, and two had liver metastases after resection of primary tumor. The response rate was 42.9% (3 PR), and the 1-year survival rate was 35.7%. No severe side effect was found. We conclude that arterial infusion chemotherapy is effective for advanced gallbladder cancer, and that a more effective regimen and adjuvant therapy should be established.

Aged↗
PubMed1993

Polychlorinated biphenyl and organochlorine insecticide residues in human adipose tissue in Poland.

Concentrations of PCBs (polychlorinated biphenyls) and organochlorine insecticides such as DDT, HCHs (hexachlorocyclohexanes), HCB (hexachlorobenzene) and chlordanes were determined in human adipose fat from the provinces of Skierniewice and Gdańsk in Poland collected during 1979 and 1990, respectively. The mean levels (microg g(-1) lipid weight basis) in Skierniewice and Gdańsk were found to be 1.2+/-0.44 and 1.5+/-1.3 for PCBs, 25+/-16 and 15+/-13 for DDT, 0.53+/-0.17 and 0.25+/-0.09 for HCHs, 0.36+/-0.22 and 0.26+/-0.23 for HCB, and 0.11+/-0.08 and 0.07+/-0.04 for chlordanes, respectively. Very high concentrations of DDT were detected, with the maximum value as high as 52 microg g(-1) lipid weight in Skierniewice and 47 microg g(-1) lipid weight in Gdańsk. PCB residues contained in the human fat from Polish cadavers are comparable to those of the levels reported for industrialized nations. It is appraised from the levels of DDT and PCBs in Poles' fat that the Polish environment is still experiencing both types of contamination, arising from agricultural and industrial activities. Contamination of humans by other organochlorine insecticides was relatively lower.

Journal Article↗