Biomedical subjects

R Ishida

Publications and source records attributed to R Ishida.

At least 55 records · Page 3Linked to original sources

PubMed1998

Induction of necrosis by zinc in prostate carcinoma cells and identification of proteins increased in association with this induction.

Zinc exhibits inhibitory effects on apoptosis, and a deficiency in this metal generally causes this type of cell death to occur. In the present study, we found that exposure to zinc results in necrosis of prostate carcinoma cells. When zinc acetate was added to LNCaP or PC-3 cells in monolayer culture, they began to detach from the culture dishes, and viability was lost after 4-8 h. Most of the cell death was found to be due to necrosis as determined by double staining with fluorescein-isothiocyanate-labeled annexin V and ethidium bromide, and by detection of hypodiploid cells. Associated with the induction of necrosis was an increase in low molecular-mass proteins, identified by HPLC analysis to be thymosin beta10, parathymosin and GAGE in LNCaP cells, and thymosin beta4, parathymosin and metallothionein in PC-3. The time course of the increase of thymosin beta10 in LNCaP cells and thymosin beta4 in PC-3 cells was consistent with that of appearance of cell detachment and dead cells. These results indicate that zinc can induce necrosis and suggest that production of proteins including beta-thymosins is involved in induction of processes leading to cell detachment.

Adenocarcinoma↗

PubMed1998

Changes in muscle strength properties caused by harvesting of autogenous semitendinosus tendon for reconstruction of contralateral anterior cruciate ligament.

A prospective study was conducted of how the muscle strength of the donor knee is affected by harvesting of the autogenous semitendinosus tendon (St) for use as a substitute graft material in cruciate ligament reconstruction. There were 25 patients from whom only the St was harvested from the contralateral (i.e., healthy/donor) knee. Using a Biodex System II (Biodex, New York, NY), the strength of the donor knee was measured during both extension and flexion, both before and 12 months after the tendon harvesting procedure. A comparative study was made of the preharvest and postharvest values for the peak torque and peak torque angle in the isokinetic contraction. There were no statistically significant differences between the preharvest and postharvest peak torque values of the donor knee. However, the peak torque angle decreased significantly after the tendon harvest; the range of the mean decrease was from 11.7 degrees to 15.0 degrees. This indicates that there was a change to a small flexion angle (P < .05). After the tendon harvest, regardless of the applied angular velocity, more than 80% of the cases showed a change of torque curve shape in which there was no peak in the latter half, and the position of the peak was shifted to the left. In conclusion, the results of this study indicate that harvesting of the autogenous St does not affect the peak torque, but the peak torque angle during flexion of the donor knee is reduced.

Adolescent↗

PubMed1997

Interaction of the DNA topoisomerase II catalytic inhibitor meso-2,3-bis(3,5-dioxopiperazine-1-yl)butane (ICRF-193), a bisdioxopiperazine derivative, with the conserved region(s) of eukaryotic but not prokaryotic enzyme.

ICRF-193 [meso-2,3-bis(3,5-dioxopiperazine-1-yl)butane], a bisdioxopiperazine compound, has been shown to be a catalytic inhibitor of DNA topoisomerase II by stabilizing the enzyme in the form of a closed "protein clamp," an intermediate form in the catalytic cycle (Roca et al., Proc Natl Acad Sci USA 91: 1781-1785, 1994). In view of its usefulness as a probe in the functional analysis of the enzyme, we tried further to define the domain(s) of the enzyme interacting with the drug by examining its inhibitory activity on type II topoisomerases from various species of eukaryotes and prokaryotes. ICRF-193 inhibited the enzyme from yeast, fly, frog, plant, and mammals at IC50 values in the range of 1-13 microM. Experiments using fission yeast truncated mutant type II enzyme lacking both amino-terminal 74 amino acids and carboxy-terminal 265 amino acids revealed that ICRF-193 interacts with the 125 kDa "core" polypeptide of the enzyme. In contrast, prokaryotic type II enzymes, Escherichia coli DNA gyrase, topo IV, and phage T4 topo, were not affected by the drug. From these results, the domain(s) common to eukaryotic but not to prokaryotic type II enzymes interacting with ICRF-193 was speculated.

Amino Acid Sequence↗

PubMed1997

DNA topoisomerase II as the cellular target of a novel antitumor agent ICRF-193, a bisdioxopiperazine derivative, in Xenopus egg extract.

We have investigated the molecular target of an antitumor agent ICRF-193, a bisdioxopiperazine derivative, in in vitro chromosome condensation system of Xenopus egg extract (XEE), where DNA topoisomerase II was previously demonstrated to play a crucial role. Demembranated Xenopus sperm head chromatin is converted to metaphase chromosome-like structure in XEE in two steps, i.e., swelling of the chromatin followed by condensation of chromosome. When ICRF-193 was added to the reaction, swelling of the chromatin was not affected but chromosome condensation was completely blocked. This blockade was reversed by exogenous supplement of calf thymus topoisomerase II, which was in turn neutralized by anti-topoisomerase II monoclonal antibody. These results demonstrate that topoisomerase II is the molecular target of the drug ICRF-193.

Animals↗

PubMed1997

ICRF-193, a catalytic inhibitor of DNA topoisomerase II, delays the cell cycle progression from metaphase, but not from anaphase to the G1 phase in mammalian cells.

We have shown previously that ICRF-193, a catalytic inhibitor of DNA topoisomerase II (topo II), delays cell cycle progression in HeLa S3 cells. We report here that the delay of the transition in M phase is observed when HeLa S3 cells were treated with ICRF-193 during metaphase, but not thereafter. ICRF-193 also delayed the degradation of cyclin B in the transition from M to G1 phase, while in Chinese hamster ovary (CHO) cells the drug did not delay the progression in M phase. Since HeLa S3 and CHO cells are 'stringent' and 'relaxed' in mitotic control, respectively, it is suggested that under topo II inhibition, the M phase checkpoint operates through an inability for chromosome segregation.

Anaphase↗

PubMed1997

Isolation and characterization of a cDNA encoding a Translin-like protein, TRAX.

Translin is a DNA binding protein which specifically binds to consensus sequences at breakpoint junctions of chromosomal translocations in many cases of lymphoid malignancies. To investigate its functional significance at such recombination hotspots, we examined whether Translin interacts with other proteins using a yeast two-hybrid system and identified an associated 33 kd protein partner, TRAX, with extensive amino acid homology. The TRAX protein was established to contain bipartite nuclear targeting sequences in its N-terminal region, suggesting a possible role in the selective nuclear transport of Translin protein lacking any nuclear targeting motifs.

Amino Acid Sequence↗

PubMed1997

Irreversible cytotoxic effect of a novel lowly immunosuppressive antitumor fluorouridine derivative, UK-21.

Previously, we reported that the 5-fluorouridine derivative, 2',3',5'-tris-O-[N(2-n-propyl-n-pentanoylglycyl]-5-fluorouridine (UK-21), is a newly synthesized lowly immunosuppressive and potent antitumor drug in comparison with other fluorouridine derivatives such as 5-fluorouracil (5-FU), 5-fluorouridine (5-FUR) and 5-fluorodeoxyuridine (5-FUDR). In order to elucidate the molecular mechanism of antitumor activity of UK-21, we compared the effect of the four drugs on cell proliferation, cell cycle progression and macromolecular syntheses. When KB cells were subjected to a colony-forming inhibition assay designed to expose the cells to the drugs for 4-96 h and wash out, UK-21 and 5-FUR inhibited the colony formation at concentrations ranging from 0.01 to 0.1 microM, whereas 1-100 microM was needed for the cytotoxicity of 5-FU and 5-FUDR. By exposure for 24-48 h, all these drugs inhibited cell growth and caused accumulation of the cells in S or G2 phase at almost the same concentrations of 0.32-8 microM. These results suggest that the cytotoxic effects of UK-21 and 5-FUR are irreversible, while those of 5-FU and 5-FUDR are reversible. To confirm this, KB cells were treated with UK-21 and/or 5-FU for 1 h, and continued to be cultured for 1-7 days, resulting in the inhibition of the cell growth by UK-21 in a dose-dependent manner at concentrations of 10-100 microM, but not by 5-FU even at 100 microM. UK-21, 5-FUR and 5-FU showed a linear relationship between exposure time and IC50 in the colony formation assay with a slope of almost -1, but 5-FUDR did not, suggesting that UK-21, 5-FUR and 5-FU are cell cycle non-specific inhibitors, while 5-FUDR is a cell cycle-specific inhibitor. UK-21 and 5-FUR, but not 5-FU and 5-FUDR inhibited the incorporation of [3H]uridine into the acid insoluble fraction, while UK-21 and 5-FUDR, but not 5-FUR and 5-FU inhibited the incorporation of [3H]thymidine. These results suggest that irreversible cytotoxic effects of UK-21 like 5-FUR are exerted through inhibition of RNA synthesis.

Antimetabolites, Antineoplastic↗

PubMed1997

Effects of ICRF-193, a catalytic inhibitor of DNA topoisomerase II, on sister chromatid exchange.

To investigate whether mammalian DNA topoisomerase II is directly involved in recombination events, the effects of ICRF-193, a specific catalytic inhibitor on sister chromatid exchange (SCE), were examined in MR-6 cells. ICRF-193 only slightly elevated SCE formation after 3 or 44 h treatments, while VP-16, a cleavable complex forming type of topoisomerase II inhibitor, caused significant enhancement. ICRF-193 had no effect on N-methyl-N'-nitro-N-nitrosoguanidine-induced SCE formation. It would thus appear that the inhibition of topoisomerase II does not affect recombinational repair, and that topoisomerase II inhibitors such as VP-16 and 4'-(9-acridinyl amino) methane sulfon-m-anisidide induce SCE through production of DNA strand breaks, rather than by inhibiting the enzyme activity.

Antineoplastic Agents, Phytogenic↗

PubMed1997

[Spectrum analysis of heart rate variability for the assessment of training effects].

The aim of our study was to investigate the effect of habitual exercise on the autonomic nervous system on the basis of spectral analysis of heart rate variability. We studied 24 healthy subjects. Eight were trained athletes (age 26.9 +/- 12.4 years, weight 58.6 +/- 7.9 kg, BMI 21.7 +/- 2.6 kg/m2) who routinely performed a minimum of 40 minutes of aerobic activity per week. The control population was represented by 16 untrained persons (age 27.1 +/- 11.9 years, weight 59.1 +/- 13.0kg, BMI 22.1 +/- 4.0kg/m2). There was no significant difference in age, gender, weight and BMI between two groups. After 10 minutes of supine resting, all subjects were instructed to perform 40W exercise on a mechanically braked bicycle ergometer for 10 minutes. Heart rate was continuously recorded according to the experimental protocol. Low and high frequency power were calculated by using fast Fourier transform. Differences of parameter values between two groups were examined by using t tests. Before loading, the athletes had a significantly lower heart rate (63.7 +/- 4.0 beats/min) than control subjects (73.0 +/- 9.1 beats/min). During ergometric loading, the coefficient of variation of heart rate (CV) and the high frequency power (HF) were significantly higher, and low frequency power (LF) was significantly lower in the athletes (CV 2.9 +/- 0.6, HF 0.5 +/- 0.2, LF 0.4 +/- 0.2) compared with the control subjects (CV 2.1 +/- 0.8, HF 0.3 +/- 0.1, LF 0.6 +/- 0.1). After loading, the athletes had a significantly lower heart rate. In conclusion, we found that there were significant differences in the spectral components of heart rate variability during exercise between athletes and control subjects. The results show that physical training could possibly increase parasympathetic activity (or decrease sympathetic activity).

Adolescent↗

PubMed1996

Threonine 1342 in human topoisomerase IIalpha is phosphorylated throughout the cell cycle.

To investigate the relationship between the modulation of topoisomerase II activity and its phosphorylation state during the cell cycle, a monoclonal antibody against C-terminal peptide (residues 1335-1350) of topoisomerase IIalpha containing a consensus sequence of casein kinase II, TDDE and its phosphorylated threonine were prepared. In an enzyme-linked immunosorbent assay, the antibody, named PT1342, recognized the immunogenic phosphopeptide but not the non-phosphorylated form of the peptide. The PT1342 antibody reacted only with a 170-kDa protein from HeLa cells and recognized anti-topoisomerase IIalpha immunoprecipitants. Furthermore, the antibody did not react with the human topoisomerase IIalpha mutated at codon 1342 from threonine to alanine, showing that PT1342 was directed against the phosphorylated threonine 1342. To examine the level of phosphorylation of threonine 1342 of topoisomerase IIalpha through the cell cycle, HeLa cells were stained simultaneously for phosphorylated topoisomerase IIalpha and DNA and analyzed by flow cytometry. Cells in the G2-M phase contained about double the PT1341-reacted topoisomerase IIalpha than did cells in G1 or S phases. The antibody stained the nuclei in interphase and mitotic chromosomes and its periphery, as seen with anti-topoisomerase IIalpha antibody. Thus, threonine 1342 in topoisomerase IIalpha is phosphorylated throughout the cell cycle.

Amino Acid Sequence↗

PubMed1996

Antitumor activity of 2-amino-4,4 alpha-dihydro-4 alpha, 7-dimethyl-3H-phenoxazine-3-one, a novel phenoxazine derivative produced by the reaction of 2-amino-5-methylphenol with bovine hemolysate.

2-Amino-4,4 alpha-dihydro-4 alpha,7-dimethyl-3H-phenoxazine-3-one (Phx) was synthesized by the reaction of 2-amino-5-methyl-phenol with bovine hemolysates. Since Phx is a phenoxazine derivative like actinomycin D, which exerts a strong anti-tumor effect by intercalating DNA, we examined the effects of Phx on cell proliferation and cell cycle progression in human epidermoid carcinoma cells (KB cells). Phx inhibited the proliferation of Kb cells in a dose-dependent manner. When KB cells were incubated for 9 h with medium containing 50 microM Phx, a transient accumulation of cells in S and G2/M phase was observed and at 24 h many of cells had lower DNA content. Although Phx had antitumor activity, the drug did not intercalate DNA, showing a different mode of action from actinomycin D.

Animals↗

PubMed1996

The prevalence of developmental anomalies of teeth and their association with tooth size in the primary and permanent dentitions of 1650 Japanese children.

The prevalence of microdontia, macrodontia, peg-shaped tooth, Carabelli's tubercle, protostylid, paramolar tubercle, central tubercle and palatal accessory cusp were examined in Japanese children. This study included 905 children with primary dentitions (mean age 4 years 7 months) and 745 high-school students with permanent dentitions (mean age 16 years 8 months). Microdontia, macrodontia, Carabelli's tubercle, protostylid and paramolar tubercle were more frequent in the primary dentition, whereas peg-shaped tooth, central tubercle and palatal accessory cusp were more frequent in the permanent dentition. The association between the presence of developmental anomalies and the size of the remaining teeth was significant in permanent dentitions. Both the literature and this study indicate that developmental anomalies of tooth number, size and morphology should be studied as a group rather than as isolates.

Adolescent↗

PubMed1996

Induction of cardiac angiotensinogen mRNA and angiotensin converting enzyme (ACE) activity in isoproterenol-induced heart injury.

Effects of isoproterenol (ISO) on the expression of cardiac angiotensinogen mRNA, angiotensin converting enzyme (ACE) activity, and mechanical functions in spontaneously hypertensive rats were investigated. In the acute phase, defined as within 24 h after the subcutaneous injection of ISO 85 mg/kg, cardiac angiotensinogen mRNA was slightly induced, but ACE activity was not. In the subacute phase, defined as within 8 d after ISO treatment on 2 successive d, both angiotensinogen mRNA expression and ACE activity in the heart were markedly induced. ACE activity in serum was not affected by ISO in either phase. In the subacute phase, ISO reduced body weight and blood pressure, increased ventricular weight and calcium content, and impaired cardiac mechanical function. Oral treatment with imidapril (10 mg/kg/d), an ACE inhibitor, 1 h before each ISO treatment and on the following 6 d, improved ventricular hypertrophy, the elevation of the left ventricular end diastolic pressure, the reduction in contractility, and the prolongation of the time constant. Imidapril significantly suppressed both serum and cardiac ACE activity but did not affect cardiac angiotensinogen mRNA expression in the subacute phase. These results indicate that enhancement of cardiac angiotensinogen mRNA and ACE activity is involved in ISO-induced cardiac dysfunction. Imidapril improved ISO-induced cardiac dysfunction, possibly by suppression of the local ACE activity as well as circulating ACE activity.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1995

DNA topoisomerase II is the molecular target of bisdioxopiperazine derivatives ICRF-159 and ICRF-193 in Saccharomyces cerevisiae.

Bisdioxopiperazines such as ICRF-159 and ICRF-193 have been shown to inhibit DNA topoisomerase II. To determine the molecular target of these compounds in vivo, we utilized a yeast genetic system in which the topoisomerase II activity can be modulated. To reduce topoisomerase II activity, we used top2-1 mutant yeast cells that have normal DNA topoisomerase II activity at 25 degrees C but greatly reduced enzyme activity at 30 degrees C, a temperature that is semipermissive for growth. At 25 degrees C top2-1 cells are as sensitive to the ICRF compounds as the wild-type strain; at 30 degrees C the cells became hypersensitive to these agents. In contrast, top2-1 strains become very resistant to the class of topoisomerase II inhibitors such as amsacrine and etoposide, which stabilize the covalent enzyme-DNA intermediate of the enzyme reaction. Overexpression of topoisomerase II from a plasmid-born TOP2 gene results in lower susceptibility to ICRF compounds and higher susceptibility to amsacrine than the parental strain exhibits. These results support the hypothesis that the main cellular target of ICRF compounds is DNA topoisomerase II, and that these compounds, unlike amsacrine and etoposide, inhibit topoisomerase II activity without stabilizing an enzyme-DNA covalent complex.

Amsacrine↗

PubMed1995

Synthesis of simian virus 40 C-family catenated dimers in vivo in the presence of ICRF-193.

The effect of ICRF-193, a non-cleavable, complex-stabilizing type of topoisomerase II inhibitor, on SV40 DNA replication in vivo was examined. As analyzed by one and two-dimensional gel electrophoresis, C-family catenated dimers, each composed of two intertwined, covalently closed SV40 DNAs, were mainly synthesized in the presence of the drug. On removal of the drug these C-family dimers were segregated into monomers. These results indicate that topoisomerase II is required for the segregation of replicated daughter molecules, but it is not absolutely required for the replication of DNA molecules up to the C-family dimers.

DNA Replication↗

PubMed1995

Single- and multiple-dose pharmacokinetics of AM-1155, a new 6-fluoro-8-methoxy quinolone, in humans.

The pharmacokinetics of AM-1155, a new 6-fluoro-8-methoxy quinolone, was examined in healthy male volunteers after the oral administration of a single dose of 100, 200, 400, or 600 mg and multiple doses of 300 mg twice daily for 6.5 days (13 total doses). Throughout the whole study period, AM-1155 was well tolerated in every subject. In the single-dose study, the concentrations in serum reached a peak between 1 and 2 h, and the peak concentrations were 0.873, 1.71, 3.35, and 5.41 micrograms/ml at the doses of 100, 200, 400, and 600 mg, respectively. The elimination half-life was 7 to 8 h, independently of the doses. The unchanged drug was excreted mainly in the urine, with 82 to 88% of the doses appearing for 72 h. The fecal recovery of the unchanged drug amounted to 5.7% for 72 h after a single oral administration of a 400-mg dose. Urinary excretion of metabolites was minimal. The serum protein binding was 20%, independently of the concentrations in serum. The concentrations in saliva were approximately 80% of those in serum. The intake of food had no effect on the pharmacokinetic parameters and urinary excretion of AM-1155 except the slight decrease in area under the concentration-time curve. The concurrent administration of probenecid prolonged the elimination half-life, increased the area under the concentration-time curve, and decreased the apparent total body clearance, renal clearance, urinary recovery of unchanged drug, and the excretion ratio (intrinsic renal clearance of AM-1155/creatinine clearance). This indicated that the tubular secretion contributed to the renal excretion of AM-1155. In the multiple-dose study, the concentrations of AM-1155 in serum and urine reached a steady state within 2 to 3 days. The measured concentrations in serum fitted well the simulation curve, which reflected the persistence of linear pharmacokinetics of AM-1155. In conclusion, AM-1155 is expected to be clinically useful because of its potent antibacterial activity and favorable pharmacokinetics.

Adult↗

PubMed1994

Antitumor bisdioxopiperazines inhibit yeast DNA topoisomerase II by trapping the enzyme in the form of a closed protein clamp.

The mechanism of inhibition of eukaryotic DNA topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] by a member of the bisdioxopiperazine family of anticancer drugs, ICRF-193, was investigated by using purified yeast DNA topoisomerase II. In the absence of ATP, ICRF-193 has little effect on the binding of the enzyme to various forms of DNA. In the presence of ATP, the drug converts the enzyme to a form incapable of binding circular DNA. Incubation of a preformed circular DNA-enzyme complex with ICRF-193 and ATP converts the complex to a form stable in molar concentrations of salt. These results can be interpreted in terms of the ATP-modulated protein-clamp model of type II DNA topoisomerases [Roca, J. & Wang, J. C. (1992) Cell 71, 833-840]; ICRF-193 can bind to the closed-clamp form of the enzyme and prevents its conversion to the open-clamp form. This interpretation is further supported by the finding that whereas both ATP and the drug are needed to form the salt-stable circular DNA-enzyme complex, ATP is not needed for maintaining this complex; furthermore, a signature of the closed-clamp form of the enzyme, Staphylococcus aureus strain V8 endoproteinase cleavage site at Glu-680, is observed if the enzyme is incubated with both ATP and ICRF-193. Inhibition of interconversion between the open- and closed-clamp forms of type II DNA topoisomerases offers a new mechanism in the selection and design of therapeutics targeting this class of enzymes.

Adenosine Triphosphate↗

PubMed1994

Inhibition of DNA topoisomerase II by ICRF-193 induces polyploidization by uncoupling chromosome dynamics from other cell cycle events.

ICRF-193, a novel noncleavable, complex-stabilizing type topoisomerase (topo) II inhibitor, has been shown to target topo II in mammalian cells (Ishida, R., T. Miki, T. Narita, R. Yui, S. Sato, K. R. Utsumi, K. Tanabe, and T. Andoh. 1991. Cancer Res. 51:4909-4916). With the aim of elucidating the roles of topo II in mammalian cells, we examined the effects of ICRF-193 on the transition through the S phase, when the genome is replicated, and through the M phase, when the replicated genome is condensed and segregated. Replication of the genome did not appear to be affected by the drug because the scheduled synthesis of DNA and activation of cdc2 kinase followed by increase in mitotic index occurred normally, while VP-16, a cleavable, complex-stabilizing type topo II inhibitor, inhibited all these processes. In the M phase, however, late stages of chromosome condensation and segregation were clearly blocked by ICRF-193. Inhibition at the stage of compaction of 300-nm diameter chromatin fibers to 600-nm diameter chromatids was demonstrated using the drug during premature chromosome condensation (PCC) induced in tsBN2 baby hamster kidney cells in early S and G2 phases. In spite of interference with M phase chromosome dynamics, other mitotic events such as activation of cdc2 kinase, spindle apparatus reorganization and disassembly and reassembly of nuclear envelopes occurred, and the cells traversed an unusual M phase termed "absence of chromosome segregation" (ACS)-M phase. Cells then continued through further cell cycle rounds, becoming polyploid and losing viability. This effect of ICRF-193 on the cell cycle was shown to parallel that of inactivation of topo II on the cell cycle of the ts top2 mutant yeast. The results strongly suggest that the essential roles of topo II are confined to the M phase, when the enzyme decatenates intertwined replicated chromosomes. In other phases of the cycle, including the S phase, topo II may thus play a complementary role with topo I in controlling the torsional strain accumulated in various genetic processes.

Animals↗