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K Kishi

Publications and source records attributed to K Kishi.

At least 181 records · Page 10Linked to original sources
PubMed1999

Effect of docetaxel on the therapeutic ratio of fractionated radiotherapy in vivo.

The aim of this investigation was to determine whether docetaxel increases the therapeutic ratio of fractionated radiotherapy in vivo. Two tumor types were chosen based on their sensitivity to docetaxel as a single agent: (a) docetaxel-sensitive MCa-4 mammary adenocarcinoma, which responds to docetaxel by G2-M-phase cell cycle arrest, apoptosis, and subsequent reoxygenation of surviving tumor cells; and (b) docetaxel-resistant SCC-VII squamous cell carcinoma, which responds to docetaxel treatment only by G2-M-phase arrest. Response of the normal jejunal mucosa in mice was compared to the response of both tumor types to confirm therapeutic gain. We conducted micromorphometric analysis of tumor cell mitosis, assayed apoptosis by its histological appearance in tissue sections, and determined tumor response by tumor growth delay. Normal tissue response of the jejunum was assayed by micromorphometric analysis of mitotic and apoptotic indices, and clonal crypt stem cell survival was measured using the microcolony assay. Two clinically relevant treatment schedules were tested for both antitumor efficacy and normal tissue toxicity: (a) a single bolus of docetaxel (33 mg/kg i.v.) 24 h before five daily fractions of radiation; and (b) daily administration of docetaxel (8 mg/kg i.v.) with radiation delivered at the peak of mitotic arrest (9 h for MCa-4 and 6 h for SCC-VII tumors). The best therapeutic gain for docetaxel-sensitive MCa-4 was achieved with a single bolus of drug 24 h before the start of fractionated radiotherapy (therapeutic gain = 2.04). This schedule takes advantage of reoxygenation of hypoxic tumor cells during the interval between drug treatment and radiation delivery. The best therapeutic gain for docetaxel-resistant SCC-VII was achieved with intermittent multiple doses of docetaxel given during the course of fractionated radiotherapy. This schedule maximized the exposure of cells to radiation while they were arrested by docetaxel in the radiosensitive G2-M phases of the cell cycle (enhancement factor = 2.0). Final therapeutic gain was reduced to 1.59 because of increased normal tissue toxicity in mice treated with multiple intermittent doses of docetaxel in combination with fractionated radiotherapy. Thus, docetaxel greatly enhanced tumor response to fractionated radiotherapy, but the magnitude of therapeutic efficacy depended on drug-radiation scheduling. The greatest therapeutic gain in the treatment of docetaxel-sensitive tumors was achieved by a single large dose of docetaxel administered 1 day before the initiation of fractionated radiotherapy and in the treatment of docetaxel-resistant tumors by daily concomitant docetaxel-radiation treatments.

Adenocarcinoma↗
PubMed1999

Deoxyribonuclease I phenotyping from saliva stains.

Good typing results were obtained using a newly developed method for extraction and purification of deoxyribonuclease I (DNase I) from saliva stains. Previously, DNase I phenotyping from saliva stains has been unsuccessful because of low enzyme activity and heavy contamination. Salivary DNase I was extracted from stains using phosphate buffer containing Nonidet P-40. Extracts were purified using Phenyl Sepharose CL-4B gel. Electrophoresis was performed, and DNase I was successfully phenotyped. All of the DNase I phenotypes, which were obtained from saliva stains using this new method, were identical to the phenotypes determined from urine samples. Moreover, DNase I was correctly phenotyped from saliva stains that had been stored for over three months at room temperature or at 37 degrees C. These results suggest that DNase I polymorphisms provide valuable information for forensic characterization of saliva stains.

Coloring Agents↗
PubMed1998

Whole body hyperthermia selectively induces heat shock protein 72 in neurons of the rat spinal cord.

The aims of this study were to examine whether the heat shock response is operative in the spinal cord, and to identify the type of responsible cell. Immunoblot analysis using an antibody specific for a highly stress-inducible heat shock protein with a molecular mass of 72 kDa (HSP72) showed that exposing rats to whole body hyperthermia remarkably induced HSP72 protein in the spinal cord within 2 h. Northern blot analysis with a cDNA probe for human HSP72 demonstrated that whole body hyperthermia induced the expression of HSP72 mRNA within 30 min in the spinal cord. Immunohistochemical analysis showed that neurons in the gray matter appear to be a preferential target of the heat shock response, suggesting that the heat shock response might have a therapeutic implication for protection against spinal cord injury.

Animals↗
PubMed1998

Macrophage colony-stimulating factor stimulates synthesis and secretion of a mouse homolog of a human IgE-dependent histamine-releasing factor by macrophages in vitro and in vivo.

Treatment of murine resident peritoneal macrophages with macrophage-CSF (M-CSF) up-regulated the synthesis of a discrete set of proteins, including a 26-kDa protein (p26). The sequence of 20 NH2-terminal amino acids of the purified p26 was identical with the mouse homolog of a human IgE-dependent histamine-releasing factor (HRF). Among macrophage activators tested (M-CSF, granulocyte-macrophage-CSF, IL-3, TNF-alpha, IFN-gamma, and LPS), only M-CSF could up-regulate the p26 HRF synthesis by cultured macrophages. M-CSF not only increased the levels of p26 HRF mRNA and protein, but also stimulated the secretion of an N-glycosylated p26 HRF with a m.w. of 30 kDa. Repeated injections of M-CSF into mouse peritoneal cavity for 4 days elicited macrophages expressing abundant p26 HRF. A single i.p. injection of M-CSF failed to increase the p26 HRF level in peritoneal macrophages of thioglycollate-, LPS-, or adjuvant-treated mice, while M-CSF challenge to OVA-immunized mice caused macrophage infiltration and overproduction of p26 HRF, similarly as did OVA challenge. The Ag-specific priming for enhanced synthesis and secretion of p26 HRF by M-CSF was also demonstrated in cultured macrophages prepared from OVA-immunized mice. An i.p. injection of M-CSF or recombinant p26 HRF triggered eosinophil recruitment, even in the absence of the Ag, in the sensitized mice, but not in normal mice. Furthermore, recombinant p26 HRF could induce eosinophilia without marked macrophage and lymphocyte infiltrations. Our results suggest that p26 HRF secreted by M-CSF-stimulated macrophages may be an important mediator for the late phase allergic inflammation.

Animals↗
PubMed1998

Identification of the three non-identical subunits constituting human deoxyribonuclease II.

We purified DNase II from human liver to apparent homogeneity. The N-terminal amino acid sequences of each of three components constituting the purified mature enzyme were then separately determined by automatic Edman degradation. A combination of this chemical information and the previously reported nucleotide sequence of the cDNA encoding human DNase II [Yasuda et al. (1998) J. Biol. Chem. 273, 2610-2626] allowed detailed elucidation of the enzyme's subunit structure: human DNase II was composed of three non-identical subunits, a propeptide, proprotein and mature protein, following a signal peptide. Expression analysis of a series of deletion mutants derived from the cDNA of DNase II in COS-7 cells suggested that although a single large precursor protein may not be necessary for proteolytic maturation, the propeptide region L17-Q46 may play an essential role in generating the active form of the enzyme.

Amino Acid Sequence↗
PubMed1998

Mouse placental cells secrete soluble leptin receptor (sOB-R): cAMP inhibits sOB-R production.

The aims of this study were to identify whether mouse placenta secretes soluble OB-R (sOB-R) and to find the regulating factor of OB-R expression. Total RNAs were extracted from placenta and decidua, and OB-R expression was assessed by Northern blot analysis. Decidua did not express OB-R mRNA. However, OB-R mRNA expression was detectable in the placenta on day 13 of pregnancy, and then it increased and reached a peak on day 17 of pregnancy. Mouse placental cells from day 12 of pregnancy were cultured and OB-R gene expression was assessed by Northern blot analysis. OB-R mRNA expression was detectable from the second day of culture and reached a peak on the third day of culture. To determine whether placental cells release sOB-R, supernatant of cultured placental cells was subjected to Western blot analysis. sOB-R was detected in the medium by the second day of culture and sOB-R release increased up to the fourth day of culture. Addition of leptin to the medium did not affect expression of OB-R mRNA. However, 8-bromo cAMP inhibited both steady-state levels of OB-R mRNA and the amount of sOB-R protein in the medium in a dose- and time-dependent manner. These results suggest that trophoblast cells differentiate, express, and release sOB-R both in vivo and in vitro and that cAMP is one of several potent regulators of sOB-R secretion by the mouse placenta.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

CD4(+) cytotoxic T-cell clones specific for bcr-abl b3a2 fusion peptide augment colony formation by chronic myelogenous leukemia cells in a b3a2-specific and HLA-DR-restricted manner.

Although it is well known that CD8(+) cytotoxic T lymphocytes (CTLs) play an important role in the suppression of cancer cell growth, the significance of CD4(+) CTLs in resistance to cancer is obscure. In an attempt to elucidate the role of CD4(+) CTLs in immunosurveillance of chronic myelogenous leukemia (CML), we examined the immunologic functions of bcr-abl b3a2 fusion peptide-specific CD4(+) CTL clones. Seven CD4(+) T-cell clones that responded to stimulation with b3a2 peptide, but not with b2a2 peptide or physiological counterparts bcr b3b4 and abl 1A-a2 peptides, were established from two healthy individuals. Restriction elements of these clones were HLA-DRB1*0901. These CD4(+) T-cell clones exhibited b3a2 peptide-specific and HLA-DRB1*0901-restricted cytotoxicity and produced interleukin-3 (IL-3), IL-4, IL-10, interferon-gamma, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor in response to bcr-abl peptide stimulation, indicating they were Th0 clones. The numbers of HLA-DRB1*0901-positive b3a2, but not those of b2a2-positive or HLA-DRB1*0901-negative CML cell colonies increased when CML cells were cultured with b3a2-specific CD4(+) CTL clones. These data suggest that bcr-abl-specific CD4(+) CTLs recognize CML cells in an antigen-specific and HLA-DR-restricted manner, and that they do not inhibit, but in fact augment, CML cell growth.

Amino Acid Sequence↗
PubMed1998

Reconstitution of insulin signaling pathways in rat 3Y1 cells lacking insulin receptor and insulin receptor substrate-1. Evidence that activation of Akt is insufficient for insulin-stimulated glycogen synthesis or glucose uptake in rat 3Y1 cells.

Rat 3Y1 cells have endogenous insulin-like growth factor-1 receptors and insulin receptor substrate (IRS)-2, but lack both insulin receptor (IR) and IRS-1. To investigate the role of IR and IRS-1 in effects of insulin, we transfected IR and IRS-1 expression plasmids into cells and reconstituted the insulin signaling pathways. 3Y1 cells stably expressing the c-myc epitope-tagged glucose transporter type 4 (3Y1-GLUT4myc) exhibit no effects of insulin, at physiological concentrations. The 3Y1-GLUT4myc-IR cells expressing GLUT4myc and IR responded to phosphatidylinositol 3,4, 5-trisphosphate (PI-3,4,5-P3) accumulation, Akt activation, the stimulation of DNA synthesis, and membrane ruffling but not to glycogen synthesis, glucose uptake, or GLUT4myc translocation. The further expression of IRS-1 in 3Y1-GLUT4myc-IR cells led to stimulation of glycogen synthesis but not to glucose uptake or GLUT4myc translocation in response to insulin, although NaF or phorbol 12-myristate 13-acetate did trigger GLUT4myc translocation in the cells. These results suggest that, in rat 3Y1 cells, (i) IRS-1 is essential for insulin-stimulated glycogen synthesis but not for DNA synthesis, PI-3,4,5-P3 accumulation, Akt phosphorylation, or membrane ruffling, and (ii) the accumulation of PI-3,4,5-P3 and activation of Akt are insufficient for glycogen synthesis, glucose uptake or for GLUT4 translocation.

Amino Acid Sequence↗
PubMed1998

An ultrastructural study of p75 neurotrophin receptor-immunoreactive fiber terminals in the reticular thalamic nucleus of young rats.

The reticular thalamic nucleus (RT) receives cholinergic fibers from both the basal forebrain and the brainstem. Recent studies have shown that the p75 neurotrophin receptor (p75NTR) is synthesized in cholinergic neurons in the basal forebrain but not in those in the brainstem. In this study, to identify cholinergic fibers originating from the basal forebrain, we used a monoclonal antibody against p75NTR (192-IgG) and characterized the ultrastructure of the immunoreactive fiber terminals in the rostral part of the RT in 3-week-old rats. Light microscopy revealed that p75NTR-immunoreactive fine fibers and varicosities were distributed throughout the nucleus. From electron micrographs, three types of labeled terminals were identified. The first type of labeled fiber terminals (63 out of 106) was consistently small, contained densely packed vesicles, and established asymmetrical synaptic contacts with heavy and bushy postsynaptic thickening on distal dendritic profiles; the second type (18 out of 106) established asymmetrical synaptic contacts with very slight postsynaptic thickening; and the third type (25 out of 106) of labeled terminals contained pleomorphic vesicles and established symmetrical synaptic contacts with more proximal dendritic surfaces than the first two types. In addition to the above, labeled dendritic profiles receiving non-labeled asymmetrical and symmetrical synaptic contacts were identified. These findings suggest that the basal forebrain cholinergic system establishes a variety of synaptic connections in the RT and influences cortical activity indirectly via thalamocortical pathways, as well as via direct projections to the cortex.

Animals↗
PubMed1998

Induction of differentiation in acute promyelocytic leukemia cells by 9-cis retinoic acid alpha-tocopherol ester (9-cis tretinoin tocoferil).

Acute promyelocytic leukemia (APL) has a specific genetic rearrangement between the retinoic acid receptor (RAR)-alpha gene and the pml nuclear protein gene. All-trans retinoic acid (ATRA) induces granulocytic differentiation of APL-derived cells and is used to treat APL patients. However, ATRA interacts with normal cells with RAR throughout the entire body, and when used at high doses or over a long duration, it induces several adverse effects. The development of drugs that selectively act on APL cells may contribute to increasing the therapeutic efficacy of APL treatment as well as elucidating the mechanisms of response to ATRA. In this study, 9-cis retinoic acid alpha-tocopherol ester (9CTT) inhibited the proliferation of APL-derived NB4 and HT93 cells and induced differentiation markers, such as granulocytic maturation, nitroblue tetrazolium reduction, and CD11b expression, in these cells. The effects of 9CTT on non-APL cells, including HL-60 and U937 cells, were much weaker than those on APL cells, and tretinoin tocoferil (TT), which is an alpha-tocopherol ester of ATRA, did not induce the differentiation of APL cells as effectively as 9CTT. The differentiation-inducing effects of 9CTT were inhibited by RAR antagonists. 9CTT and TT similarly induced the transactivating activity of RARs, but were not effective on RXRs. 9CTT downregulated the expression of PML/RAR-alpha protein more effectively than TT, which suggests that it may be involved in the selectivity of 9CTT against APL cells. Interestingly, 9CTT enhanced the differentiation of APL cells induced by ATRA, 9-cis retinoic acid, and synthetic retinobenzoic acids. Combined with 1alpha,25-dihydroxyvitamin D3 (VD3), 9CTT also more than additively induced the differentiation of APL cells. Thus, 9CTT, alone or in combination with other retinoids or VD3, may be useful for the treatment of APL.

Cell Differentiation↗
PubMed1998

pH-linked binding of Mn(II) to manganese peroxidase.

The stability of Mn(II) binding to manganese peroxidase (MnP) has been studied as a function of pH by spectrophotometric and potentiometric titrations. The sensitivity of the potentiometric titrations allows collection of data that are consistent with a high-affinity and a low-affinity Mn(II) binding site on the peroxidase. The two sites differ in affinity by 4 to 900-fold between pH 4 and 6.5. The stability of Mn(II) binding to the high-affinity site increases with increasing pH, while the stability of Mn(II) binding to the low-affinity site decreases with increasing pH. Interestingly, at pH values above 5.0, the high-affinity site appears to be partially unavailable for binding Mn(II). A pH-dependent structural change in the Mn(II) binding site is proposed to account for this partial inactivation at elevated pH.

Basidiomycota↗
PubMed1998

GLUT4 translocation by insulin in intact muscle cells: detection by a fast and quantitative assay.

We report a rapid and sensitive colorimetric approach to quantitate the amount of glucose transporters exposed at the surface of intact cells, using L6 muscle cells expressing GLUT4 containing an exofacial myc epitope. Unstimulated cells exposed to the surface 5 fmol GLUT4myc per mg protein. This value increased to 10 fmol/mg protein in response to insulin as 2-deoxyglucose (10 microM) uptake doubled. The results are substantiated by immunofluorescent detection of GLUT4myc in unpermeabilized cells and by subcellular fractionation. We further show that wortmannin and the cytoskeleton disruptors cytochalasin D and latrunculin B completely blocked these insulin effects. The rapid quantitative assay described here could be of high value to study insulin signals and to screen for potential anti-diabetic drugs.

Androstadienes↗
PubMed1998

Phenotype 2 of deoxyribonuclease I may be used as a risk factor for gastric carcinoma.

BACKGROUND: Human deoxyribonuclease I (DNase I) (EC3.1.21.1) is one of the candidate nucleases that acts in mammalian cells during apoptosis. Genetic polymorphism of DNase I has been classified into 15 phenotypes controlled by 5 autosomal codominant alleles. The purpose of this study was to determine whether DNase I polymorphism is closely related to the incidence of gastric carcinoma. METHODS: The phenotype distribution was examined using urine samples obtained from unrelated Japanese patients with gastric carcinoma (n = 97) and benign gastric diseases (n = 76). Phenotyping was performed using isoelectric focusing electrophoresis in thin polyacrylamide gel and immunoblotting with an antihuman DNase I antibody. RESULTS: A close statistical association was found between patients with gastric carcinoma and a high frequency of DNase I phenotype 2. However, there was no significant difference in the phenotype distribution between the group of patients with benign gastric diseases and the controls. CONCLUSIONS: The findings of the current study suggest that DNase I phenotype 2 may be a marker for gastric carcinoma. Therefore, compared with other phenotypes, DNase I phenotype 2 may have potential for identifying patients who are at risk of harboring or developing gastric carcinoma.

Adult↗
PubMed1998

Dietary carbohydrates enhance lactase/phlorizin hydrolase gene expression at a transcription level in rat jejunum.

We have previously shown that dietary sucrose stimulates the lactase/phlorizin hydrolase (LPH) mRNA accumulation along with a rise in lactase activity in rat jejunum [Goda, Yasutake, Suzuki, Takase and Koldovský (1995) Am. J. Physiol. 268, G1066-G1073]. To elucidate the mechanisms whereby dietary carbohydrates enhance the LPH mRNA expression, 7-week-old rats that had been fed a low-carbohydrate diet (5.5% of energy as starch) were given diets containing various monosaccharides or sucrose for 12h. Among carbohydrates examined, fructose, sucrose, galactose and glycerol elicited an increase in LPH mRNA accumulation along with a rise in lactase activity in the jejunum. By contrast, glucose and alpha-methylglucoside were unable to elicit a significant increase in LPH mRNA levels. To explore a transcriptional mechanism for the carbohydrate-induced increases in LPH mRNA levels, we employed two techniques currently available to estimate transcriptional rate, i.e. RNA protection assays of pre-mRNA using an intron probe, and nuclear run-on assays. Both assays revealed that fructose elicited an increase in transcription of the LPH gene, and that the transcription of LPH was influenced only slightly, if at all, by glucose intake. These results suggest that certain monosaccharides such as fructose or their metabolite(s) are capable of enhancing LPH mRNA levels in the small intestine, and that transcriptional control might play a major role in the carbohydrate-induced increase of LPH mRNA expression.

Animals↗
PubMed1998

Chromosomal localization of a human deoxyribonuclease II gene (DNASE2) to 19p13.2-p13.1 using both the polymerase chain reaction and fluorescence in situ hybridization analysis.

Recently obtained information on the cDNA encoding human deoxyribonuclease II (DNase II) (T. Yasuda et al., 1998, J. Biol. Chem. 273, 2610-2616) has made it possible to demonstrate the precise position of the the human DNase II gene (DNASE2) on human chromosomes. Two different sets of oligonucleotide primers specific for human DNase II cDNA sequences were used to amplify unique DNA fragments in the human DNase II gene from a panel of human x rodent hybrid cell lines carrying different human chromosomes. Based on this analysis, DNASE2 was assigned to human chromosome 19. Furthermore, regional localization of the gene to 19p13.2-p13.1 was achieved by fluorescence in situ hybridization analysis using a full-length cDNA probe corresponding to the entire open reading frame.

Chromosome Mapping↗
PubMed1998

Molecular cloning of the cDNA encoding human deoxyribonuclease II.

A rapid amplification of cDNA ends method, using degenerate oligonucleotides based upon the N-terminal amino acid sequence of human hepatic deoxyribonuclease II (DNase II), allowed a novel cDNA encoding DNase II to be constructed from thyroid gland RNA. The composite nucleotide sequence (1593 bases) included an open reading frame of 1080 bases, which encoded a single polypeptide of 360 amino acids (signal peptide, 16; propeptide, 91; mature protein, 253). Although the sequence of DNase II showed no significant homology to other mammalian proteins, its cDNA structural organization resembled those of the lysosomal cathepsin families. The two parts of the cDNA corresponding to the propeptide and the mature protein were expressed in Escherichia coli, and the recombinant polypeptides thus obtained were strongly stained with an anti-DNase II antibody on Western blotting. DNase II is ubiquitously expressed in human tissues, and the DNase II gene (DNASE2) was assigned to chromosome 19.

Amino Acid Sequence↗
PubMed1998

Detection of isozymes of deoxyribonucleases I and II on electrophoresed gels with picogram sensitivity using SYBR Green I.

A highly sensitive method for detecting deoxyribonucleases (DNases) I and II on an electrophoresed gel is described. A dried agarose film sheet containing DNA as a substrate and a buffer reagent was placed in contact with the gel surface after electrophoresis (DAFO method, Yasuda et al., Anal. Biochem. 1989, 183, 84-88). After an appropriate incubation period, the film sheet was peeled off and stained with SYBR-Green I (SG), and then the DNase isozyme bands were detected using a fluorescence image analyzer. We could detect pg levels of the DNases (DNase I, 2 pg; DNase II, 2pg), which represents a 32- to 128-fold increase in sensitivity compared with the original DAFO method using ethidium bromide (EB) as the fluorescent dye. A combination of this new detection method and isoelectric focusing electrophoresis in polyacrylamide gel allowed accurate DNase I typing from 1 microL human serum. This new technique has been named SG-DAFO, after its original dried agarose film overlay method using EB (EB-DAFO).

Benzothiazoles↗