Biomedical subjects

Y Furukawa

Publications and source records attributed to Y Furukawa.

At least 55 records · Page 3Linked to original sources

PubMed2001

In vitro cytotoxic effects of a tyrosine kinase inhibitor STI571 in combination with commonly used antileukemic agents.

The BCR/ABL tyrosine kinase has been implicated in the pathogenesis of chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL). STI571 is a novel anticancer agent that selectively inhibits the BCR/ABL tyrosine kinase. The cytotoxic effects of STI571 were studied in combination with antileukemic agents against Ph(+) leukemia cell lines, KU812, K-562, TCC-S, and TCC-Y. The cells were exposed to STI571 and to other agents simultaneously for 5 or 7 days. Cell growth inhibition was determined by MTT assay. The cytotoxic effects in combinations at the inhibitory concentration of 80% level were evaluated by the isobologram. STI571 produced synergistic effects with recombinant and natural alpha-interferons in 2 of 3 and 3 of 3 cell lines, respectively. STI571 produced additive effects with hydroxyurea, cytarabine, homoharringtonine, doxorubicin, and etoposide in all 4 cell lines. STI571 with 4-hydroperoxy-cyclophosphamide, methotrexate, or vincristine produced additive, antagonistic, and synergistic effects in 3 of 4 cell lines, respectively. These findings suggest that the simultaneous administration of STI571 with other agents except methotrexate would be advantageous for cytotoxic effects against Ph(+) leukemias. Among them, the simultaneous administration of STI571 and alpha-interferons or vincristine would be highly effective against Ph(+) leukemias and these combinations would be worthy of clinical trials. In contrast, the simultaneous administration of STI571 with methotrexate would have little therapeutic efficacy. Although there are gaps between in vitro studies and clinical trials, the present findings provide useful information for the establishment of clinical protocols involving STI571. (Blood. 2001;97:1999-2007)

Antineoplastic Agents↗

PubMed2001

CD8+ T cell subsets TC1 and TC2 cause different histopathologic forms of murine cardiac allograft rejection.

BACKGROUND: CD4+ T cell effector function is sufficient to mediate allograft rejection, and it is suggested that CD8+ T cell-mediated effects are dependent on CD4+ T cell help. CD8+ T cells can be classified into at least two functional subsets: Tc1, producing high amounts of interferon (IFN)-gamma and Tc2, producing interleukin (IL)-4, -5, -10, and -13 and low levels of IFN-gamma. Because these subsets express different chemokine receptors, they may have different capabilities of migrating into grafts. Once in the graft, each subset may perform different effector functions dependent on the cytokines it produces. We asked whether allospecific CD8+ T cells, in the absence of CD4+ T cells, are capable of mediating rejection of a primarily vascularized allograft, and if Tcl and Tc2 cells differ in their ability to mediate rejection. METHODS: Hearts from H-2d mice were transplanted into H-2b RAG 1-/- recipients. Without manipulation, these fully mismatched allografts would survive indefinitely due to the absence of mature T and B cells. We adoptively transferred allo-(H-2d)-reactive Tcl or Tc2 cells from H-2b mice into each recipient. Grafts were harvested and analyzed on predefined timepoints, rejection was graded on a modified ISHLT scale. RESULTS: On day 7, grafts from Tc1- or Tc2-injected animals showed grade 1-2 parenchymal rejection with stable phenotype and comparable distribution of graft infiltrating CD8+ T cells. Adoptive transfer of IFN-gammahigh Tc1, but not of IFN-gammalow Tc2 cells was followed by the development of graft vasculitis, as well as graft arteriopathy. Adoptive transfer of IL-4high IL-5high Tc2, but not of IL-4low IL-5low Tc1 cells lead to extensive infiltration of eosinophils and formation of giant cells. CONCLUSIONS: Both Tc1 and Tc2 cells can mediate murine cardiac allograft rejection in the absence of CD4+ T cell help, although each subset elicits a different type of inflammatory response. In this model, cytokine secretion of either functional CD8+ T effector cell subset is an important effector mechanism in the process of allograft rejection: IFN-gammahigh Tc1 cells are important in early graft vasculitis, although IL-4high IL-5high Tc2 cells promote recruitment of secondary effectors like eosinophils.

Adoptive Transfer↗

PubMed2001

Genome-wide analysis of gene expression in human hepatocellular carcinomas using cDNA microarray: identification of genes involved in viral carcinogenesis and tumor progression.

To disclose detailed genetic mechanisms in hepatocellular carcinoma (HCC) with a view toward development of novel therapeutic targets, we analyzed expression profiles of 20 primary HCCs and their corresponding noncancerous tissues by means of cDNA microarrays consisting of 23,040 genes. Up-regulation of mitosis-promoting genes was observed in the majority of the tumors examined. Some genes showed expression patterns in hepatitis B virus-positive HCCs that were different from those in hepatitis C virus-positive HCCs; most of them encoded enzymes that metabolize carcinogens and/or anticancer agents. Furthermore, we identified a number of genes associated with malignant histological type or invasive phenotype. Accumulation of such data will make it possible to define the nature of individual tumors, to provide clues for identifying new therapeutic targets, and ultimately to optimize treatment of each patient.

Carcinoma, Hepatocellular↗

PubMed2001

Regional distribution of methamphetamine in autopsied brain of chronic human methamphetamine users.

We measured levels of methamphetamine and those of its metabolite amphetamine in 15 autopsied brain regions of 14 human methamphetamine users. Only slight regional differences were observed in drug concentrations among the brain areas. Although, some redistribution of the drugs probably occurred postmortem, these data suggest that methamphetamine might not be preferentially retained in dopamine-rich brain areas but is heterogenously distributed in brain of chronic human users of the drug. The possible pharmacological actions of methamphetamine in both dopamine-rich and poor brain areas of chronic drug users need to be considered.

Adult↗

PubMed2001

Mutations in the NMMHC-A gene cause autosomal dominant macrothrombocytopenia with leukocyte inclusions (May-Hegglin anomaly/Sebastian syndrome).

Macrothrombocytopenia with leukocyte inclusions is a rare autosomal dominant platelet disorder characterized by a triad of giant platelets, thrombocytopenia, and characteristic Döhle body-like leukocyte inclusions. A previous study mapped a locus for the disease on chromosome 22q12.3-q13.2 by genome-wide linkage analysis. In addition, the complete DNA sequence of human chromosome 22 allowed a positional candidate approach, and results here indicate that the gene encoding nonmuscle myosin heavy chain-A, NMMHC-A, is mutated in this disorder. Mutations were found in 6 of 7 Japanese families studied: 3 missense mutations, a nonsense mutation, and a one-base deletion resulting in a premature termination. Immunofluorescence studies revealed that NMMHC-A distribution in neutrophils appeared to mimic the inclusion bodies. These results provide evidence for the involvement of abnormal NMMHC-A in the formation of leukocyte inclusions and also in platelet morphogenesis.

Amino Acid Sequence↗

PubMed2001

Existence of escape mutant in HTLV-I tax during the development of adult T-cell leukemia.

Although Tax protein is the main target of cytotoxic T lymphocyte (CTL) on human T-cell lymphotropic virus type I (HTLV-I)-infected cells, and Tax peptide 11 through 19 binding to HLA-A*02 has been shown to elicit a strong CTL response, there are patients with adult T-cell leukemia (ATL) bearing HLA-A*02. To explore whether there is genetic variation in HTLV-I tax that can escape CTL recognition during the development of ATL, the HTLV-I tax gene was sequenced in 55 patients with ATL, 61 patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and 62 healthy carriers, and it was correlated with the presence of HLA-A*02. First, a premature stop codon in the 5' half of the tax gene that looses transactivation activity on the viral enhancer was observed in 3 patients with acute and 1 patient with chronic ATL. This stop codon was revealed to emerge after the viral transmission to the patient from sequence analysis in family members with ATL. Second, amino acid change in Tax peptide 11-19 was observed in 3 patients with ATL. CTL assays demonstrated that this altered Tax 11-19 peptide, observed in ATL patients with HLA-A*02, was not recognized by Tax 11-19-specific CTL. Two patients with ATL had large deletions in tax by sequencing, and 5 patients with ATL had deletions in HTLV-I by Southern blotting. These findings suggest that at some stage of ATL development, HTLV-I-infected cells that can escape the host immune system are selected and have a chance to accumulate genetic alterations for further malignant transformation, leading to acute ATL.

Codon, Terminator↗

PubMed2001

Direct transcriptional activation of human caspase-1 by tumor suppressor p53.

The tumor suppressor protein p53 is a sequence-specific DNA-binding protein, and its biological responses are very often mediated by transcriptional activation of various target genes. Here we show that caspase-1 (interleukin-1beta converting enzyme), which plays a role in the production of proinflammatory cytokines and in apoptosis, is a transcriptional target of p53. Caspase-1 mRNA levels increased upon overexpression of p53 by transfection in MCF-7 cells. Human caspase-1 promoter showed a sequence homologous to the consensus p53-binding site. This sequence bound to p53 in gel shift assays. A caspase-1 promoter-reporter construct was activated 6-8-fold by cotransfection with normal p53 but not by mutant p53 (His(273)) in HeLa, as well as MCF-7, cells. Mutation of the p53-binding site in caspase-1 promoter abolished transactivation by p53. Treatment of p53-positive MCF-7 cells with the DNA-damaging drug, doxorubicin, which increases p53 levels, enhanced caspase-1 promoter activity 4-5-fold, but similar treatment of MCF-7-mp53 (a clone of MCF-7 cells expressing mutant p53) and p53-negative HeLa cells with doxorubicin did not increase caspase-1 promoter activity. Doxorubicin treatment increased caspase-1 mRNA levels in MCF-7 cells but not in MCF-7-mp53 or HeLa cells. These results show that endogenous p53 can regulate caspase-1 gene expression.

Caspase 1↗

PubMed2001

Dopa-responsive dystonia simulating spastic paraplegia due to tyrosine hydroxylase (TH) gene mutations.

Spastic paraplegia is not widely recognized to occur in dopa-responsive dystonia (DRD). The authors found a compound heterozygote for novel mutations of the human tyrosine hydroxylase (TH) gene (TH). The patient was initially diagnosed as having spastic paraplegia, but responded completely to levodopa therapy. Exercise-induced stiffness in the patient's father, who had a TH deletion, also responded to levodopa. The data expand the clinical spectrum of TH deficiency and suggest that TH mutations may account for some patients with DRD simulating spastic paraplegia.

Child↗

PubMed2001

Downregulation of an AIM-1 kinase couples with megakaryocytic polyploidization of human hematopoietic cells.

During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3-dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-ras(G12V)), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes.

Animals↗

PubMed2001

An HMG-CoA reductase inhibitor, cerivastatin, suppresses growth of macrophages expressing matrix metalloproteinases and tissue factor in vivo and in vitro.

BACKGROUND: Unstable atherosclerotic plaques that cause acute coronary events usually contain abundant macrophages expressing matrix metalloproteinases (MMPs) and tissue factor (TF), molecules that probably contribute to plaque rupture and subsequent thrombus formation. Lipid lowering with HMG-CoA reductase inhibitors reduces acute coronary events. METHODS AND RESULTS: To test whether lipid lowering with an HMG-CoA reductase inhibitor retards macrophage accumulation in rabbit atheroma, we administered cerivastatin to immature Watanabe heritable hyperlipidemic rabbits (cerivastatin group, n=10, cerivastatin 0.6 mg x kg(-1) x d(-1); control group, n=9, saline 0.6 mL x kg(-1) x d(-1)) for 32 weeks and measured macrophage accumulation and expression of MMPs and TF. Serum cholesterol levels after 32 weeks were 809+/-40 mg/dL (control group) and 481+/-24 mg/dL (treated group). Cerivastatin diminished accumulation of macrophages in aortic atheroma. Macrophage expression of MMP-1, MMP-3, MMP-9, and TF also decreased with cerivastatin treatment. Cerivastatin reduced the number of macrophages expressing histone mRNA (a sensitive marker of cell proliferation) detected by in situ hybridization but did not alter macrophages bearing a marker of death (TUNEL staining). Cerivastatin treatment (>or=0.01 micromol/L) also reduced growth, proteolytic activity due to MMP-9, and TF expression in cultured human monocyte/macrophages. CONCLUSIONS: These results suggest that lipid lowering with HMG-CoA reductase inhibitors alters plaque biology by reducing proliferation and activation of macrophages, prominent sources of molecules responsible for plaque instability and thrombogenicity.

Animals↗

PubMed2001

The role of water molecules in the association of cytochrome P450cam with putidaredoxin. An osmotic pressure study.

We have investigated the osmotic pressure dependence of the association between ferric cytochrome P450cam and putidaredoxin (Pdx) to gain an insight into the role of water molecules in the P450cam-reduced Pdx complexation amenable to physiological electron transfer. The association constant was evaluated from the electron transfer rates from reduced Pdx to P450cam. The natural logarithm of the association constant K(a) was linearly reduced by the osmotic pressure, and osmotic stress yields uptake of 25 waters upon association. In contrast, uptake of only 13 waters is observed from the osmotic pressure dependence of the association in the nonphysiological redox partners P450cam and oxidized Pdx. Although general protein-protein associations proceed through dehydration around the complex interface, the interfacial waters could mediate hydrogen-bonding interactions. Therefore, about 10 more interfacial waters imply an additional water-mediated hydrogen-bonding network in the P450cam.reduced Pdx complex, which does not exist in the complex with oxidized Pdx. It is also possible that the water-mediated hydrogen-bonding interactions support a high P450cam affinity for reduced (K(a) = 0.83 microm(-1)) relative to oxidized (K(a) = 0.058 microm(-1)) Pdx. This study points to a novel role of solvents in assisting redox state-dependent interaction between P450cam and Pdx.

Bacterial Proteins↗

PubMed2001

Purification and some properties of alanine racemase from a bivalve mollusc Corbicula japonica.

A brackish-water mollusc, Corbicula japonica, uses large quantities of D- and L-alanine as intracellular osmotically active solutes, osmolytes, for regulation of intracellular osmolarity. We purified alanine racemase from the mantle of C. japonica to characterize its enzymological properties. The molecular masses of the enzyme were estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 140 kDa by gel filtration on high-performance liquid chromatography, suggesting the trimeric or tetrameric nature of the enzyme. Neither dialysis nor chromatographic procedures in the absence of pyridoxal 5'-phosphate led to loss of enzyme activity, although carbonyl reagents, hydroxylamine and phenylhydrazine, inhibited the activity. These results suggest that alanine racemase of the animal may bind pyridoxal 5'-phosphate tightly as a cofactor. Kinetic experiments using the partially purified enzyme revealed that alanine was the sole substrate among 17 kinds of L-amino acids tested. The Lineweaver-Burk plot for L-alanine as substrate resulted in Km value of 22.6 mM, and the value for D-alanine was 9.2 mM. Together with the previous evidence that D- and L-alanine levels of this animal change with the external salinity maintaining the D-/L-alanine ratio at unity, the present results seem to indicate that the physiological role of alanine racemase in this animal is to supply D-alanine as a main intracellular osmolyte. J. Exp. Zool. 289:1-9, 2001.

Alanine↗

PubMed2001

Detection of dichromate (VI)-induced DNA strand breaks and formation of paramagnetic chromium in multiple mouse organs.

DNA single-strand breaks (and/or alkali-labile sites) induced by Cr(VI) were evaluated with the alkaline single cell gel electrophoresis (SCG) (Comet) assay in five organs (liver, kidney, spleen, lung, and brain) of male mice dosed with K(2)Cr(2)O(7) (20 mg Cr/kg) by a single ip injection in vivo, and the formation of paramagnetic Cr(V) in these organs was investigated by electron spin resonance (ESR) spectrometry. Furthermore, the in vivo effects of deferoxamine (DFO), an iron chelator, and dimethylthiourea (DMTU), a hydroxyl radical scavenger, on the formation of Cr(V) and DNA strand breaks induced by the metal in the liver and kidney were examined. SCG assay detected DNA strand breaks were detected in the liver and kidney at 15 min and showed that they were being repaired at 3 h after Cr(VI) injection. The ESR spectra of paramagnetic Cr(V) were also observed in the liver and kidney for 15 min to 24 h after Cr(VI) injection. In contrast, there were no significant levels of DNA strand breaks and Cr(V) in the spleen, lung, or brain. The pretreatment of mice with DFO reduced the formation of Cr(VI)-induced DNA strand breaks and Cr(V) complexes as well as the total contents of Cr in the liver and kidney at 15 min after the metal injection. In the case of the pretreatment with DMTU, DNA strand breaks induced by Cr(VI) were suppressed in the liver and kidney at 15 min, without any influence on the levels of Cr(V) complexes and total Cr contents in the organs. The in vitro study showed that DFO decreased the levels of Cr(V)-GSH complexes and Cr(V)-mediated hydroxyl radicals, while DMTU reduced only the levels of Cr(V)-mediated hydroxyl radicals without affecting the formation of Cr(V)-GSH complexes. These results demonstrated that the SCG assay may be useful for detecting DNA strand breaks and/or alkali-labile sites caused by Cr(VI) in vivo. The results also indicated that the in vivo formation of hydroxyl radicals during the reduction of Cr(VI) may play an important role in the induction of the DNA strand breaks caused by this metal and implied that the levels of Cr(V) inside the cells may not always be related to the induction of DNA strand breaks.

Animals↗

PubMed2001

Fixed-height exit bender of synchrotron X-rays above 40 keV.

A crystal bender for sagittal focusing has been designed for standard monochromators at SPring-8. The bender does not move the position of the crystal center when the bending radius is changed. Sagittal focusing from 40 keV to 60 keV was achieved by using Si(311) double crystals. The flux density of the focused beam measured at 40 keV was 15 times higher than that of the unfocused beam. The height deviation of the focused beam throughout the measured energy range was within +/- 0.15 mm.

Crystallization↗

PubMed2001

Stimulation of GATA-2 as a mechanism of hydrogen peroxide suppression in hypoxia-induced erythropoietin gene expression.

Hydrogen peroxide (H2O2) has previously been shown to inhibit the DNA binding activity of hypoxia inducible factor-1 (HIF-1), the accumulation of HIF-1alpha protein and erythropoietin (Epo) gene expression. Epo gene expression has been previously shown to be down-regulated through a GATA binding site at its promoter region. In this study, the effect of H2O2 on Epo gene expression under hypoxic conditions through a GATA transcription factor was investigated. Hypoxic induction was found to be inhibited upon the addition of H2O2, and this effect could be reversed through the addition of catalase. Hypoxic induction was found to be suppressed by co-transfection with a human GATA-2 cDNA expression plasmid. Transfection of Hep3B cells with a reporter gene bearing a mutation at the promoter GATA binding site was found to be only mildly affected by the addition of H2O2. Electrophoretic gel mobility shift assays (EMSAs), using the Epo promoter GATA site as a probe and the GATA-2 protein extracted from Hep3B cells, showed that addition of H2O2 enhanced the binding of GATA-2 while addition of catalase inhibited this binding. From these results, we conclude that H2O2 increases the binding activity of GATA-2 in a specific manner, thereby suppressing the activity of the Epo promoter and thus inhibiting Epo gene expression.

Binding Sites↗

PubMed2001

Heat shock protein (HSP) 47 and collagen are upregulated during neointimal formation in the balloon-injured rat carotid artery.

Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is thought to be essential for the proper processing and secretion of procollagen molecules. We investigated the time course and localization of HSP47 and collagen expression after balloon catheter angioplasty in the rat carotid artery, based on the premise that accumulation of extracellular matrix components is a main feature of intimal hyperplasia in humans and in laboratory animals. Low levels of HSP47 expression were evident in uninjured carotid arteries. Northern blot analysis revealed that HSP47 mRNA expression was markedly stimulated 1--3 days after the induced injury and a high level was maintained for 7 days, followed by a gradual decline for up to 21 days after the injury. These changes in HSP47 expression paralleled changes in alpha 1(I) collagen expression. Immunohistochemical staining revealed colocalization of HSP47 and collagen in smooth muscle cells (SMCs) of the media and intima. In situ hybridization analysis showed that activated SMCs, which proliferated and migrated into the intima, expressed high levels of HSP47. In cultured human aortic SMCs, similar upregulation of HSP47 and alpha1(I) collagen by TGF-beta was noted. These results show that SMCs activated after balloon injury express high levels of HSP47 and collagen during cell proliferation and migration, hence an overproduction of collagen and development of intimal thickening. Thus, HSP47 plays a role in the formation and progression of neointima after angioplasty.

Animals↗

PubMed2001

NdWFamide: a novel excitatory peptide involved in cardiovascular regulation of Aplysia.

Although diverse peptides are known to affect invertebrate cardiac activity, the peptidergic regulation of the cardiovascular system of Aplysia is still poorly understood. Asn-D-Trp-Phe-NH(2) (NdWFamide) is a recently purified cardioactive peptide in Aplysia. Pharmacological experiments showed that NdWFamide was one of the most potent cardioexcitatory peptides among the known endogenous cardioactive peptides in Aplysia. NdWFamide-immunopositive neuronal processes were abundant in the cardiovascular region of Aplysia, and many of them originated from neurosecretory cells in the abdominal ganglion (R3-R13 cells). The data suggest that NdWFamide is a cardioexcitatory peptide utilized by R3-R13 cells of Aplysia.

Animals↗

PubMed2001

Striatal dopaminergic and serotonergic markers in human heroin users.

To establish whether chronic opiate exposure might impair brain dopaminergic or serotonergic function in humans, we assessed biochemical indices of monoaminergic neurotransmitter activity and integrity in post mortem striatum of nine chronic heroin users and 14 control subjects. Striatal levels of the vesicular monoamine transporter were normal, suggesting that the density of dopamine nerve terminals is not reduced in heroin users. In nucleus accumbens, levels of tyrosine hydroxylase protein (-25%) and those of the dopamine metabolite homovanillic acid (-33%) were reduced significantly together with a trend for decreased dopamine (-32%) concentration. These changes could reflect either a compensatory downregulation of dopamine biosynthesis in response to prolonged dopaminergic stimulation caused by heroin, or reduced axoplasmic transport of tyrosine hydroxylase. Striatal levels of serotonin were either normal or elevated whereas concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid were decreased by 27-38%. Our data suggest that chronic heroin exposure might produce a modest reduction in dopaminergic and serotonergic activity that could affect motivational state and impulse control, respectively.

Adult↗