Biomedical subjects

H Mizukami

Publications and source records attributed to H Mizukami.

At least 19 recordsLinked to original sources

PubMed2006

Ganglioside GM3 promotes cell migration by regulating MAPK and c-Fos/AP-1.

Gangliosides have been proposed as modulators of transmembrane signaling. Recently, GM3, a glycosphingolipid containing monosaialic acids, is thought to be one of the key molecules of signal transduction in mammalian cells. In this study, we used mouse embryonic fibroblast cell lines (MEFs) established from sialyltransferase-I knockout mice (GM3 synthase KO mice) to evaluate the regulation of mitogenic signals by gangliosides. Cell proliferation assay revealed a higher growth potential of GM3 KO MEFs. Immunoblots showed upregulation of Ras/Raf/MEK/ERK pathway in GM3 KO MEFs, and these signals resulted in enhanced translocation of ERK into the nuclei. Further, both exogenous and endogenous add-back of GM3 decreased the activities of MAPK in GM3 KO MEFs. In addition, GM3 KO MEFs formed foci in high-density culture condition, and analyses of cell cycle modulators revealed the resistance of GM3 KO MEFs for entering cell cycle arrest. Finally, sustained expressions of c-Fos in GM3 KO MEFs were shown to correlate with DNA-binding activity between c-Fos and AP-1. These results demonstrate that the deletion of sialyltransferase-I changes the character of MEFs to a highly activated state of the MAPK pathway, indicating the critical role of GM3 as a regulator of membrane-transmitted signals.

Animals↗

PubMed2006

Spindle cell metaplasia arising in thyroid adenoma: characterization of its pathology and differential diagnosis.

Spindle cell metaplasia in thyroid adenoma or carcinoma is rare and its pathological features are not well characterized. Distinction of this entity from medullary or anaplastic carcinoma has an important clinical implication. We encountered a case of thyroid follicular adenoma associated with spindle cell metaplasia. It showed "tumor in tumor appearance" and neoplastic spindle cells were positive for thyroglobulin, thyroid transcription factor-1, vimentin and focally chromogranin A and somatostatin (SS). MIB-1 index was <1%. Ultrastructure of the spindle cells was reminiscent of follicular cell origin. From the findings from our case, spindle cell metaplasia appears to be a benign clinical entity, suggestive of multidirectional differentiation of follicular cells.

Adenoma↗

PubMed2006

Increased expression and activation of matrix metalloproteinase-2 (MMP-2) in O-1N: hamster oral squamous cell carcinoma with high potential lymph node metastasis.

Oral squamous cell carcinoma (OSCC) is one of the most common head and neck cancers with poor clinical outcome. MMP-2 was implied to contribute to the invasiveness and metastatization of various malignancies because of the degradation of type IV collagen. In this experiment, ELISA, Western blot and Q-RT-PCR was performed to investigate the expression and activation of MMP-2 in serum and tumor from hamster oral cancer model with high lymph node metastasis based on the various stages of OSCC development. Active MMP-2 in the serum was found elevated during oral cancer progression. In the metastatic stage, total MMP-2 level was 46% higher than it in the expansive stage, while active MMP-2 level increased 6-fold than it in the expansive stage. MMP-2 serum activation ratio was significantly enhanced in the metastatic stage than before tumor transplantation and that in the expansive stage. MMP-2 protein and MMP-2 mRNA was found to be increased during oral cancer development in hamster models. The increase of MMP-2 expression and activation starts prior to the metastasis occurrence, indicating the important role of MMP-2 in the early phase of oral cancer progression. Active MMP-2 level in serum may be a useful indicator for monitoring oral cancer progression.

Animals↗

PubMed2005

Double blockade of cell cycle progression by coptisine in vascular smooth muscle cells.

Coptisine, an isoquinoline alkaloid isolated from rhizome of Coptis japonica, inhibits proliferation of vascular smooth muscle cells (VSMCs). The aim of this study was to evaluate the action of coptisine, along with berberine (a structurally similar isoquinoline alkaloid), on progression of the cell cycle in VSMCs. Coptisine displayed antiproliferative action against VSMCs by blocking the cell cycle at G(1) and G(2)/M phases. The G(1) block was shown by inhibition of [(3)H]thymidine incorporation into VSMCs at coptisine concentrations higher than 15 microM. The mechanism underlying the G(1) arrest involved a decrease in cyclin D1 protein, although cyclin E, A, and B were not affected by coptisine treatment. The selective reduction in cyclin D1 protein was mainly attributable to accelerated proteolysis via proteasome-dependent pathway, since it was inhibited by a proteasome inhibitor, N-carbobenzoxy-L-leucinyl-L-leucinyl-L-norleucinal (MG132) and further the mRNA level of cyclin D1, protein synthesis, and mitogen-activated protein kinase (MAPK) activity remained unaltered. The mechanism underlying the G(2)/M arrest involved partial inhibition of tubulin polymerization, which was apparent at coptisine concentration of 3 microM. Berberine arrested the cell cycle at G(1) phase via a mechanism identical with coptisine, but did not cause block at G(2)/M phase. The results demonstrate that a small difference in the structure between isoquinoline alkaloids produces a big difference in activity, and that coptisine has a unique double action in arresting the cell cycle of VSMCs.

Animals↗

PubMed2005

Selective inhibition of vascular smooth muscle cell proliferation by coptisine isolated from Coptis rhizoma, one of the crude drugs composing Kampo medicines Unsei-in.

Acceleration of vascular smooth muscle cell (VSMC) proliferation is closely linked to the pathogenesis of vascular diseases. We, therefore, focused on traditional Japanese herbal medicines (Kampo medicines) used to ameliorate the impairment of microcirculation or blood stasis and screened them for their ability to inhibit rat VSMC proliferation. Among them, Unsei-in was found to effectively suppress VSMC proliferation, and Coptis rhizome was the responsible constituent crude drug. The extract of Coptis rhizome inhibited VSMC proliferation with the GI(50) value of 4.4 microg/ml, which was much lower than those against the proliferation of 3Y1, dRLh-84, B16, and HeLa cells. The Coptis rhizome extract inhibited the progression of VSMC arrested at G(0)/G(1) phase from G(0)/G(1) to S phase, but not that of 3Y1 cells. Biological assay-guided fractionation revealed that an alkaloid of Coptis rhizome, coptisine, was the active ingredient in selectively preventing VSMC proliferation with GI(50) of 3.3 microM (1.2 microg/ml). When the structurally-related isoquinoline alkaloids of protoberberine class were studied for their inhibitory activities, berberine decreased the VSMC proliferation with GI(50) of 95.1 microM (35.4 microg/ml), about 30 times higher concentration than coptisine, while palmatine failed to show any activity. This study provides evidence that coptisine, an ingredient of Unsei-in, prevents VSMC proliferation selectively at lower concentrations compared with various cells or other structurally related alkaloids.

Animals↗

PubMed2004

Long-term correction of hyperphenylalaninemia by AAV-mediated gene transfer leads to behavioral recovery in phenylketonuria mice.

Classical phenylketonuria (PKU) is a metabolic disorder caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). If untreated, accumulation of phenylalanine will damage the developing brain of affected individuals, leading to severe mental retardation. Here, we show that a liver-directed PAH gene transfer brought about long-term correction of hyperphenylalaninemia and behavioral improvement in a mouse model of PKU. A recombinant adeno-associated virus (AAV) vector carrying the murine PAH cDNA was constructed and administered to PAH-deficient mice (strain PAH(enu2)) via the portal vein. Within 2 weeks of treatment, the hyperphenylalaninemic phenotype improved and completely normalized in the animals treated with higher vector doses. The therapeutic effect persisted for 40 weeks in male mice, while serum phenylalanine concentrations in female animals gradually returned to pretreatment levels. Notably, this long-term correction of hyperphenylalaninemia was associated with a reversal of hypoactivity observed in PAH(enu2) mice. While locomotory activity over 24 h and exploratory behavior were significantly decreased in untreated PAH(enu2) mice compared with the age-matched controls, these indices were completely normalized in 12-month-old male PKU mice with lowered serum phenylalanine. These results demonstrate that AAV-mediated liver transduction ameliorated the PKU phenotype, including central nervous system dysfunctions.

Animals↗

PubMed2004

Expansion of genetically corrected neutrophils in chronic granulomatous disease mice by cotransferring a therapeutic gene and a selective amplifier gene.

Hematopoietic stem cell gene therapy has not provided clinical success in disorders such as chronic granulomatous disease (CGD), where genetically corrected cells do not show a selective advantage in vivo. To facilitate selective expansion of transduced cells, we have developed a fusion receptor system that confers drug-induced proliferation. Here, a 'selective amplifier gene (SAG)' encodes a chimeric receptor (GcRER) that generates a mitotic signal in response to estrogen. We evaluated the in vivo efficacy of SAG-mediated cell expansion in a mouse disease model of X-linked CGD (X-CGD) that is deficient in the NADPH oxidase gp91phox subunit. Bone marrow cells from X-CGD mice were transduced with a bicistronic retrovirus encoding GcRER and gp91phox, and transplanted to lethally irradiated X-CGD recipients. Estrogen was administered to a cohort of the transplants, and neutrophil superoxide production was monitored. A significant increase in oxidase-positive cells was observed in the estrogen-treated mice, and repeated estrogen administration maintained the elevation of transduced cells for 20 weeks. In addition, oxidase-positive neutrophils were increased in the X-CGD transplants given the first estrogen even at 9 months post-transplantation. These results showed that the SAG system would enhance the therapeutic effects by boosting genetically modified, functionally corrected cells in vivo.

Animals↗

PubMed2004

Adeno-associated virus vector-mediated interleukin-10 gene transfer inhibits atherosclerosis in apolipoprotein E-deficient mice.

Inflammation is a major contributor to atherosclerosis by its effects on arterial wall biology and lipoprotein metabolism. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that may modulate the atherosclerotic disease process. We investigated the effects of adeno-associated virus (AAV) vector-mediated gene transfer of IL-10 on atherogenesis in apolipoprotein E (ApoE)-deficient mice. A murine myoblast cell line, C2C12, transduced with AAV encoding murine IL-10 (AAV2-mIL10) secreted substantial amounts of IL-10 into conditioned medium. The production of monocyte chemoattractant protein-1 (MCP-1) by the murine macrophage cell line, J774, was significantly inhibited by conditioned medium from AAV2-mIL10-transduced C2C12 cells. ApoE-deficient mice were injected with AAV5-mIL10 into their anterior tibial muscle at 8 weeks of age. The expression of MCP-1 in the vascular wall of the ascending aorta and serum MCP-1 concentration were decreased in AAV5-mIL10-transduced mice compared with AAV5-LacZ-transduced mice. Oil red-O staining of the ascending aorta revealed that IL-10 gene transfer resulted in a 31% reduction in plaque surface area. Serum cholesterol concentrations were also significantly reduced in AAV5-mIL10-transduced mice. To understand the cholesterol-lowering mechanism of IL-10, we measured the cellular cholesterol level in HepG2 cells, resulting in its significant decrease by the addition of IL-10 in a dose-dependent manner. Furthermore, IL-10 suppressed HMG-CoA reductase expression in the HepG2 cells. These observations suggest that intramuscular injection of AAV5-mIL10 into ApoE-deficient mice inhibits atherogenesis through anti-inflammatory and cholesterol-lowering effects.

Adenoviridae↗

PubMed2004

Specific detection of human coagulation factor IX in cynomolgus macaques.

After screening for species-specific antihuman factor (F)IX monoclonal antibodies, we found that antibody 3A6 did not bind to cynomolgus FIX. The 3A6 epitope was found to include Ala262 of human FIX. The 3A6 antibody was used as a catching antibody in an enzyme immunoassay (EIA) for specific detection of human FIX in cynomolgus macaque plasma. No significant increase of substrate hydrolysis was observed when EIA buffer containing cynomolgus macaque plasma was subjected to the 3A6-based EIA. Addition of up to 30% cynomolgus macaque plasma or canine plasma to the assay did not alter detection of human FIX. Three cynomolgus macaques were injected with human FIX (10 U kg-1; i.v.) and the circulating human FIX was quantified in the macaque plasma. The FIX level in the circulation increased to 470 +/- 37.6 ng mL-1 at 1 h after the injection and gradually decreased to 1.79 +/- 1.1 ng mL-1 by day 5, which is approximately 0.06% of the normal human plasma FIX concentration. These data suggest that the cynomolgus macaque can be used as a primate model for studying hemophilia B gene therapy by transduction of macaque organs with vectors to express human FIX in vivo and detection of human FIX using the 3A6 monoclonal antibody.

Animals↗

PubMed2003

Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice.

The application of adeno-associated virus (AAV) vectors to cancers is limited by their low transduction efficiency. Previously, we reported that gamma-ray enhanced the second-strand synthesis, leading to the improvement of the transgene expression, and cytocidal effect of the herpes simplex virus type-1 thymidine kinase (HSVtk) and ganciclovir (GCV) system. In this study, we extended this in vitro findings to in vivo. First, the laryngeal cancer cell line (HEp-2) and HeLa were treated with AAVtk/GCV, the number of surviving cells was reduced as the concentration of GCV increased. Furthermore, the 4 Gy irradiation enhanced the killing effects of AAVtk/GCV by four-fold on HeLa cells and 15-fold on HEp-2 cells. Following the in vitro experiments, we evaluated the transgene expression and the antitumor activity of the AAV vectors in combination with gamma-ray in nude mice inoculated with HEp-2 subcutaneously. The LacZ expression was observed in the xenografted tumors and significantly increased by gamma-ray. The AAVtk/GCV system suppressed the tumors growth, and gamma-ray augmented the antitumor activity by five-fold. These findings suggest that the combination of AAVtk/GCV system with radiotherapy is significantly effective in the treatment of cancers and may lead to reduction of the potential toxicity of both AAVtk/GCV and gamma-ray.

Animals↗

PubMed2002

Reduced beta-cell mass and expression of oxidative stress-related DNA damage in the islet of Japanese Type II diabetic patients.

AIMS/HYPOTHESIS: We examined the pancreatic islet lesions in Japanese patients with Type II diabetes mellitus to determine if the damage was related to oxidative stress. METHODS: Morphometric analyses were performed on immunostained sections of the tail portion of the pancreas from 14 diabetic and 15 non-diabetic patients. Amyloid deposition and oxidative stress-induced tissue damage were evaluated by Congo-red staining and immunostaining. Resistance to oxidative stress was assessed from immunostaining results for Cu, Zn-superoxide dismutase (SOD). Expression of (pro)insulin mRNA was assessed by in situ hybridisation. RESULTS: The pancreas from diabetic patients had amyloid deposition in about 15 % of the islets, intensified reactions of 8-OHdG and HNE, as well as reduced expression of SOD. Islet volume density of beta cells and total beta-cell mass in the pancreas from diabetic patients were reduced by 22 % (p < 0.001) and 30 % (p < 0.05). Islet volume density and total mass of (pro)insulin mRNA-positive cells were similarly reduced in diabetic patients by 22 % (p < 0.001) and 39 % (p < 0.05), respectively. Islet volume density of A cells was increased by 20 % (p < 0.001) but total mass did not change. There were no changes in volume densities of islet, D and PP cells. Reduced beta-cell volume density correlated with increased positive staining of 8-OHdG. CONCLUSION/INTERPRETATION: Japanese Type II diabetic patients show a reduction of beta-cell mass and evidence of increased oxidative stress-related tissue damage that is correlated with the extent of the beta-cell lesions.

Aged↗

PubMed2002

Selective growth advantage of wild-type lymphocytes in X-linked SCID recipients.

The cytokine receptor common gamma chain (gamma c) plays a pivotal role in multiple interleukin signaling, and gamma c gene mutations cause an X-linked form of SCID (X-SCID). Recently, gamma c gene transfer into the autologous X-SCID BM achieved appreciable lymphocyte reconstitution, contrasting with the limited success in previous gene therapy trials targeting hematopoietic stem cells. To understand the mechanisms underlying this success, we examined the repopulating potential of the wild-type (WT) BM cells using an X-SCID mouse model. Limited numbers of WT cells were infused into non-ablated WT and X-SCID hosts. Whereas no appreciable engraftment was observed in WT recipients, donor-derived lymphocytes repopulated well in X-SCID, reaching 37% (10(6)cells given) and 53% (10(7) cells given) of the normal control value 5 months post BMT. A lineage analysis showed a predominance of the donor-derived lymphocytes (CD4(+) T, CD8(+) T, B and NK cells) in X-SCID while the donor-derived granulocytes and monocytes engrafted poorly. These results showed a selective advantage of WT cells in X-SCID, and that the advantage was restricted to lymphocytes. In human gene therapy for X-SCID, an analogous growth advantage would greatly enhance the repopulation of lymphocytes derived from a very small number of gamma c gene-supplemented precursors.

Animals↗

PubMed2002

Delayed delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) is a strong candidate agent in the neuroprotective treatment of Parkinson's disease (PD). We investigated whether adeno-associated viral (AAV) vector-mediated delivery of a GDNF gene in a delayed manner could prevent progressive degeneration of dopaminergic (DA) neurons, while preserving a functional nigrostriatal pathway. Four weeks after a unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA), rats received injection of AAV vectors expressing GDNF tagged with FLAG peptide (AAV-GDNFflag) or beta-galactosidase (AAV-LacZ) into the lesioned striatum. Immunostaining for FLAG demonstrated retrograde transport of GDNFflag to the substantia nigra (SN). The density of tyrosine hydroxylase (TH)-positive DA fibers in the striatum and the number of TH-positive or cholera toxin subunit B (CTB, neuronal tracer)-labeled neurons in the SN were significantly greater in the AAV-GDNFflag group than in the AAV-LacZ group. Dopamine levels and those of its metabolites in the striatum were remarkably higher in the AAV-GDNFflag group compared with the control group. Consistent with anatomical and biochemical changes, significant behavioral recovery was observed from 4-20 weeks following AAV-GDNFflag injection. These data indicate that a delayed delivery of GDNF gene using AAV vector is efficacious even 4 weeks after the onset of progressive degeneration in a rat model of PD.

Animals↗

PubMed2001

Development and characterization of an antisense-mediated prepackaging cell line for adeno-associated virus vector production.

One of the limitations of recombinant adeno-associated virus (rAAV) vector systems for gene therapy applications has been the difficulty in producing the vector in sufficient quantity for adequate evaluation. Since the AAV Rep proteins are cytotoxic, it is not easy to establish stable cell lines that express them constitutively. We describe a novel 293-derived prepackaging cell line which constitutively expresses the antisense rep/cap driven by a loxP-flanked CMV promoter. This cell line was converted into a packaging cell line expressing Rep/Cap for rAAV vector production through adenovirus-mediated introduction of a Cre recombinase gene. Without the introduction of the Cre recombinase gene, the cell line was shown to produce neither Rep nor Cap. rAAV vector was produced (1 x 10(9) genome copies/3.5-cm dish) 4 days after the transduction with Cre-expression adenovirus vector together with transfection of AAV vector plasmid. We further showed that the addition of Cap-expression adenovirus vector caused a 10-fold increase in the yield of rAAV vector. This system is also capable of producing rAAV as a transfection-free system by using a small amount of rAAV instead of vector plasmid.

Adenoviridae↗

PubMed2001

Reversible integration of the dominant negative retinoid receptor gene for ex vivo expansion of hematopoietic stem/progenitor cells.

Since hematopoietic stem cells (HSCs) differentiate readily ex vivo resulting in the loss of self-renewal and engraftment abilities, the transient block of differentiation is essential to maintain those abilities during their ex vivo expansion culture. To this end, we developed a method of reversible integration of the dominant negative retinoic acid receptor (DN-RAR) gene, a differentiation-blocking gene, into cells utilizing the Cre/loxP-dependent gene recombination system. The murine immature hematopoietic 32D cells differentiate into mature neutrophils upon G-CSF treatment. However, 32D cells transduced with a retroviral vector expressing the DN-RAR gene put between two loxP sites continued to proliferate without showing differentiation even in the presence of G-CSF. After the cells were fully amplified, the cells were transduced with the Cre recombinase gene. The cells then restored the ability to differentiate into mature neutrophils upon G-CSF treatment. PCR analysis showed that the DN-RAR gene was efficiently removed from the genome by introduction of the Cre gene. This system may eventually be applicable to the ex vivo expansion of HSCs.

Animals↗

PubMed2001

A novel recombinant adeno-associated virus vaccine induces a long-term humoral immune response to human immunodeficiency virus.

Recombinant adeno-associated virus (AAV) has attracted tremendous interest as a promising vector for gene delivery. In this study we have developed an HIV-1 vaccine, using an AAV vector expressing HIV-1 env, tat, and rev genes (AAV-HIV vector). A single injection of the AAV-HIV vector induced strong production of HIV-1-specific serum IgG and fecal secretory IgA antibodies as well as MHC class I-restricted CTL activity in BALB/c mice. The titer of HIV-1-specific serum IgG remained stable for 10 months. When AAV-HIV vector was coadministered with AAV-IL2 vector, the HIV-specific cell-mediated immunity (CMI) was significantly enhanced. Boosting with AAV-HIV vector strongly enhanced the humoral response. Furthermore, the mouse antisera neutralized an HIV-1 homologous strain, and BALB/c mice immunized via the intranasal route with an AAV vector expressing the influenza virus hemagglutinin (HA) gene showed protective immunity against homologous influenza virus challenge. These results demonstrate that AAV-HIV vector immunization may provide a novel and promising HIV vaccination strategy.

AIDS Vaccines↗

PubMed2001

Glutamate is not involved in the MPP+-induced dopamine overflow in the striatum of freely moving C57BL/6 mice.

The role of glutamate in the N-methyl-4-phenyldihydropyridinium (MPP+) toxicity has been argued in the past decade. However, the effects of glutamate efflux and NMDA antagonist on MPP+-induced dopamine overflow have not been documented. To clarify this, we perfused MPP+ through a microdialysis probe in the striatum of freely moving mature C57BL/6 mice. The 60-min perfusion of 10 and 100 microM MPP+ strikingly increased dopamine levels to 28- and 93-fold of the basal values, respectively. In contrast, an administration of MPP+ did not induce marked glutamate release: the MPP+-perfusion slightly increased the glutamate level at 100 microM, but not at 10 microM. The addition of 100 microM (+)-MK-801 or 200 microM (+/-)-AP-7 to the perfusate did not attenuate MPP+-induced dopamine overflow. The extent of dopamine release only depended on the amount of MPP+ accumulation into the cells. These results indicated that, at least in the striatum, neither glutamate release nor the NMDA antagonist, including (+)-MK-801, could regulate MPP+-evoked dopamine overflow.

1-Methyl-4-phenylpyridinium↗

PubMed2001

Norharman, an indoleamine-derived beta-carboline, but not Trp-P-2, a gamma-carboline, induces apoptotic cell death in human neuroblastoma SH-SY5Y cells.

Carbolines, azaheterocyclic amines derived from indoleamines, have various biological activities, such as neurotoxicity of beta-carbolines and potent mutagenicity of gamma-carbolines. In this study, structural significance among these carbolines was investigated in relation to the types of cell death, apoptosis and necrosis, using human neuroblastoma SH-SY5Y cells. DNA damage was quantitatively analyzed by a single-cell gel electrophoresis assay. DNA damage was induced by both beta-carbolines, harman and norharman, and gamma-carbolines, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-4-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), in a dose dependent manner. Gamma-carbolines were more potent to damage DNA than beta-carbolines. Alkaline lysis of the cells prevented DNA damage induced by beta-carboline, and pre-treatment of the cells with cycloheximide, an inhibitor of protein synthesis, reduced DNA damage caused by norharman. Morphological observation showed condensed and fragmented nuclei typical for apoptosis, in the cells treated with norharman. Thus, DNA damage induced by norharman was proved to be apoptotic. However, harman, which had a methyl substitution at the position 1, might induce necrosis in the cells. On the other hand, gamma-carbolines, Trp-P-1 and Trp-P-2, directly damaged DNA. Thus, the nitrogen atom at the gamma-position and/or an amino group in carboline structure would be required to induce the direct DNA cleavage.

Apoptosis↗