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Hiroyuki Mizuguchi

Publications and source records attributed to Hiroyuki Mizuguchi.

90 records · Page 5Linked to original sources
PubMed2003

Role of H1-calponin in pancreatic AR42J cell differentiation into insulin-producing cells.

Basic or h1-calponin is a smooth muscle-specific, actin-binding protein that is involved in the regulation of smooth muscle contractile activity. We found in this study the expression of mRNA and protein for h1-calponin in AR42J-B13 cells, which is a useful model for investigating islet beta-cell differentiation from pancreatic common precursor cells. Following treatment of AR42J cells with activin A and hepatocyte growth factor, the protein levels of h1-calponin decreased in a time-dependent manner during the course of the cell differentiation. When h1-calponin was continuously overexpressed by utilizing recombinant adenovirus-mediated gene transfer, the percentage of cell differentiation in h1-calponin overexpressing cells was markedly suppressed as compared with that in the cells without overexpression (6.7 +/- 2.5 vs. 28.6 +/- 3.2%, P < 0.001, Student's t test). Finally, overexpression of h1-calponin (65.6 +/- 3.4), or that lacking actin-binding domain (55.9 +/- 3.4%), significantly (P < 0.001) suppressed the activin A-stimulated transcriptional activity of activin responsive element (ARE), whereas calponin homology-domain disruption mutant did not (100.6 +/- 1.9%). These results suggest that regulation of h1-calponin is involved in the regulation of differentiation of AR42J cells into insulin-producing cells at least partly through modulating ARE transcriptional activity.

Actins↗
PubMed2002

Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis.

Aspartate aminotransferase has been known to undergo a significant conformational change, in which the small domain approaches the large domain, and the residues at the entrance of the active site pack together, on binding of substrates. Accompanying this conformational change is a two-unit increase in the pK(a) of the pyridoxal 5'-phosphate-Lys(258) aldimine, which has been proposed to enhance catalysis. To elucidate how the conformational change is coupled to the shift in the aldimine pK(a) and how these changes are involved in catalysis, we analyzed structurally and kinetically an enzyme in which Val(39) located at both the domain interface and the entrance of the active site was replaced with a bulkier residue, Phe. The V39F mutant enzyme showed a more open conformation, and the aldimine pK(a) was lowered by 0.7 unit compared with the wild-type enzyme. When Asn(194) had been replaced by Ala in advance, the V39F mutation did not decrease the aldimine pK(a), showing that the domain rotation controls the aldimine pK(a) via the Arg(386)-Asn(194)-pyridoxal 5'-phosphate linkage system. The maleate-bound V39F enzyme showed the aldimine pK(a) 0.9 unit lower than that of the maleate-bound wild-type enzyme. However, the positions of maleate, Asn(194), and Arg(386) were superimposable between the mutant and the wild-type enzymes; therefore, the domain rotation was not the cause of the lowered aldimine pK(a) value. The maleate-bound V39F enzyme showed an altered side-chain packing pattern in the 37-39 region, and the lack of repulsion between Gly(38) carbonyl O and Tyr(225) Oeta seemed to be the cause of the reduced pK(a) value. Kinetic analysis suggested that the repulsion increases the free energy level of the Michaelis complex and promotes the catalytic reaction.

Aspartate Aminotransferases↗
PubMed2002

Enhancement of phage-mediated gene transfer by nuclear localization signal.

The cell membrane and the nuclear membrane are two major barriers hindering the free movement of various macromolecules through animal cells. Nevertheless, some proteins can actively bypass these barriers by dint of intrinsic peptidic signals, so incorporation of these signals might improve the efficacy of artificial gene delivery vehicles. We examined the role of the nuclear localization signal (NLS) in gene transfer, using recombinant lambda phage as a model of the polymer/DNA complexes. We prepared a lambda phage displaying a 32-mer NLS of SV40 T antigen on its surface (NLS phage), and found that this NLS phage, delivered into the cytoplasm by appropriate devices, has higher affinity for the nucleus and induces the expression of encapsulated marker genes more efficiently than does the wild-type phage. This suggests that the 32-mer NLS peptide will become a practical tool for artificial gene delivery vehicles with enhanced nuclear targeting activity.

Amino Acid Sequence↗
PubMed2002

Strength evaluation of transcriptional regulatory elements for transgene expression by adenovirus vector.

In studies of both gene function and gene therapy, transgene expression may be assisted considerably through the use of transcriptional regulatory elements with high activity. In this study, we evaluated the strength of various transcriptional regulatory elements both in vitro (six types of cell line) and in vivo (mouse heart, lung, kidney, spleen, and liver) by adenovirus-mediated gene transfer. In the case of the promoter/enhancer (P/E), the activity of CMV P/E (from the human cytomegalovirus immediate-early 1 gene) and hybrid CA P/E (composed of the CMV enhancer and chicken beta-actin promoter) were investigated, both of which are known to be strong and widely used. While hybrid CA P/E showed a higher transgene expression activity than CMV P/E, the addition of the intron A sequence (the largest intron of CMV) to CMV P/E increased the activity of CMV P/E to the same or higher level than that of hybrid CA P/E. Concerning the polyadenylation signal (P(A)) sequence, one from the bovine growth hormone (BGH) gene was about two times more efficient than that from the Simian virus 40 (SV40) late gene, both in vitro and in vivo. In the context of the CMV P/E containing the intron A sequence, a further increase in transgene expression was obtained by the addition of a SV40 enhancer downstream from the P(A) sequence. The combination of the SV40 P(A) and a SV40 enhancer showed almost comparable activity to BGH P(A). This information would be helpful for the construction of adenovirus vectors for studies regarding both gene function and gene therapy.

Adenoviridae↗
PubMed2002

Modification of a fiber protein in an adenovirus vector improves in vitro gene transfer efficiency to the mouse microglial cell line.

In microglia, it is difficult to introduce exogenous genes of interest even by recombinant adenovirus vectors (Ad) which can infect with high efficiency only to the cells expressing coxackievirus and adenovirus receptors (CAR). We found a lack of CAR expression in primary cultured murine microglia (PCMG) and its immortalized cell line MG5 by reverse transcription-polymerase chain reaction. In order to improve the efficiency of gene transfer, we generated a novel Ad (Ad-RGD) by an incorporation of the Arg-Gly-Asp motif (RGD) containing peptide in the HI loop of the viral fiber knob domain, which enables the virus to contact target cells through alpha V integrins which are known to be ubiquitously expressed on the surface of mammalian cells. Ad-RGD showed a remarkable improvement (13-18-fold) in the delivery of Escherichia coli LacZ gene in MG5 cells and a moderate increase in PCMG cells under the treatment with granulocyte-macrophage colony stimulating factor. These results suggest that Ad-RGD may be a potent tool for the delivery of genes to microglia activated by optimum stimulation, and thus analyzing the function of microglia with utilization of MG5 and PCMG cells.

Adenoviridae↗
PubMed2002

Identification of the p33(ING1)-regulated genes that include cyclin B1 and proto-oncogene DEK by using cDNA microarray in a mouse mammary epithelial cell line NMuMG.

The candidate tumor suppressor p33(ING1) plays an important role in inducinggrowth arrest at G(0)-G(1) phase of the cell cycle and/or promoting apoptosis in cancerous cells. p33(ING1) is reported to act as a transcriptional cofactor by associating with tumor suppressor p53, HAT, or histone deacetyltransferase, suggesting that p33(ING1) is involved in chromatin-mediated transcriptional regulation. However, the molecular mechanism of p33(ING1)-mediated transcriptional regulation is poorly understood. Here we analyzed expression profiles in mouse mammary epithelial cells (NMuMG) by using a cDNA microarray consisting of 2304 mouse cDNAs after inducing transformation with antisense inhibitor of growth 1 (ING1) in retrovirus vector. The subsequent confirmation of the altered expression levels of the selected genes by semiquantitative reverse transcription-PCR demonstrated that overexpression of the antisense ING1 stimulated expression of 14 genes, which included cyclin B1, 12-O-tetradecanoylphorbol-13-acetate-inducible sequence 11, proto-oncogene DEK, and osteopontin, whereas we have detected transcriptional repression of 5 genes, including TPT1. In addition, adenovirus-mediated overexpression of ING1 in NMuMG cells resulted in down-regulation of cyclin B1, 12-O-tetradecanoylphorbol-13-acetate-inducible sequence 11, DEK, and osteopontin, whereas the levels of TPT1 expression were increased. The further analysis using p53(-/-) SAOS2 cells showed that the p33(ING1)-induced cyclin B1 down-regulation was p53 dependent. Thus, our cDNA microarray analysis suggested that p33(ING1) targets the multiple genes, including proto-oncogene DEK and cyclin B1, at least some of which are regulated in a p53-dependent manner, in the cells undergoing cell growth or apoptosis.

Animals↗
PubMed2002

A novel murine model of Graves' hyperthyroidism with intramuscular injection of adenovirus expressing the thyrotropin receptor.

In this work we report a novel method to efficiently induce a murine model of Graves' hyperthyroidism. Inbred mice of different strains were immunized by i.m. injection with adenovirus expressing thyrotropin receptor (TSHR) or beta-galactosidase (1 x 10(11) particles/mouse, three times at 3-wk intervals) and followed up to 8 wk after the third immunization. Fifty-five percent of female and 33% of male BALB/c (H-2(d)) and 25% of female C57BL/6 (H-2(b)) mice developed Graves'-like hyperthyroidism with elevated serum thyroxine (T(4)) levels and positive anti-TSHR autoantibodies with thyroid-stimulating Ig (TSI) and TSH-binding inhibiting Ig (TBII) activities. In contrast, none of female CBA/J (H-2(k)), DBA/1J (H-2(q)), or SJL/J (H-2(s)) mice developed Graves' hyperthyroidism or anti-TSHR autoantibodies except SJL/J, which showed strong TBII activities. There was a significant positive correlation between TSI values and T(4) levels, but the correlations between T(4) and TBII and between TSI and TBII were very weak. TSI activities in sera from hyperthyroid mice measured with some chimeric TSH/lutropin receptors suggested that their epitope(s) on TSHR appeared similar to those in patients with Graves' disease. The thyroid glands from hyperthyroid mice displayed diffuse enlargement with hypertrophy and hypercellularity of follicular epithelia with occasional protrusion into the follicular lumen, characteristics of Graves' hyperthyroidism. Decreased amounts of colloid were also observed. However, there was no inflammatory cell infiltration. Furthermore, extraocular muscles from hyperthyroid mice were normal. Thus, the highly efficient means that we now report to induce Graves' hyperthyroidism in mice will be very useful for studying the pathogenesis of autoimmunity in Graves' disease.

Adenoviridae↗
PubMed2002

Fiber-mutant technique can augment gene transduction efficacy and anti-tumor effects against established murine melanoma by cytokine-gene therapy using adenovirus vectors.

Melanoma cells are relatively resistant to adenovirus vector (Ad)-mediated gene transfer due to the low expression of Coxsackie-adenovirus receptor (CAR), which acts as a primitive Ad-receptor. Therefore, extremely high doses of Ad are required for effective gene therapy against melanoma. In the present study, we investigated whether fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob could promote gene delivery and anti-tumor effects in the murine B16 BL6 tumor model. B16 BL6 cells (in vitro) and tumors (in vivo) infected with RGD fiber-mutant Ad containing a tumor necrosis factor alpha gene (Ad-RGD-TNFalpha) produced more TNFalpha than those infected with conventional Ad-TNFalpha. In addition, Ad-RGD-TNFalpha required about one-tenth the dosage of Ad-TNFalpha for induction of equal therapeutic effects upon intratumoral injection into established B16 BL6 tumors. Furthermore, the combination of both TNFalpha- and interleukin 12-expressing RGD fiber-mutant Ads exhibited more effective tumor regression than the Ad expressing each alone. These results suggested that the fiber-mutant for altering Ad-tropism is a very potent technology for advancing gene therapy for melanoma.

Adenoviridae↗
PubMed2002

Adenovirus vectors containing chimeric type 5 and type 35 fiber proteins exhibit altered and expanded tropism and increase the size limit of foreign genes.

Adenovirus (Ad) fiber proteins are responsible for the initial attachment of the virion to the cell membrane. Most Ad vectors currently in use are based on the Ad type 5 (Ad5), which belong to subgroup C, and use the coxsackievirus and adenovirus receptors (CAR) as the initial receptor. Ad35, which belongs to subgroup B, recognizes unknown receptor(s) other than CAR. In this study, the feasibility of the Ad vector containing Ad5/35 chimeric fiber protein was examined in a wide variety of cell types, such as CAR-positive or -negative human tumor cells, rodent cells, and blood cells (a total of 20 cell types), and in mice in vivo. Transduction data suggested that the Ad vectors containing the Ad5/35 chimeric fiber protein exhibited altered and expanded tropism when compared with the Ad5-based vector. The chimeric vector also allows the packaging of larger foreign DNAs than the conventional Ad5-based vector, which can package approximately 8.1-8.2 kb of foreign DNA. The chimeric vector containing approximately 8.8 kb of foreign DNA was generated without affecting the viral growth rate and titer. These results suggested that inclusion of the Ad35 fiber protein into the Ad5-based vector could lead to an improved efficiency in gene therapy and in gene transfer experiments, especially for the cells lacking in sufficient CAR expression.

Adenoviridae↗
PubMed2002

Receptor-independent augmentation of adenovirus-mediated gene transfer with chitosan in vitro.

Recombinant adenovirus is one of the most widely used viral vectors for gene delivery. This study was designed to evaluate the ability of chitosan, a cationic, linear polysaccharide composed of beta(1,4) linked glucosamine partly containing N-acetyl-glucosamine, to enhance the in vitro infectivity of adenovirus to mammalian cells. Wild type and a fiber-mutant replication-defective recombinant adenoviruses expressing beta-galactosidase were used. In the latter, an RGD peptide, the binding site for alpha(v)beta3 and alpha(v)beta5 integrin, was introduced in the fiber knob enabling adenovirus receptor-independent viral infection. Enhanced effect of chitosan on the infectivity of both adenoviruses was observed in Chinese hamster ovary cells that do not express the receptor for adenovirus with beta-galactosidase activity assay and x-gal staining. These data indicate the receptor-independent mechanism(s) for this enhancement effect. In addition, we found that pH of the culture medium, and molecular mass and concentration of chitosan are also critical factors. Thus, the highest effect was obtained with 0.1-1 microg/ml of chitosan with molecular mass of 19K and 40K in the culture medium of pH 6.4; on the other hand, the effect was negligible with the higher chitosan concentrations (10 microg/ml or more), lower or higher molecular mass (11K and 110K) of chitosan, or at pH of 7.4. Studies using several cell lines with variable levels of adenoviral infectivity revealed that this enhanced effect is evident in the cells with poor infectivity to adenovirus. Since chitosan is biocompatible and inexpensive, these data indicate that chitosan may be a potential candidate for a non-viral vector to safely increase adenoviral infectivity to mammalian cells, particularly those with poor susceptibility to adenoviral infection.

Adenoviridae↗
PubMed2002

Enhanced antitumor effect and reduced vector dissemination with fiber-modified adenovirus vectors expressing herpes simplex virus thymidine kinase.

There are at least two hurdles confronting the use of the adenovirus (Ad)-mediated herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system for the treatment of cancer. One is inefficient Ad vector-mediated gene transfer into tumor cells lacking the primary receptor, i.e., the coxsackievirus and adenovirus receptor (CAR). The other is hepatotoxicity due to unwanted vector spread into the liver, even when Ad vectors are injected intratumorally. Herein, we present an attractive strategy for overcoming such limitations based on use of a fiber-modified Ad vector containing an RGD peptide motif in the fiber knob. HSVtk-expressing Ad vectors containing mutant fiber (AdRGD-tk) or wild-type fiber (Ad-tk) were injected intratumorally into CAR-negative B16 melanoma cells inoculated into mice, after which GCV was injected intraperitoneally for 10 days. AdRGD-tk showed approximately 25 times more antitumor activity than Ad-tk. Histopathological studies suggested that liver damage in mice injected with AdRGD-tk was significantly lower than that in mice injected with Ad-tk. Intratumoral administration of luciferase-expressing Ad vectors containing the mutant fiber (AdRGD-L2) resulted in nearly 40 times more luciferase production in the tumor, but 8 times less production in the liver than the conventional Ad vectors (Ad-L2). These results indicate that combination of fiber-modified vectors and a HSVtk/GCV system is a potentially useful and safe approach for the treatment of tumors lacking CAR expression, and that fiber-modified vectors could be of great utility for gene therapy and gene transfer experiments.

Adenoviridae↗
PubMed2002

Structure of imidazole glycerol phosphate synthase from Thermus thermophilus HB8: open-closed conformational change and ammonia tunneling.

Imidazole glycerol phosphate synthase (IGPs) catalyzes the fifth step in the histidine biosynthetic pathway located at the branch point to de novo purine biosynthesis. IGPs is a multienzyme comprising glutaminase and synthase subunits. The glutaminase activity, which hydrolyzes glutamine to give ammonia, is coupled with substrate binding to the synthase subunit. The three-dimensional structure of the IGPs from Thermus thermophilus HB8 has been determined at 2.3 A resolution, and compared with the previously determined structures for the yeast and Thermotoga maritima enzymes. The structure of each subunit is similar to that of the corresponding domain in the yeast enzyme or subunit in the T. maritima enzyme. However, the overall structure is significantly different from the yeast and T. maritima enzymes, indicating that IGPs may change the relative orientation between the two subunits and close the glutaminase site upon glutamine binding. The putative ammonia tunnel, which carries nascent ammonia from glutaminase to the synthase site, has a closed gate comprising a cyclic salt bridge formed by four charged residues of the synthase subunit. The side chain of Lys100 in the cyclic salt bridge might change its side chain direction to form new interactions with the main chain carbonyl group of glutamine from the synthase subunit and the hydoxyl group of tyrosine from the glutaminase subunit, resulting in the opening of the gate for ammonia transfer.

Aminohydrolases↗
PubMed2002

Tumor necrosis factor alpha-gene therapy for an established murine melanoma using RGD (Arg-Gly-Asp) fiber-mutant adenovirus vectors.

Although adenovirus vectors (Ad) provide high-level transduction efficacy to many cell types, extremely high doses of Ad are required for sufficient gene transduction into several tumors, including melanoma. Here, we demonstrated that the expression of coxsackie-adenovirus receptor, a primitive Ad-receptor, was very low in murine and human melanoma cells. We also found that fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob remarkably augmented gene transduction efficacy in melanoma cells by targeting alpha(v)-integrins. In addition, intratumoral injection of RGD fiber-mutant Ad containing the tumor necrosis factor alpha gene (Ad-RGD-TNFalpha) revealed dramatic anti-tumor efficacy through hemolytic necrosis in an established murine B16 BL6 melanoma model. Ad-RGD-TNFalpha required one-tenth the dosage of Ad-TNFalpha to induce an equal therapeutic effect. These results suggest that alpha(v)-integrin-targeted Ad will be a very powerful tool for the advancement of melanoma gene therapy.

Adenoviridae↗
PubMed2002

Autoinhibitory regulation of p73 by Delta Np73 to modulate cell survival and death through a p73-specific target element within the Delta Np73 promoter.

p73 is a p53-related tumor suppressor but is also induced by oncogene products such as E2F-1, raising a question as to whether p73 is a tumor suppressor gene or oncogene. Unlike p53, p73 has several variants, including Delta Np73, which lacks the NH(2)-terminal transactivation domain. Although, in developing neurons, Delta Np73 is expressed abundantly and seems to inhibit the proapoptotic function of p53, the role of p73 and Delta Np73 and their regulatory mechanism in cell growth and differentiation are poorly understood. Here we report that p73, but not p53, directly activates the transcription of endogenous Delta Np73 by binding to the p73-specific target element located at positions -76 to -57 within the Delta Np73 promoter region. The activation of Delta Np73 promoter by p63 was marginal. Delta Np73 was associated with p73 alpha, p73 beta, and p53, as demonstrated by immunoprecipitation assays, and inhibited their transactivation activities when we used reporters of Mdm2, Bax, or Delta Np73 itself in SAOS-2 cells. Furthermore, induction or overexpression of Delta Np73 promoted cell survival by competing with p53 and p73 itself. Thus, our results suggest that the negative feedback regulation of p73 by its target Delta Np73 is a novel autoregulatory system for modulating cell survival and death.

Animals↗
PubMed2002

Comparison of the efficiency and safety of non-viral vector-mediated gene transfer into a wide range of human cells.

Non-viral gene transfer into a wide range of human cells was examined in order to clarify the factors that affect the efficiency and safety of non-viral vectors and to optimize the conditions so that high efficiency and low toxicity could be achieved. Six non-viral vectors (Lipofectin, LipofectAMINE PLUS, SuperFect, Effectene, DMRIE-C and DOTAP) were used to transfect a mammalian expression plasmid pCMVbeta into 16 types of human primary cells and cultured cell lines. Transfection efficiency was quantified using a galactosidase assay. Cytotoxic effects were measured by lactate dehydrogenase (LDH) assay and WST-8 assay. In serum-free conditions, LipofectAMINE PLUS, Effectene and SuperFect, on average, transfected DNA more successfully than Lipofectin, DMRIE-C, and DOTAP, although the levels of gene expression with these vectors varied remarkably in different cells. The most effective vector also differed depending on the cell type. Serum was found to inhibit gene transfer and reduce the cytotoxicity of all of these vectors except Effectene. The efficiency and toxicity of the non-viral vectors used depended on the type of vector, the DNA/vector ratio, the type of cell, and the presence of serum. These results provided useful information for the optimization of transfer conditions of these non-viral vectors.

Cell Line↗
PubMedDate not supplied

The tet-off system is more effective than the tet-on system for regulating transgene expression in a single adenovirus vector.

BACKGROUND: Control of transgene expression in mammalian cells is desirable for gene therapy and the study of gene function in basic research. This study evaluates the functionality of single adenovirus (Ad) vectors containing a tetracycline-controllable expression system (tet-off or tet-on system). METHODS: An Ad-mediated binary transgene expression system was generated containing a tet-off or a tet-on system, which introduced the gene of interest with a tetracycline-regulatable promoter and the tetracycline-responsive transcriptional activator gene into the E1 and E3 deletion regions, respectively. The functionality of the Ad-mediated tet-off and tet-on systems was compared under various conditions in vitro and in vivo. RESULTS: The Ad vector containing the tet-off system provided negative control of gene expression ranging from 20- to 500-fold, depending on the cell type and condition. In contrast, the Ad vector containing the tet-on system increased gene expression by only 2- to 28-fold and required about two-log orders higher concentration of an inducer (doxycycline) to switch on the gene expression, compared with the Ad vector containing the tet-off system. CONCLUSION: Ad vectors containing the tet-off system are a better choice for regulated gene expression than Ad vectors containing the tet-on system. Single Ad vectors containing the tetracycline-controllable expression system will greatly facilitate in vitro and in vivo analyses of gene function and may be useful for gene therapy.

Adenoviridae↗
PubMedDate not supplied

Adenovirus vector-mediated gene transfer into stem cells.

Stem cells, including embryonic stem (ES) cells, mesenchymal stem cells (MSCs), and hematopoietic stem cells (HSCs), are defined by their capacity for self-renewal and multilineage differentiation. Efficient gene transfer into stem cells is essential for the basic research in developmental biology and for therapeutic applications in gene-modified regenerative medicine. Adenovirus (Ad) vectors, based on Ad type 5, can efficiently and transiently introduce the exogenous gene into many cell types via the primary receptor, coxsackievirus, and adenovirus receptor (CAR). However, some kinds of stem cells, such as MSCs and HSCs, cannot be efficiently transduced with conventional Ad vectors based on Ad serotype 5 (Ad5), because of the lack of CAR expression. To overcome this problem, fiber-modified Ad vectors and an Ad vector based on another serotype of Ad have been developed. Here, we review the advances in the development of Ad vectors suitable for stem cells and discuss their application in basic biology and clinical medicine.

Adenoviridae↗