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Toshiharu Yamashita

Publications and source records attributed to Toshiharu Yamashita.

At least 19 recordsLinked to original sources
PubMed2006

The real-time, three-dimensional analyses of benign and malignant skin tumors by confocal laser scanning microscopy.

BACKGROUND: In obtain images of skin tumors non-invasively with real-time, confocal laser scanning microscope (CLSM) is introduced. OBJECTIVE: Reconstructed images of given horizontal sections were converted into three-dimensions using the data set of a large number of tomograms in the horizontal directions. METHODS: To develop the multiplaner reconstruction images of skin tumors in vertical directions and three-dimensionally reconstructed images of tumors will be obtained from the continuously collected horizontal image data sets. RESULTS: Three-dimensional analyses of the skin tumors from reconstructed images of the CLSM scanning have provided the information as to their physiological characteristics as well as the extent of deep invasion in real-time with non-invasive manner. High performance three-dimensional conversion software was effective in displaying three-dimensional construction of skin tumors. CONCLUSION: The CLSM scanning images followed by three-dimensional reconstruction using them can provide the real-time and non-invasive diagnoses of skin tumors and analyze the radial growth phase of tumors and the three-dimensional growth characteristics.

Aged↗
PubMed2005

Levels of blood and urine chemicals associated with longer duration of having arsenicosis in Bangladesh.

Arsenicosis is presently one of the significant public health problems in Bangladesh. Employing household screening of over 3.6 million people living in 6 arsenic-affected Upzilas of Bangladesh, 1,503 arsenicosis patients were identified at first and then blood and urine were collected from some of them and analyzed through laboratory techniques. As the relation between blood and urine chemicals with duration of having arsenicosis (DHA) is not clear, this study presented all findings by shorter versus longer DHA. Complications namely chronic bronchitis, conjunctivitis/congestions, weakness, and wasting were common, with relatively higher rates in longer group. Logistic regression analysis adjusted for age, sex, education, smoking, duration of drinking tube-well water, and whether any arsenicosis patients were in the family-indicated higher odds ratio (OR) of longer DHA (LDHA) in 3rd tertile with respect to GOT (OR = 2.12; 95%CI: 1.09-4.13), and blood glucose (OR = 2.00; 95%CI: 1.07-3.72) than 1st tertile. The OR of LDHA was significantly lower (OR = 0.48; 95%CI: 0.25-0.93) in 3rd tertile for triglycerides compared with 1st tertile. Albumin/globulin (A/G) ratio of 2nd tertile showed significantly lower OR of LDHA (OR=0.51; 95%CI: 0.28-0.95) than 1st tertile. Further epidemiological investigations based on a large sample, through cohort or case control studies, may be useful for validating and generalizing the results in Bangladesh.

Adult↗
PubMed2004

Identification of SCN3B as a novel p53-inducible proapoptotic gene.

Tumor suppressor p53 is a transcription factor that induces growth arrest and/or apoptosis in response to cellular stress. To identify novel p53-inducible genes, we compared the expression of genes in normal mouse embryo fibroblasts (MEFs) to p53-null cells by cDNA representational difference analysis. We report here that expression of endogenous sodium channel subunit beta 3 (SCN3B) is upregulated in mouse embryonic fibroblasts by DNA damage in a p53-dependent manner. In addition, we found that SCN3B levels are upregulated in human cancer cell lines by DNA damaging agents, as well as by overexpression of p53, but not significantly by p63 or p73. Furthermore, we identified two putative p53-binding sites upstream of the first exon (RE1) and in the third intron (RE2). The p53 protein can directly interact with the putative p53-binding sites in vivo, as assessed by chromatin immunoprecipitation. A reporter gene assay revealed that these two p53-binding sites are functional response elements. The SCN3B protein appears to be localized to the endoplasmic reticulum (ER). Introduction of the SCN3B gene into T98G and Saos2 cells potently suppressed colony formation. Furthermore, we found that adenovirus-mediated transfer of SCN3B induced apoptosis when combined with anticancer agents. The results presented here suggest that SCN3B mediates a p53-dependent apoptotic pathway and may be a candidate for gene therapy combined with anticancer drugs.

Animals↗
PubMed2004

Tyrosinase-related proteins suppress tyrosinase-mediated cell death of melanocytes and melanoma cells.

The synthesis of melanin intermediates through tyrosinase (TYR) involves the production of cytotoxic free radicals. By using recombinant adenoviruses that express TYR, tyrosinase-related protein 1 (TYRP1) or DOPAchrome tautomerase (DCT), we analyzed the biological function of these proteins with regard to melanin production and the growth of melanocytes, fibroblasts, melanoma cells and nonmelanoma cancer cells. High-level expression of TYR produced newly synthesized melanin and induced cell death in all of these cells. However, when TYRP1 or DCT was coexpressed with TYR in melanocytes and melanoma cells, TYR-mediated cell death was clearly decreased. This decrease was not observed in nonmelanocytic cells. Western blot analysis and measurement of enzyme activity revealed that the expression of TYRP1 or DCT had little effect on the amount or activity of cointroduced TYR in either the melanocytic or nonmelanocytic cells. In cells expressing both TYR and TYRP1 or TYR and DCT, the total amount of melanin and/or eumelanin increased substantially more than that in cells expressing TYR alone. On the other hand, the level of pheomelanin was similar in these three cell types. These findings suggest that TYRP1 and DCT play an important role in suppressing TYR-mediated cytotoxicity in melanocytic cells without decreasing TYR expression and/or activity. These biological activities of TYRP1 and DCT may work through the interaction with TYR in melanosomal compartment.

Cell Death↗
PubMed2004

Regulation of immature protein dynamics in the endoplasmic reticulum.

The quality of nascent protein folding in vivo is influenced by the microdynamics of the proteins. Excessive collisions between proteins may lead to terminal misfolding, and the frequency of protein interactions with molecular chaperones determines their folding rates. However, it is unclear how immature protein dynamics are regulated. In this study, we analyzed the diffusion of immature tyrosinase in the endoplasmic reticulum (ER) of non-pigmented cells by taking advantage of the thermal sensitivity of the tyrosinase. The diffusion of tyrosinase tagged with yellow fluorescence protein (YFP) in living cells was directly measured using fluorescent correlation spectroscopy. The diffusion of folded tyrosinase in the ER of cells treated with brefeldin A, as measured by fluorescent correlation spectroscopy, was critically affected by the expression level of tyrosinase-YFP. Under defined conditions in which random diffusional motion of folded protein was allowed, we found that the millisecond-order diffusion rate observed for folded tyrosinase almost disappeared for the misfolded molecules synthesized at a nonpermissive high temperature. This was not because of enhanced aggregation at the high temperature, as terminally misfolded tyrosinase synthesized in the absence of calnexin interactions showed comparable, albeit slightly slower, diffusion. Yet, the thermally misfolded tyrosinase was not immobilized when measured by fluorescence recovery after photobleaching. In contrast, terminally misfolded tyrosinase synthesized in cells in which alpha-glucosidases were inhibited showed extensive immobilization. Hence, we suggest that the ER represses random fluctuations of immature tyrosinase molecules while preventing their immobilization.

Bacterial Proteins↗
PubMed2004

Basic fibroblast growth factor promotes apoptosis and suppresses granulation tissue formation in acute incisional wounds.

Cytokines are thought to play an important role in cellular loss and apoptosis during the repair of granulation tissue. In order to investigate the role of apoptosis following the administration of basic fibroblast growth factor (bFGF) to a wound, the present study examined the relationship between the degree of granulation tissue formation and the level of apoptosis in rat skin incisional wounds, following treatment with an intradermal injection of bFGF (0.1 microg and 1 microg per cm of wound). Histological assessment of the width of the wound tissue showed that the degree of granulation tissue in the 1 microg-bFGF-treated group had increased by day 7, but then subsequently diminished by days 14 and 28. The TUNEL index increased rapidly from day 1, peaking on day 7, with the index being higher in the 1 microg-bFGF-treated group on days 4, 7, and 14, when compared with a control group. In parallel with a marked increase in the TUNEL index over the first 14 days, the number of cells positive for vimentin and CD3 in the 1 microg-bFGF-treated wounds had decreased by day 14. The number of PCNA-positive cells, an indicator of cell proliferation, peaked on day 4 in the bFGF-treated wounds and then declined rapidly. On the basis of these results, it is suggested that the suppression of granulation tissue formation in bFGF-treated wounds is mainly due to an early and persistent increase in apoptosis in the granulation tissue cells. The expression of both transforming growth factor (TGF)-beta1 and bFGF was also elevated in the bFGF-treated wounds on days 4 and 7, suggesting that fibroblast apoptosis was induced by bFGF treatment. Unexpectedly, on day 28, the wound breaking strength was not reduced in the bFGF-treated wounds. These results indicate that apoptosis regulation following bFGF administration to an incisional wound may lead effectively to granulation tissue formation and promote a scar-less repair process.

Acute Disease↗
PubMed2004

Combination of porous hydroxyapatite and cationic liposomes as a vector for BMP-2 gene therapy.

The clinical significance of hydroxyapatite (HAP) as a bone substitute has become apparent in recent years and bone morphogenetic protein (BMP) a substance which induces bone has attracted much attention. In this study, a 1.2 cm diameter bone defects created on rabbit cranium were treated with the BMP-2 gene (cDNA plasmid) introduced with porous HAP after completion of hemostasis and the resultant bone formation was analyzed histopathologically. The amounts of bone formation was compared BMP-2 cDNA plasmids were not combined with cationic liposomes as a vector. Four groups of rabbits were compared. In the HAP group the cranial bone defect was treated with HAP containing 40 microg of liposomes and a dummy gene (PU). The BMP gene HAP group was treated with HAP soaked in liposomes and 10 microg of the BMP-2 gene. In addition, a group was treated with the gene without implanting HAP. Bone formation on the cranial defects was evaluated 3, 6 and 9 weeks after the operation, by X-ray and histopathological examinations. Three weeks after the operation there was vigorous bone formation in the cranial defect in the group which received the BMP-2 gene without HAP, and complete ossification was observed at 9 weeks. In the group which received HAP containing the BMP-2 gene, although new bone formation was evident surrounding the scaffold 3 weeks post-operation, the induced bone tissue did not fill all the pores of the scaffold even at 9 weeks post-operation. These results confirm the clinical usefulness of gene therapy for bone formation, using the BMP-2 gene combined with cationic liposomes as a vector. It is possible that the effects of administering the BMP-2 gene will be improved by specializing the microstructure of scaffold for gene therapy.

Animals↗
PubMed2004

Local administration of hepatocyte growth factor gene enhances the regeneration of dermis in acute incisional wounds.

Hepatocyte growth factor (HGF) has a number of biological activities, e.g., mitogenic, motogenic, antiapoptotic, antifibrous, and morphogenic. It also has angiogenic and angioprotective activities for endothelial cells. The aim of this study was to characterize the role of HGF in wound healing by administering the HGF gene locally to acute incisional skin wounds created on the backs of rats. To create wounds, the backs of Wistar rats were clipped and three 2-cm-long incisional wounds were made deep to the fascia. The wounds contained pannicrus carnosum and were created at intervals of 2 cm. After suturing, the HGF gene was then administered intradermally. Apoptotic cells in wound lesions were identified by TUNEL method as well as by immunological detection of active caspase-3. In the HGF-treated animals, we found almost complete suppression of apoptosis and well-organized wound healing. Histopathological examination revealed that the proliferation of fibroblasts was suppressed and that scar formation was less apparent in the HGF-treated animals compared to the controls. It is thought that administration of the HGF gene immediately after surgery may enhance the healing process through suppressing apoptosis, which occurred in the controls 1 week after suturing the incisional wound. In addition, locally increased HGF expression due to the introduction of the HGF gene to cells around wounds enhances dermal regeneration, possibly by promoting regeneration of dermal tissue, which results in less scarring due to its antifibrotic effect. Thus, HGF supplementation through gene therapy may be an effective strategy for treating wounds, as it increases the regeneration of the dermis to allow for "scarless wound healing."

Animals↗
PubMed2004

Ectopic expression of tyrosinase increases melanin synthesis and cell death following UVB irradiation in fibroblasts from familial atypical multiple mole and melanoma (FAMMM) patients.

Patients with familial atypical multiple mole and melanoma (FAMMM) [so-called familial dysplastic naevus syndrome (FDNS)] have a high risk for the development of malignant melanoma. The underlying gene defect has an autosomal dominant inheritance with variable expression and incomplete penetrance. Fibroblasts derived from FAMMM patients have high sensitivity to UVC and mutagens, e.g. 4-nitroquinoline-1-oxide. We were interested in identifying how the combination of inherent sensitivity to UV light and abnormal melanin synthesis interacts in the development of melanoma in FAMMM patients. Intermediates of melanin synthesis produce free radicals that are toxic to cells. Atypical moles (dysplastic naevi) are engaged in the biosynthesis of abnormal melanin pigments. This study examined whether there was any abnormal melanin pigmentation or cell damage after the ectopic expression of tyrosinase in fibroblasts from FAMMM patients when compared with fibroblasts from normal subjects. Fibroblasts from FAMMM patients (3012T and 3072T) were associated with a higher sensitivity than normal human fibroblasts to the toxicity of UVB. When cells were infected with tyrosinase-expressing adenovirus (Ad-HT) and irradiated with UVB, FAMMM fibroblasts showed higher tyrosinase activity, produced more melanin pigments and were degraded more significantly than normal human fibroblasts. Western blot analysis revealed that Ad-HT-infected 3072T produced a larger amount of tyrosinase protein than did Ad-HT-infected normal fibroblasts after UVB irradiation. Our findings suggest: (1) that FAMMM fibroblasts have an unknown machinery which enhances tyrosinase expression by UVB irradiation; and (2) that the resulting increase in melanin synthesis affects the cytotoxicity of UVB to FAMMM fibroblasts. All of these processes may be involved in the genomic instability and development of melanoma in FAMMM patients.

Adenoviridae↗
PubMed2004

A case of systemic osteomyelitides due to Mycobacterium avium.

A case of multiple osteomyelitides due to Mycobacterium avium (M. avium) infection with osteosclerotic bone lesions is reported. A 67-year-old male had been suffering from persistent fever and back pain since October 1999, and 20.0-2.5 mg prednisolone per day was prescribed for continuous inflammatory symptoms in January 2000. Six months later, computed tomography revealed osteosclerotic lesions in the left femur and thoracic vertebrae, but no skin lesion associated with mastocytosis or internal malignancy was identified. In September of 2002, a dome-shaped, soft subcutaneous tumor developed on the upper sternum. Histopathological findings revealed subcutaneous adipose tissue with several foci of tiny abscesses. Two weeks later, creamy pus was discharged through a draining sinus at the center of the wound. M. avium was demonstrated in the pus by Zeel-Nielsen staining and microplate hybridization.

Aged↗
PubMed2004

Over-expression of the testis-specific gene TSGA10 in cancers and its immunogenicity.

The TSGA10 gene was originally isolated in normal testis by differential mRNA display. TSGA10 is located on chromosome 2q11.2 and consists of 19 exons extending over 3 kb. TSGA10 mRNA expression was investigated in normal and malignant tissues using quantitative real-time RT-PCR. It was predominantly expressed in the testis in adult normal tissues. In malignant tissues, TSGA10 was over-expressed in 4 of 20 hepatocellular carcinomas (HCC), 1 of 20 colon cancers, 7 of 20 ovarian cancers, 3 of 20 prostate cancers, 1 of 21 malignant melanomas, and 8 of 21 bladder cancers. Serological analysis revealed that 3 out of 346 patients with various types of cancer possessed antibody against recombinant TSGA10 protein. They included 2 patients with hepatocellular carcinoma and a patient with malignant melanoma.

Antibodies↗
PubMed2003

Epigenetic inactivation of CHFR and sensitivity to microtubule inhibitors in gastric cancer.

Mitotic checkpoints prevent errors in chromosome segregation that can lead to neoplasia. Therefore, it is notable that gastric cancers often show impaired checkpoint function. In the present study, we examined the functional consequences of epigenetic inactivation of the mitotic checkpoint gene CHFR in gastric cancers. CHFR expression was silenced by DNA methylation of the 5' region of the gene in 20% of the gastric cancer cell lines tested and in 39% of primary gastric cancers; expression could be restored by treatment with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor. In addition, histones H3 and H4 were found to be deacetylated in cell lines showing aberrant methylation, indicating a role for histone deacetylation in the methylation-dependent gene silencing. Cells not expressing CHFR showed impaired checkpoint function, which led to nuclear localization of cyclin B1 after treatment with docetaxel or paclitaxel, two microtubule inhibitors. Apparently, the absence of CHFR is associated with sensitivity of cells to mitotic stress caused by microtubule inhibition, and restoration of CHFR expression by 5-aza-2'-deoxycytidine or adenoviral gene transfer restored the checkpoint. By affecting mitotic checkpoint function, CHFR inactivation likely plays a key role in tumorigenesis in gastric cancer. Moreover, the aberrant methylation of CHFR appears to be a good molecular marker with which to predict the sensitivity of gastric cancers to microtubule inhibitors.

Acetylation↗
PubMed2003

Identification of the interleukin 4 receptor alpha gene as a direct target for p73.

p73 has a high degree of structural homology to p53 and can activate transcription of p53-responsive genes. However, analysis of p73-deficient mice revealed a marked divergence in the physiological activities of p53 family genes and distinguishes p73 from p53. Mice deficient for p73 exhibit profound defects, including hippocampal dysgenesis, chronic infection, and inflammation, as well as abnormalities in pheromone sensory pathways. p73 plays important roles in neurogenesis, sensory pathways, and homeostatic regulation. Here, we found that the interleukin 4 receptor alpha (IL-4Ralpha) gene is up-regulated by p73 but not significantly by p53 in several human cancer cell lines. IL-4Ralphatranscription is also activated in response to cisplatin, a DNA-damaging agent known to induce p73. By using small interference RNA designed to target p73, we demonstrated that silencing endogenous p73 abrogates the induction of the IL-4Ralpha gene after cisplatin treatment. Furthermore, we identified a p73-binding site in the first intron of the IL-4Ralpha gene that can directly interact with the p73 protein in vivo. This p73-binding site consists of eight copies of a 10-bp consensus p53-binding motif and is a functional response element that is relatively specific for p73 among the p53 family. p73beta promoted localized nucleosomal acetylation through recruitment of coactivator p300, indicating that p73 regulates transcription of IL-4Ralpha through the unique p73-binding site. We also found that p73beta-transfected tumor cells are sensitive to IL-4-mediated apoptosis. Our data suggest that IL-4Ralpha could mediate, in part, certain immune responses and p73-dependent cell death.

Acetylation↗
PubMed2003

deltaNp63alpha functions as both a positive and a negative transcriptional regulator and blocks in vitro differentiation of murine keratinocytes.

deltaNp63 is overexpressed in squamous carcinomas where it is associated with proliferation and is believed to enhance cell growth by blocking p53-mediated transactivation. In normal epithelium, deltaNp63alpha protein expression is abundant in basal cells and decreases with differentiation. To explore the biological consequences of deltaNp63alpha overexpression in relation to squamous carcinogenesis, we evaluated its effect on normal squamous differentiation and p53 transactivation function in keratinocytes. Forced overexpression of deltaNp63alpha in primary murine keratinocytes in vitro inhibits morphological differentiation induced by elevated extracellular [Ca(2+)], abrogates Ca(2)(+)-induced growth arrest, and blocks expression of maturation-specific proteins keratin 10 and filaggrin. This suggests that deltaNp63 overexpression in squamous carcinomas may serve to maintain the basal cell phenotype and promote cell survival. deltaNp63alpha blocks transactivation of p53 responsive reporter constructs mediated by endogenous or exogenous p53 at 17 h postinfection, as expected. However, at 41 h, when p53-mediated transactivation is diminished, deltaNp63alpha enhances transactivation of these reporter constructs by 2.2-12-fold over control. Maximal deltaNp63alpha-induced transactivation requires intact p53 responsive elements, but is independent of cellular p53 status. This positive transcriptional function of deltaNp63alpha appears to be cell-type specific, as it is not observed in primary dermal fibroblasts or Saos-2 cells. These findings support deltaNp63alpha as a master regulator of keratinocyte differentiation, and suggest a novel function of this protein in the maintenance of epithelial homeostasis.

Adenoviridae↗
PubMed2003

HLF/HIF-2alpha is a key factor in retinopathy of prematurity in association with erythropoietin.

An HLF (HIF-1alpha-like factor)/HIF-2alpha-knockout mouse is embryonic lethal, preventing investigation of HLF function in adult mice. To investigate the role of HLF in adult pathological angiogenesis, we generated HLF-knockdown (HLF(kd/kd)) mice by inserting a neomycin gene sandwiched between two loxP sequences into exon 1 of the HLF gene. HLF(kd/kd) mice expressing 80-20% reduction, depending on the tissue, in wild-type HLF mRNA were fertile and apparently normal. Hyperoxia-normoxia treatment, used as a murine model of retinopathy of prematurity (ROP), induced neovascularization in wild-type mice, but not in HLF(kd/kd) mice, whereas prolonged normoxia following hyperoxic treatment caused degeneration of retinal neural layers in HLF(kd/kd) mice due to poor vascularization. Cre-mediated removal of the inserted gene recovered normal HLF expression and retinal neovascularization in HLF(kd/kd) mice. Expression levels of various angiogenic factors revealed that only erythropoietin (Epo) gene expression was significantly affected, in parallel with HLF expression. Together with the results from intraperitoneal injection of Epo into HLF(kd/kd) mouse, this suggests that Epo is one of the target genes of HLF responsible for experimental ROP.

Animals↗