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Biomedical subjects

Shin Sasaki

Publications and source records attributed to Shin Sasaki.

At least 19 recordsLinked to original sources
PubMed2007

Muscle creatine kinase/SV40 hybrid promoter for muscle-targeted long-term transgene expression.

Gene therapy for congenital protein deficiencies requires lifelong expression of a deficient protein. Current gene therapy approaches preferentially employ the strong cytomegalovirus (CMV) promoter/enhancer or its derivative CAG promoter; however, these promoters provide only temporary transgene expression. To create a promoter that enables long-lasting expression in muscle, hybrid promoters were constructed by coupling the muscle creatine kinase (MCK) enhancer to various strong promoters for enhancement of tissue specificity and improved transcriptional activity. A hybrid promoter containing the MCK enhancer and the simian virus 40 promoter (MCK/SV40 promoter) yielded long-term (>6 months) expression of a human secretory alkaline phosphatase (huSEAP) reporter gene following electrotransfer of the plasmid into mice, whereas expression using a conventional CMV or CAG promoter faded away within a few weeks. To explore the mechanism behind the sustained expression obtained with the MCK/SV40 promoter, mice were immunized with a LacZ expression plasmid driven by MCK/SV40 or a conventional promoter. Minimal cellular and humoral responses to LacZ were observed in MCK/SV40 promoter-treated animals, and mouse SEAP gene expression in vivo was successfully maintained by both the MCK/SV40 and conventional promoters. These results suggest that the lower immunogenicity of the MCK/SV40 promoter contributed to long-lasting gene expression in mice. Therefore, the MCK/SV40 promoter may provide the basis for development of an effective transgene expression cassette for treatment of congenital protein deficiencies in which therapeutic proteins are recognized as foreign by the host immune system.

Animals↗
PubMed2006

Distal colorectal cancers with microsatellite instability (MSI) display distinct gene expression profiles that are different from proximal MSI cancers.

Promoter methylation of the mismatch repair gene plays a key role in sporadic microsatellite instability (MSI) colorectal cancers. However, promoter methylation often occurs in proximal colon cancers, and molecular phenotypes underlying MSI cancers in distal colon have not been fully clarified. Our goal was to clarify the difference between MSI and microsatellite stability (MSS) cancers and, furthermore, to determine distinct characteristics of proximal and distal MSI cancers. By DNA microarray analysis of 84 cancers (33 MSI and 51 MSS), we identified discriminating genes (177 probe sets), which predicted MSI status with a high accuracy rate (97.6%). These genes were related to phenotypic characteristics of MSI cancers. Next, we identified 24 probe sets that were differentially expressed in proximal and distal MSI cancers. These genes included promoter methylation-mediated genes, whose expression was significantly down-regulated in proximal MSI cancers. Among discriminating genes between MSI and MSS, nine methylation-mediated genes showed down-regulation in MSI cancers. Of these, 7 (77.8%) showed down-regulation in proximal MSI cancers. Furthermore, methylation-specific PCR confirmed that frequency of hMLH1 promoter methylation was significantly higher in proximal MSI cancers (P = 0.0317). These results suggested that there is a difference between proximal and distal MSI cancers in methylation-mediated influence on gene silencing. In conclusion, using DNA microarray, we could distinguish MSI and MSS cancers. We also showed distinct characteristics of proximal and distal MSI cancers. The inactivation form of hMLH, per se, differed between proximal and distal MSI cancers. These results suggested that distal MSI cancers constitute a distinct subgroup of sporadic MSI cancers.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Multivalent DNA vaccine protects mice against pulmonary infection caused by Pseudomonas aeruginosa.

For efficacious vaccine development against Pseudomonas aeruginosa (P. aeruginosa), the immunogenicity of multivalent DNA vaccine was evaluated. Three different plasmids each targeting a fusion of outer membrane proteins (OprF/OprI), a protein regulating type III secretion system (PcrV), or an appendage (PilA) were prepared and mice were immunized with single (monovalent) or a combination of these plasmids (multivalent) via intramuscular electroporation (imEPT) or gene gun. Immunization with multivalent DNA vaccine via imEPT induced the most potent protection against lethal pneumonia. Although the serum levels of IgG binding to whole bacteria cells were comparable between groups, the strongest immune protection was associated with the serum levels of Th1-dominated multivalent IgG, the bronchoalveolar levels of macrophage inflammatory protein 2 (MIP-2) and IFN-gamma, and the number of neutrophils and macrophages in the bronchoalveolar lavage following intranasal challenge. These results implied the possible clinical application of multivalent DNA vaccine against P. aeruginosa.

Animals↗
PubMed2006

Toll-like receptor adaptor molecules enhance DNA-raised adaptive immune responses against influenza and tumors through activation of innate immunity.

Toll-like receptors (TLRs) recognize microbial components and trigger the signaling cascade that activates the innate and adaptive immunity. TLR adaptor molecules play a central role in this cascade; thus, we hypothesized that overexpression of TLR adaptor molecules could mimic infection without any microbial components. Dual-promoter plasmids that carry an antigen and a TLR adaptor molecule such as the Toll-interleukin-1 receptor domain-containing adaptor-inducing beta interferon (TRIF) or myeloid differentiation factor 88 (MyD88) were constructed and administered to mice to determine if these molecules can act as an adjuvant. A DNA vaccine incorporated with the MyD88 genetic adjuvant enhanced antigen-specific humoral immune responses, whereas that with the TRIF genetic adjuvant enhanced cellular immune responses. Incorporating the TRIF genetic adjuvant in a DNA vaccine targeting the influenza HA antigen or the tumor-associated antigen E7 conferred superior protection. These results indicate that TLR adaptor molecules can bridge innate and adaptive immunity and potentiate the effects of DNA vaccines against virus infection and tumors.

Adaptor Proteins, Signal Transducing↗
PubMed2006

hRFI overexpressed in HCT116 cells modulates Bcl-2 family proteins when treated with 5-fluorouracil.

Exogenous overexpression of hRFI, originally isolated in our laboratory, inhibits not only death receptor-mediated apoptosis but also the mitochondrial apoptosis induced by several chemotherapeutic agents including 5-fluorouracil (5-FU). Recently, it has become clear that hRFI targets and degradates caspase-8 and -10 in death receptor-mediated apoptosis by E3 ubiquitin activity in a ring finger domain homologous to that of X-chromosome-linked inhibitor of apoptosis protein (XIAP). However, the cellular mechanism of the inhibition of mitochondrial apoptosis by hRFI has not been fully elucidated. We prepared HCT116 overexpressing hRFI (HCT116/hRFI) cells and comprehensively analyzed the expression changes of 51 apoptosis-related genes with or without 5-FU treatment between HCT116/hRFI and mock cells using microfluidic low-density arrays. As a result, we identified four genes (Bcl-2, Bcl-XL, cIAP2, and CFLAR) whose expression was four or more times higher in HCT116/ hRFI cells than in HCT116/LacZ cells, and found that Bcl-2 and the ratio of Bcl-2/Bax or Bcl-2/Bak were upregulated when HCT116/hRFI cells were treated with 5-FU. Furthermore, we also validated the up-regulation of Bcl-2 and Bcl-XL in HCT116/hRFI cells treated with 5-FU by Western blot analysis. Such evidence suggests that the modulation of Bcl-2 family proteins seen in 5-FU treatment plays an important role in the anti-apoptotic function of HCT116/hRFI cells.

Antimetabolites, Antineoplastic↗
PubMed2006

Activator protein 1-mediated transcriptional regulation strategy sustains long-term expression of a xenogeneic gene product in vivo: an implication for gene therapy targeting congenital protein deficiencies.

Maintenance of high-level transgene expression is the main challenge in current gene therapy. Although the cytomegalovirus (CMV) promoter/enhancer or its derivative the CAG promoter has been harnessed in current gene therapy vectors, transgene expression by these vectors is often transient and remains at suboptimal levels due to undefined mechanisms, possibly including the shortage of transcriptional machinery. To overcome this drawback, we designed a novel transcriptional control system, designated here as transcription factor-supercharging promoter system, in which transgene expression is regulated by the positive feedback circuit consisting of cis- and trans-acting elements of gene expression machinery. Among combinations of these elements, a plasmid composed of a target gene expression cassette driven by the chimeric CMV promoter containing repetitive 12-O-tetradecanoylphorbol-13-acetate-responsive elements as cis-acting elements (CMV-TTT) and expression cassettes for c-Fos and c-Jun genes as trans-acting elements facilitated high and long-term (>10 months) expression of a transgene after its intramuscular electroporation-mediated delivery in mice. Since human secretory alkaline phosphatase was used as a reporter, it was suggested that the immune evasion mechanism elicited by the CMV-TTT and/or c-Fos/ c-Jun expression also contributed to the sustained expression in mice. Our strategy may open a new avenue for a gene therapy that involves lifelong supplementation of a deficient protein that could be targeted by the host's immune system.

Animals↗
PubMed2006

[A case of pulmonary aspergillosis accompanied with eosinophilic pneumonia].

An infiltrative shadow was observed in the upper lobe of the left of a 60-year-old man lung during the course of paroxysmal tachycardia. Fever and coughing occurred one month later, and ground-glass opacities (GGOs) were found extensively in the lower left lung field. Although his condition strongly suggested allergic bronchopulmonary aspergillosis (ABPA) due to significantly increased eosinophil count in the peripheral blood and bronchoalveolar lavage fluid of the lower lobe of the left lung, anti-aspergillus antibody precipitation, and positive immediate intradermal test, the Rosenberg diagnostic criteria for ABPA were not satisfied. He was given a diagnosis of eosinophilic pneumonia, and GGO disappeared when itraconazole and prednisolone were administered. However, since the shadow in the upper lobe of the left lung later increased and percutaneous thoracocentesis revealed the presence of Aspergillus fumigatus, prednisolone treatment was discontinued and micafungin was administered. Nonetheless, the shadow spread into both lungs and regardless of treatment with amphotericin-B, the patient died. Although the present case is different from a typical ABPA, it indicates that allergic reaction can be induced by Aspergillus infection in the lung field, apart from the central airway. In addition, the development of an appropriate therapeutic strategy against both infection and allergy in such cases is essential.

Aspergillosis, Allergic Bronchopulmonary↗
PubMed2006

[A case of myelodysplastic syndrome complicated with organizing pneumonia].

A 74-year-old man had been given a diagnosis of myelodysplastic syndrome (MDS), and had been treated with granulocyte-colony stimulating factor (G-CSF). 1 year later, he suffered from fever and his chest X-ray lung biopsy did not provide a diagnosis, video-assisted thoracoscopic lung biopsy was performed, which yielded a histological diagnosis of organizing pneumonia. His pulmonary disease was diagnosed as secondary organizing pneumonia due to MDS, and was treated successfully with steroids. Vigorous efforts to establish a histological diagnosis is needed for the antibiotics-resistant pneumonia in the case of MDS.

Aged↗
PubMed2005

Effect of cystathionine beta-synthase variant 844ins68bp and methylenetetrahydrofolate reductase A1298C polymorphisms in xenografts on 5-FU efficacy and doubling time.

The association of MTHFR and CBS variants with the doubling time and responsiveness to several chemodrugs was analyzed in 26 human cancer xenografts. The tumors homozygous for the absence of insertion (NN) for the CBS 844ins68bp were more chemosensitive than those with insertion (NI) to TS-1 (P=0.0048), suggesting a potential effect of this variant on fluoropyrimidine efficacy. Furthermore, the doubling time of tumors with a variant C allele (AC or CC) in MTHFR-A1298C was significantly longer than that of tumors with a normal allele (AA) (P=0.0008). Twenty-nine cellular proliferation-related genes were associated with MTHFR-A1298C genotyping and with the doubling time.

Antimetabolites, Antineoplastic↗
PubMed2005

TRAF4 acts as a silencer in TLR-mediated signaling through the association with TRAF6 and TRIF.

Toll-like receptors (TLR) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase play an essential role in intracellular eradication of engulfed pathogens. Here, we demonstrate the physical and functional association between components of the cytosolic NADPH oxidase and TLR-mediated signaling molecules. Cytosolic components of NADPH oxidase suppressed TLR-mediated NF-kappaB activation as well as IFN-beta promoter activation. We demonstrate that TNF-associated factor (TRAF) 4 associates with p47(phox), a component of cytosolic NADPH oxidase, and physically interacts and functionally counteracts with TRAF6 and Toll-IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-beta (TRIF) molecules that critically regulate TLR-mediated signaling. TRAF4 mRNA expression was elicited in RPMI 8226 cells following LPS or CpG DNA treatment. These results suggest that TRAF4 participates in the molecular mechanism underlying silencing of TLR-mediated signaling through the interaction with molecules harboring phagosome/endosome membrane.

Adaptor Proteins, Vesicular Transport↗
PubMed2005

Epigallocatechin gallate induces apoptosis of monocytes.

BACKGROUND: Monocytes are the main effector cells of the immune system, and the regulation of their survival and apoptosis is essential for monocyte-involved immune responses. Green tea polyphenol catechin has been reported to have antiallergic and anti-inflammatory activities, but its effect on monocytes has not yet been explored. OBJECTIVE: To elucidate the mechanisms of the anti-inflammatory effect of catechin, we studied the effect of catechin, especially epigallocatechin gallate (EGCG), on the apoptosis of monocytes. METHODS: Isolated peripheral blood monocytes were incubated without or with catechin, and apoptosis was evaluated by annexin V and propidium iodide double-staining or terminal deoxynucleotidyl assay. The activation of caspases 3, 8, and 9 was also evaluated by flow cytometry. The influence of GM-CSF or LPS, the known monocyte survival factors, on the EGCG-induced apoptosis of monocytes was investigated. RESULTS: Among the 4 catechin derivatives tested, EGCG and epicatechin gallate induced apoptosis of monocytes. Caspases 3, 8, and 9, which play a central role in the apoptotic cascade, were dose-dependently activated by EGCG treatment. The EGCG-induced apoptosis of monocytes was not affected by GM-CSF or LPS. CONCLUSION: Catechin, especially EGCG, by promoting monocytic apoptosis, may be a new promising anti-inflammatory agent, and should be tested in clinical trials.

Anti-Inflammatory Agents↗
PubMed2005

Overexpression of hRFI (human ring finger homologous to inhibitor of apoptosis protein type) inhibits death receptor-mediated apoptosis in colorectal cancer cells.

The acquisition of antiapoptotic properties is one of the essential mechanistic steps in colorectal carcinogenesis and is closely correlated with a loss of chemosensitivity and radiosensitivity. Human ring finger homologous to inhibitor of apoptosis protein type (hRFI) is a newly discovered gene encoding a ring finger domain highly homologous to that of X chromosome-linked inhibitor of apoptosis protein. Immunohistochemistry has revealed that the expression of hRFI increased in transition from normal colorectal mucosas to adenomas and from adenomas to carcinomas, suggesting an essential role in the early stage of colorectal carcinogenesis. However, the function role of hRFI in colorectal carcinoma has not been elucidated. To determine whether hRFI possesses an antiapoptotic function in colorectal cancer cells, HCT116 colorectal cancer cells stably overexpressing hRFI were established. The hRFI transfectant exhibited significant resistance to apoptosis induced by tumor necrosis factor-alpha or tumor necrosis factor-related apoptosis-inducing ligand compared with control. This antiapoptotic response was associated with decreased activity of caspase-3, -8, and -9. We also established an antisense down-regulation of hRFI, which effectively reversed the antiapoptotic activity of the hRFI transfectant. This confirmed that the antiapoptotic property of the hRFI transfectant was not due to the clonal effect but in fact dependent on hRFI function. In conclusion, hRFI possesses an antiapoptotic function in HCT116 colorectal cancer cells. Considering the progressive increase of hRFI expression in the advance of the colorectal adenoma-carcinoma sequence, hRFI is one of the important players in colorectal carcinogenesis through its effect on apoptosis regulation.

Apoptosis↗
PubMed2004

Transcriptional regulation of the human TLR9 gene.

To clarify the molecular basis of human TLR9 (hTLR9) gene expression, the activity of the hTLR9 gene promoter was characterized using the human myeloma cell line RPMI 8226. Reporter gene analysis and EMSA demonstrated that hTLR9 gene transcription was regulated via four cis-acting elements, cAMP response element, 5'-PU box, 3'-PU box, and a C/EBP site, that interacted with the CREB1, Ets2, Elf1, Elk1, and C/EBPalpha transcription factors. Other members of the C/EBP family, such as C/EBPbeta, C/EBPdelta, and C/EBPepsilon, were also important for TLR9 gene transcription. CpG DNA-mediated suppression of TLR9 gene transcription led to decreased binding of the trans-acting factors to their corresponding cis-acting elements. It appeared that suppression was mediated via c-Jun and NF-kappaB p65 and that cooperation among CREB1, Ets2, Elf1, Elk1, and C/EBPalpha culminated in maximal transcription of the TLR9 gene. These findings will help to elucidate the mechanism of TLR9 gene regulation and to provide insight into the process by which TLR9 evolved in the mammalian immune system.

Animals↗
PubMed2004

The genome sequence of silkworm, Bombyx mori.

We performed threefold shotgun sequencing of the silkworm (Bombyx mori) genome to obtain a draft sequence and establish a basic resource for comprehensive genome analysis. By using the newly developed RAMEN assembler, the sequence data derived from whole-genome shotgun (WGS) sequencing were assembled into 49,345 scaffolds that span a total length of 514 Mb including gaps and 387 Mb without gaps. Because the genome size of the silkworm is estimated to be 530 Mb, almost 97% of the genome has been organized in scaffolds, of which 75% has been sequenced. By carrying out a BLAST search for 50 characteristic Bombyx genes and 11,202 non-redundant expressed sequence tags (ESTs) in a Bombyx EST database against the WGS sequence data, we evaluated the validity of the sequence for elucidating the majority of silkworm genes. Analysis of the WGS data revealed that the silkworm genome contains many repetitive sequences with an average length of <500 bp. These repetitive sequences appear to have been derived from truncated transposons, which are interspersed at 2.5- to 3-kb intervals throughout the genome. This pattern suggests that silkworm may have an active mechanism that promotes removal of transposons from the genome. We also found evidence for insertions of mitochondrial DNA fragments at 9 sites. A search for Bombyx orthologs to Drosophila genes controlling sex determination in the WGS data revealed 11 Bombyx genes and suggested that the sex-determining systems differ profoundly between the two species.

Animals↗
PubMed2004

Improvement of DNA vaccine immunogenicity by a dual antigen expression system.

This study examined whether increased antigen expression resulted in enhanced antigen-specific immune responses in the context of DNA vaccines. To increase antigen expression, two copies of antigen expression cassettes were arranged in a plasmid pDX. BALB/c mice were intramuscularly immunized with various constructs that express influenza antigens and analysed for DNA-raised immunity. The plasmid pDX that expresses two copies of the antigen gene induced stronger antigen-specific immune responses than the plasmid pGA which expresses single antigen gene. To explore the in vivo transgene expression by pDX and pGA, luciferase activity was measured in the muscles transduced with luciferase expression plasmids. The pDX expressing two copies of luciferase induced the highest luciferase activity, which corresponded to the results from vaccination. We concluded that increasing the number of antigen expression cassettes in a vaccine construct improved antigen expression in the transduced tissue, which induced stronger DNA-raised immune responses.

Animals↗
PubMed2004

Aberrant expression of lysophosphatidic acid (LPA) receptors in human colorectal cancer.

Lysophosphatidic acid (LPA) is a simple bioactive phospholipid with diverse effects on various cells, that interacts with three G protein-coupled transmembrane receptors, LPA1, LPA2, and LPA3. The expression pattern and functions of these LPA receptors in various tumors have not been fully examined, except in ovarian cancer. To evaluate the LPA receptor expression profile in human colorectal cancer and in normal mucosa, we used real-time reverse transcription-polymerase chain reaction (RT-PCR) and measured the expression levels of LPA1, LPA2, and LPA3 messenger RNA (mRNA) in 26 colorectal cancers and 16 corresponding normal tissue samples. Normal epithelium expressed both LPA1 and LPA2 mRNA at similar levels. In comparison, colorectal cancers expressed LPA1 mRNA at a significantly lower level (0.3-fold; P<0.05), and LPA2 mRNA at a significantly higher level (three-fold; P<0.05), as compared with normal tissues. Thus, the ratio of LPA2/LPA1 increased markedly during malignant transformation (18-fold increase). LPA3 mRNA was expressed at only a low level in both normal and cancer tissues. We also assessed LPA2 expression immunohistochemically using a rat anti-LPA2 monoclonal antibody, and confirmed high expression of LPA2 in colorectal cancer at the protein level. As for LPA1, we examined Western blot analysis for 16 matched normal and cancer tissues. It revealed a significant decrease in the expression of LPA1 protein in cancer tissues compared to normal mucosa in nine of 16 cases, and in the remaining seven cases the expression levels was much the same. These results suggested that alteration of LPA receptor expression might be an important event in the development of colorectal cancer, and therefore, LPA and its receptors could be a chemopreventive target against colorectal cancer.

Adenocarcinoma↗
PubMed2004

Diffuse expression of hRFI is correlated with blood vessel invasion in gastric carcinoma.

BACKGROUND: hRFI, which has a relatively high homology to XIAP, is preferentially expressed in esophageal and colorectal carcinomas, and is involved in the initial tumor formation in the colorectal adenoma-carcinoma sequence. Furthermore, its diffuse expression is associated with colorectal carcinogenesis. However, hRFI expression in gastric carcinomas has not been evaluated so far. METHODS: We performed immunohistochemical staining on 76 gastric carcinoma samples using the antibody to hRFI and also analyzed the correlation between the staining pattern of hRFI and the clinico-pathological characteristics. RESULTS: All of the samples were stained focally (31 cases, 40.8%) or diffusely (45 cases, 59.2%) in the cancerous region. On the contrary, most of the normal gastric region showed no staining, except for a few cases that showed slight immunoreactivity in speckles. Furthermore, the proportion of blood vessel involvement was significantly higher in carcinomas with diffuse hRFI expression (28/45, 62.2%) than in carcinomas with focal expression (7/31, 22.6%) (P < 0.001). Liver metastasis was consistently observed in five cases (11.1%) in diffuse, but only one (3.3%) in focal type during the average follow up period of 5 years. However, the 3-year survival rate did not show significant difference between these different staining patterns of hRFI. CONCLUSIONS: These results suggest that the detection of the expression pattern of hRFI in gastric carcinomas can be another useful predictor of liver recurrence, especially when combined with other factors.

Adenocarcinoma↗