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Biomedical subjects

M Tachibana

Publications and source records attributed to M Tachibana.

At least 55 records · Page 3Linked to original sources
PubMed2001

Production of cloned mice from embryonic stem cells arrested at metaphase.

In mammals, cloned individuals can be produced from somatic cells. The combined use of gene targeting in embryonic stem cells and cloning contributes to the investigation of gene function in mammals. However, one of the major limitations to cloning is the low viability of cloned embryos, leading typically to high rates of pre- and postnatal death. The present study investigated whether cloning efficiency is influenced by the procedural differences involved in using transfected embryonic stem cells arrested at M phase for cloning by both single and serial transfer. In contrast to a previous study, in which fibroblasts were used, in the present study using embryonic stem cells there was no difference in the rate of production of cloned pups after the use of a single or serial nuclear transfer, although the proportion of blastocysts (70% versus 51%) was significantly higher (P < 0.001) after serial nuclear transfer. After embryo transfer of 445 blastocysts, 218 (49%) implanted and 27 (6% of blastocysts transferred) live pups were born. Of these 27 pups, 23 developed to adults of apparently normal fertility. Of these adults, 39% (n = 9) were derived from targeted embryonic stem cells, which is similar to the proportion of targeted embryonic stem cells in the population used for cloning. This study showed that cloning with embryonic stem cells is a viable procedure resulting in the production of transgenic cloned adults.

Animals↗
PubMed2001

Flow-through chemiluminescence sensor using immobilized oxidases for the selective determination of L-glutamate in a flow-injection system.

A selective and sensitive chemiluminometric flow sensor for the determination of L-glutamate in serum, based on immobilized oxidases such as glutamate oxidase (GOD), uricase (UC) and peroxidase (POD), is described herein. The principle for the selective chemiluminometric detection for L-glutamate is based on coupled reactions of four sequentially aligned immobilized oxidases, UC/POD/GOD/POD in a flow cell. The immobilized UC was employed to decompose urate, which is one of the major interfering components in serum for a luminol-H2O2 chemiluminescence reaction. The H2O2 produced from the UC reaction readily reacted with reducing components, such as ascorbate and glutathione, and then the excess H2O2 was decomposed by the immobilized POD. L-Glutamate in the sample plug was enzymatically converted to H2O2 with immobilized GOD. Subsequently, the peroxide reacts with luminol on the immobilized POD to produce chemiluminescence, proportional to glutamate concentration. The enzymes were immobilized on tresylated poly(vinyl alcohol beads). The immobilized enzymes were packed into TPFE tube (1.0 mm i.d. x 60 cm), in turn, and used as a flow cell. The sampling rate was 30 h-1. The calibration graph for L-glutamate is linear for 20 nM-5 microM; the detection limit (signal-to-noise = 3) is 10 nM.

Ascorbic Acid↗
PubMed2001

Four strains of spontaneously hyperlipidemic (SHL) mice: phenotypic distinctions determined by genetic backgrounds.

Spontaneously hyperlipidemic (SHL) mice are Japanese wild mice (KOR) with disruption of the apolipoprotein E (Apo E) gene. These mice (KOR-Apoe(shl)) are superhypercholesterolemic and develop severe xanthoma, but their atherosclerosis is relatively mild compared with Apo E knockout mice. First, we tested whether this distinction is due to additional mutation of the Apoc1 and/or Apoc2 genes in KOR-Apoe(shl). Southern blot analysis, but found no gross disruption of these genes. Next, we tested whether the phenotypic distinction is due to differences in the genetic background. To this end, we established three lines of congenic SHL mice with a genetic background of C57BL/6, BALB/c or C3H/He, and named them, respectively, C57BL/6.KOR-Apoe(shl) (B6.KOR-Apoe(shl)), BALB/c.KOR-Apoe(shl) (C.KOR-Apoe(shl)) and C3H/He.KOR-Apoe(shl) (C3.KOR-Apoe(shl)). Hypercholesterolemia was most severe in KOR-Apoe(shl) followed the by others as follows; KOR-Apoe(shl)>>C3.KOR-Apoe(shl)>C.KOR-Apoe(shl)>B6.KOR-Apoe(shl). In contrast, atherosclerosis was most severe in B6.KOR Apoe(shl) followed by the others: B6.KOR-Apoe(shl)>C.KOR-Apoe(shl)>>C3.KOR-Apoe(shl)> or =KOR-Apoe(shl). This order, however, did not match that in xanthoma, which was highly prominent in KOR-Apoe(shl) but mild in B6.KOR-Apoe(shl), C.KOR-Apoe(shl) and C3.KORApoe(shl). This order, however, did not match that in xanthoma, which was highly prominant in KOR-Apoe(shl) but mild in B6.KOR-Apoe(shl), C.KOR-Apoe(shl) and C3.KOR-Apoe(shl). These distinctions suggest that the severity of each of the phenotypes is determined by distinct genetic backgrounds which probably are composed of polymorphism of lipid metabolism-related proteins. We found that apolipoprotein A-I is decreased in each SHL strain and polymorphic between B6.KOR-Apoe(shl) and the other strains examined. This polymorphism may be related to the most severe atherosclerosis observed in B6.KOR-Apoe(shl). It is most likely that combination of such polymorphisms is due to the genetic background accountable for phenotype distinctions.

Animals↗
PubMed2001

Immunotherapy of bladder cancer using autologous dendritic cells pulsed with human lymphocyte antigen-A24-specific MAGE-3 peptide.

Recent investigations have demonstrated the efficacy of autologous dendritic cells (DCs) pulsed with tumor antigens to generate tumor-specific CTLs against cancer cells. Melanoma antigens (MAGE) are a family of tumor-specific antigens shown to be expressed in various tumors, including bladder cancers and melanoma, but not in normal tissues except for the testis. Because invasive bladder cancers are frequently reported to express MAGE, we explored the possibility of establishing a new immunotherapeutic modality against advanced bladder cancer using autologous DCs pulsed with one of the MAGE-3 epitope peptides (IMPKAGLLI), which is synthesized to bind specifically to HLA-A24. A MAGE-3-expressing bladder cancer cell line, FY, was newly established from a lymph node metastasis of bladder cancer in a HLA-A24+ patient. The FY cell-specific CTL response was significantly higher when CTL was induced by autologous DCs pulsed with IMPKAGLLI than by FY cells alone or by nonpulsed DCs in vitro. A total of four HLA-A24+ patients with advanced MAGE-3+ bladder cancers were treated with s.c. injections of autologous DCs pulsed with IMPKAGLLI every 2 weeks for a minimum of 6 and a maximum of 18 times. Three of four patients showed significant reductions in the size of lymph node metastases and/or liver metastasis. No significant untoward side effects were noted in these patients. This study indicated that, at sometime in the future, tumor-specific DC-based cancer immunotherapy may be useful as an additional treatment modality against advanced bladder cancer.

Aged↗
PubMed2001

[Detection of PSA mRNA in prostate cancer patients' blood].

A sensitive technique using reverse transcriptase-polymerase chain reaction (RT-PCR) for PSA mRNA has been used to detect circulating tumor cells in the peripheral blood of prostate cancer patients. We evaluated the clinical utility of this method for staging and monitoring for prostate cancer. The number of patients who were RT-PCR positive was increased in higher clinical stages. This technique may provide useful information for treating patients with prostate cancer, especially candidates for radical prostatectomy. Confirming the value of this modality as a prognostic factor will require further study.

Biomarkers, Tumor↗
PubMed2000

Prognostic value of alkaline phosphatase flare in patients with metastatic prostate cancer treated with endocrine therapy.

OBJECTIVES: To assess the prognostic significance of alkaline phosphatase (ALP) flare in 114 patients with metastatic prostate cancer treated with endocrine therapy. METHODS: ALP flare was defined as a transient increase of the serum ALP level to 120% or more of the pretreatment value after the initiation of endocrine therapy, followed by a subsequent decrease. RESULTS: Univariate analysis demonstrated that patients with poorly differentiated adenocarcinoma, an extent of disease of 2 or greater, a serum ALP level above twice the upper limit of normal, a serum prostate-specific antigen level greater than 100 ng/mL, an ALP flare, and a hemoglobin level of 12 g/dL or less had a significantly lower survival rate than their respective counterparts. Multivariate Cox's proportional hazards model analysis demonstrated that tumor histologic features and ALP flare were significant prognostic indicators for survival. CONCLUSIONS: The results of the present study suggest that the tumor histologic features and the ALP flare are significant prognostic indicators for survival in patients with metastatic prostate cancer treated with endocrine therapy.

Adenocarcinoma↗
PubMed2000

Prostate specific antigen adjusted for transition zone volume: the most powerful method for detecting prostate carcinoma.

BACKGROUND: Several methods for the identification of patients with prostate carcinoma have been proposed to enhance the clinical usefulness of prostate specific antigen (PSA). However, it remains unclear which method is superior in practical use. The authors attempted prospectively to identify the most powerful method with which to detect prostate carcinoma, especially among patients with intermediate PSA levels. METHODS: Between October 1997 and August 1999, systematic sextant biopsies were performed on 281 patients, including 147 with PSA levels between 4.1 ng/mL and 10.0 ng/mL. The clinical values of PSA, the free PSA to total PSA ratio (free/total PSA ratio), alpha-1-antichymotrypsin-PSA complex (PSA-ACT), the calculated derivatives, PSA density (PSAD), and PSA density of the transition zone (PSATZD) for the detection of prostate carcinoma were compared by using receiver operating characteristic (ROC) curves and logistic regression analyses. RESULTS: According to ROC curve analysis, PSATZD had the greatest area under the curve in the overall patient population and in patients with intermediate PSA levels. In patients with intermediate PSA levels, at the sensitivity of 90%, PSATZD would have prevented unnecessary biopsies in 68 of 117 patients who were without prostate carcinoma, whereas PSA, free/total PSA ratio, and PSA-ACT would have prevented unnecessary biopsies in 25, 28, and 25 patients, respectively. Stepwise logistic regression analysis showed that PSATZD and findings on digital rectal examination were significant independent predictors. CONCLUSIONS: PSATZD had the most useful validity in the differentiation between prostate carcinoma and benign prostatic enlargement in the overall patient population and in patients with intermediate PSA levels.

Aged↗
PubMed2000

Intravesical and intravenous therapy of human bladder cancer by the herpes vector G207.

G207, a conditionally replicating herpes vector, efficiently kills human bladder cancer cells in vitro. To evaluate the therapeutic potential of G207, we have established three in vivo models similar to the clinical situation. In vivo, G207 was intraneoplastically, intravesically, or intravenously inoculated in nude mice. Intraneoplastic inoculation into subcutaneous tumor caused significant tumor growth inhibition. Intravesical inoculation of G207 also caused decreased tumor growth in an orthotopic human bladder cancer model. Furthermore, multiple intravenous inoculation markedly inhibited subcutaneous tumor growth. These results suggest that intravesical therapy with G207 is effective for localized bladder tumor, especially for carcinoma in situ (CIS), and intravenous therapy with G207 is promising for invasive or metastasized bladder tumor.

Animals↗
PubMed2000

Poly(allylamine) beads as selective sorbent for preconcentration of formaldehyde and acetaldehyde in high-performance liquid chromatographic analysis.

Formaldehyde and acetaldehyde in water were determined by preconcentration with poly(allylamine) beads, derivatization with 2,4-dinitrophenylhydrazine (DPH) and analysis by HPLC. Poly(allylamine) beads (0.5 g) were used to adsorb formaldehyde and acetaldehyde at 1.2-150 microg l(-1) and 3.5-220 microg l(-1) from water (1 l). The concentration factor is 50 fold. The aldehydes were eluted and derivatized with 2 mM DPH in 0.5 M H2SO4 (10 ml). The time of analysis was 1 h. The detection limits (S/N=3) for formaldehyde and acetaldehyde were 0.6 and 2 microg l(-1), respectively.

Acetaldehyde↗
PubMed2000

Coreceptor function of mutant human CD4 molecules without affinity to gp120 of human immunodeficiency virus.

Despite extensive mutational studies on the human CD4 molecule and its affinity to human immunodeficiency virus (HIV) envelope glycoprotein gp120, coreceptor functions of such mutant molecules have only been examined by indirect measurement of their affinity to class II major histocompatibility complex (MHC) molecules. In this report, coreceptor functions of mutant human CD4 molecules, which have no or reduced affinity to an HIV envelope protein, gp120, were assessed in a murine T cell receptor/class II MHC recognition system. The substitution of human C" beta strand with the murine homologous segment resulted in the loss of the coreceptor function as well as in the complete loss of gp120 binding capacity, corroborating the consensus that Phe-43 in C" beta strand plays crucial roles in both situations. However, simultaneous replacement of the C'-C" loop along with the C" beta strand by homologous murine segments rescued the coreceptor function, whereas gp120 binding capacity remained negative. Further analysis indicated that insertion of lysine between Gly-41 and Ser-42 can partially compensate for the coreceptor function lost by the Phe-43 --> Val mutation. Although the coreceptor function of these mutant CD4 molecules in a human T cell recognition system is yet to be determined, these observations necessitate a re-evaluation of the role played by Phe-43 in coreceptor function. Examination of the sensitivities of the mutant CD4 molecules expressed on HeLa cells to infection by a T cell-tropic HIV-1 strain indicated that only those mutants that had completely lost gp120 binding capacity were resistant to the infection. All mutants having whole C" substitution, irrespective of additional substitutions or their coreceptor functions, were resistant to the infection.

Amino Acid Sequence↗
PubMed2000

Structural organization of the human microphthalmia-associated transcription factor gene containing four alternative promoters.

Microphthalmia-associated transcription factor (MITF) affects the development of many types of cells, including melanocytes and retinal pigment epithelium (RPE). MITF consists of at least three isoforms, MITF-A, MITF-H and MITF-M, differing at their amino-termini and expression patterns. Here, we characterize the structural organization of the human MITF gene. The gene contains at least four isoform-specific first exons, exons 1A, 1H, 1B and 1M in the 5' to 3' direction, each of which encodes the unique amino-terminus of a given isoform, including newly identified MITF-B. The 5'-flanking regions of these isoform-specific exons are termed promoters A, H, B and M, respectively, which showed different promoter activities, as judged by transient transfection assay. Promoter A directs the expression of a reporter gene in RPE, cervical cancer and melanoma cells, whereas promoter M is functional only in melanoma cells. Promoter H showed the significant activity in RPE and cervical cancer cells but not in melanoma cells. In contrast, the 1.7 kb 5'-flanking region of exon 1B showed no noticeable promoter activity in these cell lines. Therefore, alternative promoters provide the MITF gene with the diversity in transcriptional regulation and the capability of generating structurally different protein isoforms.

Amino Acid Sequence↗
PubMed2000

Ser298 of MITF, a mutation site in Waardenburg syndrome type 2, is a phosphorylation site with functional significance.

MITF (microphthalmia-associated transcription factor) is a basic-helix-loop-helix-leucine zipper (bHLHZip) factor which regulates expression of tyrosinase and other melanocytic genes via a CATGTG promoter sequence, and is involved in melanocyte differentiation. Mutations of MITF in mice or humans with Waardenburg syndrome type 2 (WS2) often severely disrupt the bHLHZip domain, suggesting the importance of this structure. Here, we show that Ser298, which locates downstream of the bHLHZip and was previously found to be mutated in individuals with WS2, plays an important role in MITF function. Glycogen synthase kinase 3 (GSK3) was found to phosphorylate Ser298 in vitro, thereby enhancing the binding of MITF to the tyrosinase promoter. The same serine was found to be phosphorylated in vivo, and expression of dominant-negative GSK3beta selectively suppressed the ability of MITF to transactivate the tyrosinase promoter. Moreover, mutation of Ser298, as found in a WS2 family, disabled phos-phorylation of MITF by GSK3beta and impaired MITF function. These findings suggest that the Ser298 is important for MITF function and is phosphorylated probably by GSK3beta.

Amino Acid Substitution↗
PubMed2000

Concurrent expressions of metallothionein, glutathione S-transferase-pi, and P-glycoprotein in colorectal cancers.

PURPOSE: Because the status of the inherent drug-resistance of colorectal cancers remains obscure, human colorectal cancers with no neoadjuvant therapy were retrospectively investigated regarding the expression of three drug-resistant proteins: metallothionein, glutathione S-transferase-pi, and P-glycoprotein. METHODS: Paraffin-embedded tissues of 130 colorectal cancers (Dukes A, 20; B, 49; C, 41; D, 20) obtained by surgical resections from 1982 to 1989 were used. The three proteins were immunostained by the streptavidin-biotin complex method. The immunostaining was judged to be positive if more than 5 percent of cells showed positive staining by use of cell analysis system. The data were compared with clinicopathologic features (Dukes A-D) and patients' prognosis (Dukes AC). RESULTS: Metallothionein, glutathione S-transferase-pi, and P-glycoprotein were positively expressed in 91 (70 percent), 30 (23 percent), and 98 (75 percent), respectively. A total of 120 (86 percent) expressed at least one drug-resistant protein. No intergroup differences were observed between positive and negative expressions of the proteins and their clinicopathologic features except tumor location. Rectal cancers positively expressed P-glycoprotein and three proteins more frequently. Twenty-six (20 percent), 65 (50 percent), and 21 (16 percent) cancers positively expressed one, two, and three proteins, respectively. The disease-free survival rates of patients with Dukes A through C cancer with positive staining for one, two, and three proteins were 100, 94, and 83 percent (at 1 year); 100, 72, and 51 percent (at 3 years); and 94, 66, and 38 percent (at 5 years), respectively (Kaplan-Meier with log-rank test; P = 0.016). In the multivariate Cox analysis, age, Dukes stage, tumor size, and glutathione S-transferase-pi were independent prognostic factors. CONCLUSIONS: The patients with concurrent expression of drug-resistant proteins in their cancers had worse prognoses. Examining drug-resistant proteins in colorectal cancers may be useful in selecting adjuvant chemotherapy and in predicting prognosis more accurately.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Microsatellite analysis in multiple chromosomal regions as a prognostic indicator of primary bladder cancer.

This study investigated gene abnormalities in bladder cancer patients and the relationship between chromosomal alteration and the clinical outcome using microsatellite analysis. A total of 45 human bladder tumor patients were analyzed. The microsatellite markers for 18q21.1 (D18S46, D18S363, and D18S474), 9p21-22 (D9S171, D9S747, D9S1747, and IFNA), and 17p13.1 (TP53) were used for the loss of heterozygosity (LOH) detection. The clinical outcomes were estimated with univariate and multivariate analyses. The results show that patients with LOHs in 18q21.1 and 9p21-22 exhibited a significantly poor prognosis. LOHs of these chromosomal regions had the most predictable potential compared with the other known prognostic factors, such as tumor grade, TNM stage, and age. It is concluded that microsatellite analysis for 18q21.1 and 9p21-22 is capable of predicting the clinical outcome of bladder cancer patients.

Adult↗
PubMed2000

Long-term survival of transmural advanced gastric carcinoma following curative resection: multivariate analysis of prognostic factors.

Patients with transmural (T2N0-T3N2) advanced gastric carcinoma comprise the largest group with uncertain outcome. These patients must be stratified according to the prognostic variables so the high risk group can be precisely identified. A total of 152 patients with transmural advanced gastric carcinomas were uniformly treated with a curative intent between 1979 and 1989 with at least a 7-year follow-up. Results and prognostic factors of long-term survival were analyzed by univariate and multivariate analyses. Seventy-five (49%) patients with advanced gastric carcinoma survived more than 7 years, which indicates the curative nature of surgery for gastric carcinoma. Seventeen variables were evaluated by univariate analysis. In the multivariate analysis, patient's age [>70 years, relative risk (RR) 2.4)], intraoperative blood loss (>500 ml, RR 1.7), blood vessel invasion (RR 2.3), ratio of invaded dissected lymph nodes (>0.2, RR 3.0), and tumors penetrating the serosa (RR 3.9) were the independent prognostic variables. The results of this study indicate that extensive lymphadenectomy and minimal intraoperative blood loss might be helpful for improving patient survival following a curative resection. Patients with vessel invasion, serosal involvement, and those >70 years of age should be considered at high risk and require appropriate adjuvant therapy to prolong survival.

Adenocarcinoma↗
PubMed2000

Autocrine growth promotion by multiple hematopoietic growth factors in the established renal cell carcinoma line KU-19-20.

Increasing evidence suggests that paraneoplastic syndrome may be mediated by tumor-related cytokine release, although the specific factors involved remain to be clearly defined. The cancer cells used in the present study were obtained from a 67-year-old man with metastatic renal cell carcinoma in the subcutaneous space who demonstrated marked leukocytosis (37,800/mm3). The primary tumor of the kidney was pathologically diagnosed as renal cell carcinoma consistent with the sarcomatoid type. On microscopic observation, the cultured cells exhibited an epithelial appearance with vacuole formation in their cytoplasm. Ultrastructural observations revealed relatively marked microvilli and a tight junction. Significant amounts of GM-CSF, G-CSF, IL-6, and IL-8 concentrations in the culture media were identified by an enzyme-linked immunosorbent assay. Reverse transcriptase polymerase chain reaction (RT-PCR) significantly exhibited marker protein m-RNA expression in cancer cells. In addition, GM-CSF receptor and IL-6 receptor mRNA expression was also demonstrated by RT-PCR. The administration of both IL-6 and GM-CSF induced cell-proliferation activities estimated by both [3H]-thymidine and bromodeoxyuridine labeling. Anti-IL-6 antibody and anti-GM-CSF antibody neutralized the enhanced proliferative activities generated by these cytokines. Our findings indicate that the established renal cancer cell line can be demonstrated by both the production of multiple cytokines and by their promotion of autocrine growth. These cells are thus considered to be useful as an effective model for multipotent differentiated renal cell carcinoma, as well as for studying the mechanisms of action of autocrine growth.

Aged↗
PubMed2000

Prognostic significance of Fas and Fas ligand system-associated apoptosis in gastric cancer.

BACKGROUND: Previous studies indicate that gastric carcinomas express Fas ligand and down-regulate Fas to escape from the host immune attack; however, the prognostic importance of Fas/FasL expression in this tumor is yet to be evaluated. METHODS: Specimens from 87 gastric carcinoma patients of different stages treated in a defined period with curative intent were evaluated for apoptosis, Fas, FasL, and CD8 expression using an immunohistochemical method. RESULTS: The percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive apoptotic cells expressed as apoptotic index (AI) was higher in 43 patients when the cut-off value was set at the median value. There were no significant correlations between AI and clinicopathologic parameters. Thirty-nine patients showed a high number of CD8+ cells within cancer nests. Positive FasL and Fas expression was seen in 53 and 72 patients, respectively. CD8 and FasL expressions were related only to patients' age. Fas expression had significant correlations with tumor invasion and Lauren classification. There were significant direct correlations between AI and number of nest CD8+ cells and between AI and grade of Fas expression. Apoptotic index, pT stage, CD8 expression, and Fas expression were identified as independent prognostic factors. CONCLUSIONS: Spontaneous apoptosis in gastric carcinoma may be an independent prognosticator for survival and is significantly influenced by tumor Fas expression and number of nest CD8 + cells.

Adenocarcinoma↗