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Biomedical subjects

M Seto

Publications and source records attributed to M Seto.

At least 55 records · Page 3Linked to original sources
PubMed2000

Stable transfectants of smooth muscle cell line lacking the expression of myosin light chain kinase and their characterization with respect to the actomyosin system.

We constructed a plasmid vector having a 1.4-kilobase pair insert of myosin light chain kinase (MLCK) cDNA in an antisense direction to express antisense mRNA. The construct was then transfected to SM3, a cell line from vascular smooth muscle cells, producing a few stable transfectants. The down-regulation of MLCK expression in the transfectants was confirmed by both Northern and Western blots. The control SM3 showed chemotaxic motility to platelet-derived growth factor-BB, which was supported by lamellipodia. However, the transfectants showed neither chemotaxic motility nor developed lamellipodia, indicating the essential role of MLCK in the motility. The specificity for the targeting was assessed by a few tests including the rescue experiment. Despite this importance of MLCK, platelet-derived growth factor-BB failed to induce MLC20 phosphorylation in not only the transfectants but also in SM3. The mode in which MLCK was involved in the development of membrane ruffling is discussed with special reference to the novel property of MLCK that stimulates the ATPase activity of smooth muscle myosin without phosphorylating its light chain (Ye, L.-H., Kishi, H., Nakamura, A., Okagaki, T., Tanaka, T., Oiwa, K., and Kohama, K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6666-6671).

Actomyosin↗
PubMed2000

Molecular diagnosis and gene therapy in musculoskeletal tumors.

Significant progress has occurred in the molecular analyses of bone and soft-tissue tumors, and genetic studies have led to the development of important new diagnostic tools for the clinical management of patients with sarcomas. The detection of fusion genes induced by tumor-specific translocations, such as EWS-FLI1 in Ewing's sarcoma, SYT-SSX in synovial sarcoma, and CHOP-FUS in myxoid liposarcoma, is becoming significant for clinical diagnosis, because these sarcomas are often indistinguishable from other bone and soft-tissue tumors. Gene therapies with several gene transfer systems have been employed for some incurable cancers. It has been demonstrated that a Herpes simplex virus thymidine kinase (HSV-tk) gene can convert certain nucleoside analogs, such as ganciclovir, which disrupt DNA synthesis and are toxic to nucleosides. Human chondrosarcoma cells transduced with the HSV-tk gene were more sensitive to the cytotoxity of ganciclovir than non-transduced cells. Coculture of chondrosarcoma cells with and without the HSV-tk gene showed a bystander effect. The local injection of gene transduced cells into the chondrosarcoma implanted in nude mice markedly reduced tumor size after the administration of ganciclovir. These results suggested the possibility of gene therapy for chondrosarcoma.

Animals↗
PubMed2000

API2-MALT1 chimeric transcripts involved in mucosa-associated lymphoid tissue type lymphoma predict heterogeneous products.

Recent progress in molecular analysis of low-grade B cell lymphoma has revealed that API2 at 11q21 and a novel gene, MALT1 at 18q21, are involved in t(11;18)(q21;q21), a characteristic chromosome aberration for mucosa-associated lymphoid tissue (MALT) type lymphoma. We describe here the establishment of a reverse transcription-polymerase chain reaction (RT-PCR) assay that we used to analyze 22 cases of MALT lymphoma. All five cases that were shown to possess t(11;18)(q21;q21) showed the specific amplification of API2-MALT1 chimeric transcripts. Of the remaining 17 cases for which cytogenetic data were not available, three cases demonstrated the presence of fusion transcripts, indicating that a significant percentage of MALT lymphoma cases of the present series appeared to possess t(11;18). A single fragment was observed in each of these cases, but the size varied from case to case. Sequencing analysis revealed that there are two breakpoints in API2 and three in MALT1, and that all of the fusion transcripts are in-frame. On the basis of these results, four kinds of chimeric proteins can be predicted for the present series. Thus, the RT-PCR assay used here should serve as an effective molecular tool for understanding molecular pathogenesis and the clinical significance of API2-MALT1 for MALT lymphomas.

Adult↗
PubMed2000

Cyclin D1 expression is useful as a prognostic indicator for advanced esophageal carcinomas, but not for superficial tumors.

The purpose of the present study was to define the overexpression of cyclin D1 in superficial and advanced esophageal carcinomas and to investigate whether the expression of this molecule indicates a poor prognosis. This study included 41 patients with superficial esophageal carcinomas (Tis and T1) and 48 patients with advanced esophageal carcinomas (T2, T3, and T4). The expression of cyclin D1 in surgically resected specimens was evaluated immunohistochemically with a monoclonal antibody. Positive immunoreactivity was found in 31 of 89 cases (35%). Overexpression of cyclin D1 did not correlate with TNM classification or histologic type. Of the 48 patients with advanced esophageal carcinomas, 32 patients with cyclin D1-negative tumors survived longer than did 16 patients with cyclin D1-positive tumors (P = 0.0017). In contrast, we observed no survival difference between patients with cyclin D1-positive and -negative superficial esophageal carcinoma. These results suggest that cyclin D1 indicates a poor prognosis in cases of advanced esophageal carcinoma but not in cases of superficial esophageal carcinoma.

Biomarkers, Tumor↗
PubMed2000

Identification and characterization of a molecule, BAM11, that associates with the pleckstrin homology domain of mouse Btk.

Bruton's tyrosine kinase (Btk) is required for normal B cell development and signal transduction through cell surface molecules, and its defects lead to X-linked immune deficiency in mice and X-linked agammaglobulinemia in humans. In this report, we will describe the identification and characterization of a molecule, BAM11, which binds to the pleckstrin homology domain of Btk. A sequence homology search revealed that BAM11 has 89% homology, at the amino acid level, to human LTG19/ENL, that was originally identified as one of the fusion partners involved in chromosomal translocations of 11q23, MLL/ALL-1/HRX, in leukemia cells. Deletion mutants demonstrated that the region of BAM11 required for binding to Btk was localized between amino acid residues 240 and 256. Forced expression of a truncated form of BAM11 (amino acids 246-368) inhibited IL-5-induced proliferation by 50%, whereas forced expression of full-length BAM11 in Y16 cells did not affect the IL-5 responsiveness. We have also shown that BAM11 (amino acids 246-368) inhibited the kinase activity of Btk. These results suggest that the binding of BAM11 to Btk plays a regulatory role in the Btk signal transduction pathway. A cell fractionation study and analysis using EGFP-fused Btk protein demonstrated that a proportion of Btk is present within the nucleus.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2000

Thin and thick filament regulation of contractility in experimental cerebral vasospasm.

OBJECTIVE: Cerebral vasospasm is a potentially fatal consequence of aneurysmal subarachnoid hemorrhage and influences the prognosis of the patient. The purpose of this study was to evaluate the status of thin (actin) and thick (myosin) filament regulation of smooth muscle contraction in the double-subarachnoid hemorrhage canine model of cerebral vasospasm and to determine the effects of a kinase inhibitor reported to be effective in vasospasm, HA1077, on thin and thick filament regulation. METHODS: Cerebral vasospasm was assessed by vertebral angiography. Myosin regulatory light chain phosphorylation was measured using glycerol-urea gels, whereas protein levels of the thin filament-associated protein calponin were measured by Western blot. RESULTS: The basilar arteries of dogs in which subarachnoid hemorrhage was induced narrowed to 36% +/- 2.0% of their size on the first day (n = 12). The phosphorylation of the regulatory light chain tended to increase, but the change did not reach statistical significance (35% +/- 5.9% [n = 12] versus 25% +/- 4.8% [n = 10] in control arteries). In contrast to this increase, significant degradation of calponin was observed in the samples from vasospastic dogs (85.4% +/- 5.45% [n = 5] versus 15.2% +/- 6.21% [n = 5]; P < 0.01). Prophylactic treatment with intravenous injections of HA1077 at 0.67 mg/kg b.i.d. significantly inhibited vasospasm (diameters, 65% +/- 10.2% of Day 1 diameters [n = 5]; P < 0.05), and calponin degradation (57.8% +/- 13.9% [n = 4]) was substantially reduced. CONCLUSION: These data suggest that degradation of the thin filament-associated protein calponin plays a role in cerebral vasospasm and that the antivasospastic action of HA1077 is, at least in part, due to prevention of calponin degradation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Helicobacter pylori and the t(11;18)(q21;q21) translocation in gastric low-grade B-cell lymphoma of mucosa-associated lymphoid tissue type.

The reported regression of mucosa-associated lymphoid tissue (MALT) type gastric low-grade B-cell lymphoma following treatment for Helicobacter pylori (H. pylori) infection has not yet been comprehensively analyzed, especially in relation to the recently identified c-IAP2-MALT1 / MLT gene alteration resulting from the t(11;18)(q21;q21) chromosomal translocation found in MALT lymphoma. The relationship between MALT lymphomas and H. pylori was investigated in 30 patients who received an antibacterial treatment. Patients were followed up by means of endoscopy and biopsy. Molecular genetic analyses focused on the presence or absence of the immunoglobulin heavy chain (IgH) gene and / or MALT1 / MLT gene alteration resulting from t(11;18)(q21;q21) translocation. H. pylori was positive in 26 of the 30 patients. The overall success rate of cure of H. pylori infection was 96% (25 / 26). Thirteen patients (52%) showed complete remission (CR) of lymphoma, nine (36%) partial remission (PR), and three (12%) registered no change (NC). Statistical analysis revealed significant differences between CR and PR / NC patients in age ( < 60 or 60), in lymphoma location (single or multiple sites) and in the presence or absence of gene rearrangement before eradication (P < 0.05). Endoscopy showed a cobblestone appearance only in PR cases and polypoid features predominantly in NC cases. Two NC patients with polypoid gross appearance showed rearrangements involving either c-IAP2 or MALT1 gene in Southern blot analysis, while none of seven other resected patients with non-polypoid superficial gross appearance showed rearrangement. Gastric MALT lymphoma could be pragmatically subdivided into three groups, CR (MALT-A), PR (MALT-B), and NC (MALT-C) on the basis of the reaction to eradication of H. pylori. We speculate that MALT-A may represent an incipient neoplasm or dysplasia, MALT-B a neoplasm activated by antigenic stimulation of H. pylori, and MALT-C a lymphoma independent of H. pylori. Polypoid lesions in MALT-C were associated with c-IAP2-MALT1 / MLT gene alteration resulting from t(11;18)(q21;q21). This classification is thought to be clinically significant for deciding the most appropriate mode of treatment of MALT-type lymphoproliferative disorders.

Adult↗
PubMed2000

Estimation of the yield coefficient of Pseudomonas sp. strain DP-4 with a low substrate (2,4-dichlorophenol [DCP]) concentration in a mineral medium from which uncharacterized organic compounds were eliminated by a non-DCP-degrading organism.

The yield coefficient (YC) of Pseudomonas sp. strain DP-4, a 2, 4-dichlorophenol (DCP)-degrading organism, was estimated from the number of CFU produced at the expense of 1 unit amount of DCP at low concentrations. At a low concentration of DCP, the YC can be overestimated in pure culture, because DP-4 assimilated not only DCP but also uncharacterized organic compounds contaminating a mineral salt medium. The concentration of these uncharacterized organic compounds was nutritionally equivalent to 0.7 microg of DCP-C ml(-1). A mixed culture with non-DCP-degrading organisms resulted in elimination of ca. 99.9% of the uncharacterized organic compounds, and then DP-4 assimilated only DCP as a substrate. In a mixed culture, DP-4 degraded an initial concentration of 0.1 to 10 microg of C ml of DCP(-1) and the number of CFU of DP-4 increased. In the mixed culture, DCP at an initial concentration of 0.07 microg of C ml(-1) was degraded. However, the number of CFU of DP-4 did not increase. DCP at an extremely low initial concentration of 0.01 microg of C ml(-1) was not degraded in mixed culture even by a high density, 10(5) CFU ml(-1), of DP-4. When glucose was added to this mixed culture to a final concentration of 1 microg of C ml(-1), the initial concentration of 0.01 microg of C ml of DCP(-1) was degraded. These results suggested that DP-4 required cosubstrates to degrade DCP at an extremely low initial concentration of 0.01 microg of C ml(-1). The YCs of DP-4 at the expense of DCP alone decreased discontinuously with the decrease of the initial concentration of DCP, i.e., 1.5, 0.19, or 0 CFU per pg of DCP-C when 0.7 to 10, 0.1 to 0.5, or 0.07 microg of C ml of DCP(-1) was degraded, respectively. In this study, we developed a new method to eliminate uncharacterized organic compounds, and we estimated the YC of DP-4 at the expense of DCP as a sole source of carbon.

Bacteria↗
PubMed2000

Rho kinase inhibitor HA-1077 prevents Rho-mediated myosin phosphatase inhibition in smooth muscle cells.

In smooth muscle, a Rho-regulated system of myosin phosphatase exists; however, it has yet to be established whether Rho kinase, one of the downstream effectors of Rho, mediates the regulation of myosin phosphatase activity in vivo. In the present study, we demonstrate in permeabilized vascular smooth muscle cells (SMCs) that the vasodilator 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077), which we show to be a potent inhibitor of Rho kinase, dose dependently inhibits Rho-mediated enhancement of Ca(2+)-induced 20-kDa myosin light chain (MLC(20)) phosphorylation due to abrogating Rho-mediated inhibition of MLC(20) dephosphorylation. By an immune complex phosphatase assay, we found that guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) stimulation of permeabilized SMCs caused a decrease in myosin phosphatase activity with an increase in the extent of phosphorylation of the 130-kDa myosin-binding regulatory subunit (MBS) of myosin phosphatase in a Rho-dependent manner. HA-1077 abolished both of the Rho-mediated events. Moreover, we observed that the pleckstrin homology/cystein-rich domain protein of Rho kinase, a dominant negative inhibitor of Rho kinase, inhibited GTPgammaS-induced phosphorylation of MBS. These results provide direct in vivo evidence that Rho kinase mediates inhibition of myosin phosphatase activity with resultant enhancement of MLC(20) phosphorylation in smooth muscle and reveal the usefulness of HA-1077 as a Rho kinase inhibitor.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Increased migration in late G(1) phase in cultured smooth muscle cells.

Migration and proliferation of smooth muscle cells (SMC) contribute to neointimal formation after arterial injury. However, the relation between migration and proliferation in these cells is obscure. To discriminate between migration and proliferation, we employed a migration assay of SMC at different phases of the cell cycle. Serum-deprived SMC were synchronized in different phases of the cell cycle by addition of serum for various periods of time. Migration induced by platelet-derived growth factor B-chain homodimer was maximal in SMC that were predominantly in the late G(1) (G(1b)) phase. In addition, in nonsynchronized SMC, 65-75% of SMC that had migrated were in the G(1b) phase. Phosphorylated myosin light chain was enriched around the cell periphery in SMC in the G(1b) phase compared with SMC in the other cell cycle phases. Interestingly, the Triton X-100-insoluble fraction of myosin was remarkably decreased in G(1b)-enriched SMC. These findings suggest that migratory activity of SMC may be coupled with the G(1b) phase. The phosphorylation and retention of myosin might explain some of the properties responsible for increased migration.

Actins↗
PubMed2000

Overexpression of cyclin D1 occurs frequently in human pancreatic endocrine tumors.

The molecular pathogenesis of human pancreatic endocrine tumors (PETs) is poorly understood. Three independent animal models have pointed to the pivotal role of the G1/S cell cycle transition in pancreatic endocrine cell proliferation. We thus hypothesized that the cell cycle regulator cyclin D1 may contribute to the pathogenesis of human PETs. Overexpression of cyclin D1 was identified in 43% of cases, and no correlation was observed with clinical phenotype. The novel observation of frequent overexpression of cyclin D1 suggests that this established oncogene may be implicated in the pathogenesis of human PETs. The absence of detectable alterations in cyclin D1 genomic structure suggests that the mechanism for its oncogenic activation in PETs may be transcriptional or posttranscriptional.

Adolescent↗
PubMed2000

[A long-term survivor of extensive-stage small cell lung cancer].

A 53-year-old man was admitted for evaluation of multiple hepatic tumors. A thoracic CT scan revealed a mass lesion in the right middle lobe. Transcutaneous needle aspiration cytology from the lung tumor and transcutaneous hepatic tumor biopsy were performed, from which the cytohistological diagnosis was small cell lung cancer with liver metastasis. The patient was treated with 3 courses of high dose cisplatin with vindesine and mitomycin and 5 courses of carboplatin with etoposide, and achieved a complete response. We herein report a long-term survivor of extensive-stage small cell lung cancer.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

Truncated TSG101 transcripts in human leukemia and lymphoma cell lines.

Inactivation of the TSG101 gene has been shown to induce cellular transformation of NIH3T3 fibroblasts, and aberrant TSG101 transcripts have been observed not only in various human solid tumors but also in hematopoietic malignant disorders. In the present study, we performed nested reverse transcription/polymerase chain reaction (RT-PCR) analysis to identify aberrant TSG101 transcripts in 43 human leukemia and lymphoma cell lines. We could detect only a single band of the wild-type transcript with the expected size in virtually all cell lines after the first round of PCR. As in the case with various human solid tumors, the smaller TSG101 transcripts appeared in most of these cell lines after the second round of PCR. Thus, the expression level of the variant transcripts was extremely low as compared with that of the wild-type transcript, and this finding was also confirmed by Northern blot analysis. Identification of various truncated transcripts with extensive deletions in the TSG101 coding region was confirmed by means of sequencing analysis, and expression of these transcripts did not appear to be associated with a specific type of hematopoietic malignant disorder. Southern blot analysis did not indicate any gross TSG101 gene rearrangement. The truncated transcripts were also detected in normal peripheral blood leukocytes. Our results suggest that the truncated TSG101 transcripts are definitely detectable in various human leukemia and lymphoma cell lines, but do not support the notion that the variant transcripts may have a major functional relevance in the pathogenesis of human hematopoietic malignant disorder.

Amino Acid Sequence↗
PubMed2000

Therapy-related leukemia and myelodysplastic syndrome: a large-scale Japanese study of clinical and cytogenetic features as well as prognostic factors.

It is known that alkylating agents and topoisomerase II inhibitors can cause distinct forms of therapy-related leukemia and myelodysplastic syndrome (TRL/MDS). Although several reports have been made on each of these agents separately, no study has yet been conducted to evaluate the effect of these two types of agents in the same population. In a nationwide, large-scale population study, the clinical and cytogenetic features as well as the prognostic factors in 256 patients with TRL/MDS were assessed. Median age was 61 years, and the median period of latency from primary malignancies was 47.9 months. The latency period was significantly shorter in patients undergoing chemotherapy, especially that of topoisomerase II inhibitors, for primary cancer. The morphological diagnosis of TRL/MDS was acute myeloid leukemia in 59% and MDS in 41% of patients. Chromosome abnormalities that frequently involved chromosomes 5, 7 or 11 were documented in 77% of the 189 patients examined. MLL gene rearrangements were detected in 11 of 58 subjects and were correlated with a borderline significance (P = 0.072) with topoisomerase II inhibitor administration. Overall median survival was only 9.7 months. Survival was similar in cases with or without MLL gene rearrangement. Multivariate analysis identified chromosome 5 abnormalities, hypoproteinemia, poor therapy outcomes for primary cancer, C-reactive protein, and thrombocytopenia as being significantly poor prognostic factors (P < 0.05). This large-population study provided a comprehensive update of TRL/MDS status in Japan, identified significant prognostic factors, and enabled the clinical significance of MLL gene rearrangement to be assessed.

Adult↗
PubMed2000

Analysis of genetic polymorphism in NQO1, GST-M1, GST-T1, and CYP3A4 in 469 Japanese patients with therapy-related leukemia/ myelodysplastic syndrome and de novo acute myeloid leukemia.

Several genetic polymorphisms in metabolic activation or detoxification enzymes have been associated with susceptibility to therapy-related leukemia and myelodysplastic leukemia (TRLIMDS). We analyzed gene polymorphisms of NAD(P)H:quinone oxidoreductase (NQOl), glutathione S-tranferase (GST)-MI and -TI, and CYP3A4, the enzymes of which are capable of metabolizing anticancer drugs, in 58 patients with TRL/MDS and in 411 patients with de novo acute myeloid leukemia (AML). Homozygous Ser/Ser genotype of NQOl at codon 187, causing loss of function, was more frequent in the patients with TRLIMDS (14 of 58, 24.1%; OR = 2.62) than in those with de novo AML (64 of 411, 15.6%), and control (16 of 150, 10.6%; P = 0.002). Allelic frequencies of NQOJ were different between TRL/ MDS and de novo AML (P = 0.01). In GST-MJ and -Ti, the incidence of homologous deletion was similar among the three groups. The polymorphism of the 5' promoter region of CYP3A4 was not found in persons of Japanese ethnicity. These results suggest that the NQOJ polymorphism is significantly associated with the genetic risk of TRLIMDS.

Adult↗
PubMed1999

Molecular cloning of a cDNA encoding mouse A15, a member of the transmembrane 4 superfamily, and its preferential expression in brain neurons.

A15, a member of the transmembrane 4 superfamily (TM4SF), was isolated by differential screening of the cDNAs that are preferentially expressed on immature T cells. As a first step in the study of the biological function of the A15 molecule, we isolated cDNAs encoding the entire coding region of mouse A15. Nucleotide sequence analysis of the cDNAs revealed that mouse A15 shares 97% amino acid sequence identity with its human counterpart. The mouse A15 protein product has not yet been characterized, but is predicted to be 244 amino acids with four hydrophobic domains. Northern blot analysis of the RNA samples from various mouse tissues disclosed that the A15 transcripts are expressed most strongly in the brains, and are detectable in the colon, muscle, heart, kidney, and spleen. In situ hybridization of the mouse brain with ribo-probe established that the A15 transcripts are expressed primarily in neurons of the frontal cortex, olfactory bulb, dentate gyrus, caudoputamen, and CA3 region of the hypothalamus as well as in Purkinje cells in the cerebellar cortex, which strongly suggests that A15 may have a special function in the fundamental neuronal functioning of the higher nervous system.

Amino Acid Sequence↗