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Tsutomu Tsuji

Publications and source records attributed to Tsutomu Tsuji.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of the promoter for the alpha3 integrin gene in various tumor cell lines: roles of the Ets- and Sp-family of transcription factors.

The alpha3beta1 integrin is an adhesion receptor for extracellular matrix proteins, including laminin isoforms, and plays crucial roles in the organization of epithelial and endothelial tissues. The aberrant expression of this adhesion molecule on tumor cells is associated with their invasive and metastatic potentials. In the present study, we analyzed the elements essential for alpha3 integrin gene expression in various tumor cell lines with different tissue origins by luciferase assay. An approximately 0.3 kb fragment of the 5'-flanking region of the mouse alpha3 integrin gene (-260/+84, relative to the major transcription start site) showed strong promoter activity in all six examined tumor cell lines. However, we found that these cell lines could be divided into two groups according to the level of dependency on the putative Ets-transcription factor binding motif located at -133. This motif was previously shown to be crucial for alpha3 integrin expression in MKN1 gastric carcinoma cells. The gene expression in one group of cell lines was upregulated mainly by the Ets motif, whereas that in the other group was less dependent on the Ets motif. We then postulated that additional regulatory elements were responsible for the expression of alpha3 integrin, and found that a GC-rich motif at -69 was another important element. An electrophoretic mobility shift assay using specific antibodies and a Western blot analysis of nuclear proteins revealed that the Sp3-transcription factor bound to this GC-rich motif. These results suggest that the Sp3 and Ets transcription factors cooperatively regulate alpha3 integrin gene expression and that the contribution of each element depends on the type of tumor cells.

Animals↗
PubMed2006

Transforming growth factor-beta1 upregulates transcription of alpha3 integrin gene in hepatocellular carcinoma cells via Ets-transcription factor-binding motif in the promoter region.

The invasive and metastatic potentials of hepatocellular carcinoma (HCC) are positively correlated with the expression level of alpha3beta1 integrin, a high-affinity adhesion receptor for laminin isoforms. Transforming growth factor (TGF)-beta1 stimulates non-invasive HCC cells to acquire invasive phenotypes in association with the enhanced expression of alpha3 integrin. In this study, we investigated the molecular mechanism underlying the upregulation of alpha3beta1 integrin by TGF-beta1 in non-invasive HepG2 HCC cells. The treatment of HepG2 cells with TGF-beta1 induced the expression of alpha3 integrin and potentiated these cells to adhere to laminin-5 and to migrate through laminin-5-coated membranes. The promoter activity was measured by luciferase assay with a series of deletion constructs of the 5'-flanking region of the mouse alpha3 integrin gene, and the results showed that the -260/-119 region (relative to the major transcription start site) contained elements responsive to TGF-beta1 stimulation. The introduction of mutations into the putative consensus binding sequence for the Ets-family of transcription factors located at -133 greatly decreased the promoter activity responding to TGF-beta1 stimulation. The nuclear proteins extracted from TGF-beta1-stimulated HepG2 cells yielded a larger amount of DNA-nuclear protein complexes than did those extracted from unstimulated cells, as determined by an electrophoretic mobility shift assay using an oligonucleotide containing the Ets-site as a probe. These results suggest that TGF-beta1 stimulates HepG2 cells to express a higher level of alpha3 integrin by transcriptional upregulation via Ets transcription factors and to exhibit a more invasive phenotype.

Base Sequence↗
PubMed2006

Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins.

The interactions of cells with basement membranes are primarily mediated via the engagement of laminins by a group of integrin family proteins, including integrins alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4. To explore the ligand-binding specificities of these laminin-binding integrins, we produced these integrins, including two alpha7beta1 splice variants (alpha7X1beta1 and alpha7X2beta1), as soluble recombinant proteins and determined their binding specificities and affinities toward a panel of purified laminin isoforms containing distinct alpha chains. Among the five laminin-binding integrins investigated, alpha3beta1 and alpha6beta4 exhibited a clear specificity for laminin-332 (alpha3beta3gamma2) and laminin-511 (alpha5beta1gamma1)/521 (alpha5beta2gamma1), while integrin alpha6beta1 showed a broad specificity, binding to all laminin isoforms with a preference for laminin-111 (alpha1beta1gamma1), laminin-332 and laminin-511/521. The two alpha7beta1 variants were distinct from alpha3beta1, alpha6beta1 and alpha6beta4 in that they did not bind to laminin-332. alpha7X1beta1 bound to all laminins, except laminin-332, with a preference for laminin-211 (alpha2beta1gamma1)/221 (alpha2beta2gamma1) and laminin-511/521, while alpha7X2beta1 bound preferentially to laminin-111 and laminin-211/221. Laminin-511/521 was the most preferred ligand for all the laminin-binding integrins, except for alpha7X2beta1, whereas laminin-411 was the poorest ligand, capable of binding to alpha6beta1 and alpha7X1beta1 with only modest binding affinities. These comprehensive analyses of the interactions between laminin-binding integrins and a panel of laminins clearly demonstrate that the isoforms of both integrins and laminins differ in their binding specificities and affinities, and provide a molecular basis for better understanding of the adhesive interactions of cells with basement membranes of defined laminin compositions.

Amino Acid Sequence↗
PubMed2006

Platelet activation through interaction with hemodialysis membranes induces neutrophils to produce reactive oxygen species.

The intradialytic activation of leukocytes is one of the major causes of hemodialysis-associated complications. During hemodialysis, the formation of microaggregates consisting of platelets and neutrophils has been observed to accompany the production of reactive oxygen species (ROS) by leukocytes. In this study, we investigated the interaction of platelets and neutrophils with hemodialysis membranes in vitro to elucidate the mechanism underlying microaggregate formation and its relevance to leukocyte activation. The production of ROS in neutrophils was induced by the coincubation of neutrophils with polysulfone (PS) membranes, and was increased when platelets were present in the neutrophil suspension. Neutrophils that were incubated with polymethylmethacrylate (PMMA) membranes in the presence of platelets also produced significant levels of ROS, suggesting that the presence of platelets augmented ROS production in neutrophils. Platelets adhered more firmly to hydrophobic membranes such as PS and PMMA membranes than to hydrophilic membranes, such as those composed of regenerated cellulose (RC) or ethylene vinylalcohol copolymer (EVAL). The adhesion of platelets to dialysis membranes composed of different materials was correlated with those membranes' ability to induce platelet activation as assessed by the cell surface expression of P-selectin. Moreover, coincubation of neutrophils with platelets that had been treated with hydrophobic membranes induced a higher level of superoxide anion relative to those treated with hydrophilic membranes in association with the P-selectin-mediated microaggregate formation. These results suggest that platelets activated through interaction with hemodialysis membranes stimulate neutrophils to produce ROS via P-selectin-mediated adhesion, and that this property of adhesion to platelets is critical for the biocompatibility of hemodialysis membranes.

Animals↗
PubMed2005

Homotypic dimerization of the actin-binding protein p57/coronin-1 mediated by a leucine zipper motif in the C-terminal region.

The actin-binding protein p57/coronin-1, a member of the coronin protein family, is selectively expressed in immune cells, and has been implicated in leucocyte migration and phagocytosis by virtue of its interaction with F-actin (filamentous actin). We previously identified two sites in the N-terminal region of p57/coronin-1 by which it binds actin, and in the present study we examine the role of the leucine zipper motif located in the C-terminal coiled-coil domain in mediating the homotypic association of p57/coronin-1. Recombinant p57/coronin-1 protein in solution formed a homodimer, as analysed by Superose 12 column chromatography and by sucrose density gradient centrifugation. In vivo, a truncated form consisting of the C-terminal coiled-coil domain co-precipitated with full-length p57/coronin-1 when both were co-expressed in COS-1 cells. A chimaeric construct composed of the C-terminal domain of p57/coronin-1 (which lacks the actin-binding sites) fused with green fluorescent protein co-localized with cortical F-actin-rich regions in COS-1 cells only when full-length p57/coronin-1 was expressed simultaneously in the cells, suggesting that the C-terminal region is required for the homotypic association of p57/coronin-1. Furthermore, p57LZ, a polypeptide consisting of the C-terminal 90 amino acid residues of p57/coronin-1, was sufficient for dimerization. When two leucine residues out of the four that constitute the leucine zipper structure in p57LZ or full-length p57 were replaced with alanine residues, the mutants failed to form homodimers. Taken together, these results demonstrate that p57/coronin-1 forms homodimers, that the association is mediated by the leucine zipper structure in the C-terminal region, and that it plays a role in the cross-linking of F-actin in the cell.

Animals↗
PubMed2005

Blood group A glycosphingolipid accumulation in the hair of patients with alpha-N-acetylgalactosaminidase deficiency.

In the hair of individuals with blood group AB, the level of blood group A glycosphingolipids is much lower than that of blood group B. We hypothesized that in hair, blood group A determinants are converted by alpha-N-acetylgalactosaminidase (alpha-NAGA, E.C.3.2.1.49) to H determinants. To address our hypothesis, the relative amount of ABH glycosphingolipids in hairs and nails of normal subjects, patients with Kanzaki disease, and heterozygous carriers of alpha-NAGA deficiency were analyzed by dot-blotting and enzyme-linked immunosorbent assay. In hair from normal subjects with blood group B, ABH glycosphingolipids consisted of 88% blood group B- and 12% blood group H glycosphingolipids. In blood group A subjects, 14% were group A- and 86% were group H glycosphingolipids. In Kanzaki patients, 81% were blood group A- and 19% were blood group H glycosphingolipids. In 2 alpha-NAGA deficiency carriers, the ABH glycosphingolipids consisted of 67% blood group A- and 33% blood group H glycosphingolipids. These results indicate that blood group A glycosphingolipids are catabolized to H glycosphingolipids by alpha-NAGA, resulting in lower levels of blood group A glycosphingolipids in the hair of normal subjects, and alpha-NAGA deficiency causes accumulation of blood group A glycosphingolipids in the hair of Kanzaki patients. This finding is of clinical relevance because it suggests that hair may be used to diagnose and assess the alpha-NAGA status of individuals.

ABO Blood-Group System↗
PubMed2005

On a salmon (Oncorhynchus [corrected] keta) liver RNase, belonging to RNase T2 family: primary structure and some properties.

A base-nonspecific and acid ribonuclease (RNase Ok2) was purified from the liver of a salmon (Oncorhnchus keta) to a homogeneous state by SDS-PAGE. The primary structure of RNase Ok2 was determined by protein chemistry and molecular cloning. The RNase Ok2 was a glycoprotein and consisted of 216 amino acid residues. Its molecular mass of protein moiety was 25,198, and its amino acid sequence showed that it belongs to the RNase T2 family of enzymes. The optimal pH of RNase Ok2 was around 5.5. The base preferences at the B1 and B2 sites were estimated from the rates of hydrolysis of 16 dinucleoside phosphates to be G>>A>U, C, and G>A>U>C respectively. In this enzyme, one of the three histidine residues which have been thought to be important for catalysis of RNase Rh, a typical RNase of this family of enzymes, His104 was replaced by tyrosine residue. Based on the results, the role of H104, which has been proposed to be a phosphate binding site with a substrate, was reconsidered, and we proposed a revised role of this His residue in the hydrolysis mechanism of RNase T2 family enzymes.

Amino Acid Sequence↗
PubMed2004

p51/p63 Controls subunit alpha3 of the major epidermis integrin anchoring the stem cells to the niche.

p51/p63, a member of the tumor suppressor p53 gene family, is crucial for skin development. We describe here identification of ITGA3 encoding integrin alpha(3) as a target of its trans-activating function, proposing that p51/p63 allows epidermal stem cells to express laminin receptor alpha(3)beta(1) for anchorage to the basement membrane. When activated by genotoxic stress or overexpressed ectopically in non-adherent cells, p51/p63 transduced a phenotype to attach to extracellular matrices, which was accompanied by expression of ITGA3. Motifs matching the p53-binding consensus sequence were located in a scattered form in intron 1 of human ITGA3, and served as p51/p63-responsive elements in reporter assays. In addition to the trans-activating ability of the TA isoform, we detected a positive effect of the DeltaN isoform on ITGA3. The high level alpha(3) production in human keratinocyte stem cells diminished upon elimination of p51/p63 by small interfering RNA or by Ca(2+)-induced differentiation. Furthermore, a chromatin immunoprecipitation experiment indicated a physical interaction of p51/p63 with intron 1 of ITGA3. This study provides a molecular basis for the standing hypothesis that p51/p63 is essential for epidermal-mesenchymal interactions.

DNA-Binding Proteins↗
PubMed2004

Adhesion of gastric carcinoma cells to peritoneum mediated by alpha3beta1 integrin (VLA-3).

The interaction between gastric carcinoma cells and the peritoneal lining is a key step in peritoneal dissemination. In this study, we examined the roles of the beta1 family of integrin receptors in the adhesion of such cells to the peritoneum. The adhesion of several gastric carcinoma cell lines to peritonea excised from mice was inhibited most by an anti-alpha3 integrin antibody and to a lesser extent by an anti-alpha2 integrin antibody. In the peritoneal implantation of NUGC-4 human gastric carcinoma cells in athymic mice, treatment of the cells with anti-alpha2 or anti-alpha3 integrin antibody reduced the number of disseminated nodules; suppression by the anti-alpha3 integrin antibody was stronger than that by the anti-alpha2 integrin antibody. The cDNAs to human alpha2 and alpha3 integrins were introduced into K562 leukemic cells, which were positive for the integrin beta1 subunit but negative for the alpha2 or alpha3 subunit. The alpha3 integrin-transfected cells adhered to excised peritoneum and to a monolayer of peritoneal mesothelial cells more firmly than did the alpha2 integrin-transfected cells or the mock transfectant. Reverse transcription-PCR was used to analyze the expression of laminin-5 and laminin-10/11, which have been reported to serve as high-affinity ligands for alpha3beta1 integrin. mRNA for these laminin isoforms was found in mesothelial cells from the diaphragm and parietal peritoneum. These results strongly suggest that alpha3beta1 integrin plays an essential role in mediating the initial attachment of cancer cells to the peritoneum, leading to the formation of peritoneal metastasis.

Antibodies↗
PubMed2004

Acetylcholine-induced translocation of RhoA in freshly isolated single smooth muscle cells of rat bronchi.

By using immunofluorostaining and confocal laser microscopy, acetylcholine-induced translocation of RhoA was visualized in freshly isolated bronchial smooth muscle cells of the rat. The cellular distribution of RhoA at rest was observed uniformly in the cytosolic space with no staining in the nucleus, whereas acetylcholine stimulation induced a relocalization of RhoA to the cell periphery. From the results of line scans and surface plots, the peripheral to cytosolic ratio of RhoA was significantly increased by acetylcholine stimulation. Thus, the present study clearly demonstrated an acetylcholine-induced translocation of RhoA to the plasma membrane in single bronchial smooth muscle cells of the rat.

Acetylcholine↗
PubMed2004

Physiological and pathological roles of alpha3beta1 integrin.

Alpha3beta1 integrin has been considered to be a mysterious adhesion molecule due to the pleiotropy in its ligand-binding specificity. However, recent studies have identified laminin isoforms as high-affinity ligands for this integrin, and demonstrated that alpha3beta1 integrin plays a number of essential roles in development and differentiation, mainly by mediating the establishment and maintenance of epithelial tissues. Furthermore, alpha3beta1 integrin is also implicated in many other biological phenomena, including cell growth and apoptosis, angiogenesis and neural functions. This integrin receptor forms complexes with various other membrane proteins, such as the transmembrane-4 superfamily proteins (tetraspanins), cytoskeletal proteins and signaling molecules. Recently, lines of evidence have been reported showing that complex formation regulates integrin functions in cell adhesion and migration, signal transduction across cell membranes, and cytoskeletal organization. In addition to these roles in physiological processes, alpha3beta1 integrin performs crucial functions in various pathological processes, especially in wound healing, tumor invasion and metastasis, and infection by pathogenic microorganisms.

Animals↗
PubMed2003

Inhibition of P-selectin-mediated cell adhesion by a sulfated derivative of sialic acid.

P-selectin, a carbohydrate-binding cell adhesion molecule expressed on activated endothelial cells and platelets, plays a key role in the recruitment of leukocytes to inflammatory and hemorrhagic sites. It simultaneously recognizes a sialic acid-containing carbohydrate chain and the sulfated tyrosine residues of a specific counter-receptor expressed on the leukocyte surface. We examined the inhibitory effects of a synthetic sulfated derivative of sialic acid (NMSO3) on P-selectin-mediated cell adhesion and found the following: (1) P-selectin/IgG chimera bound to immobilized NMSO3. (2) The binding of P-selectin/IgG chimera to purified P-selectin glycoprotein ligand-1 was inhibited by soluble NMSO3. (3) The adhesion of HL60 cells to P-selectin-expressing CHO cells was inhibited by NMSO3. (4) NMSO3 inhibited P-selectin-induced tumor necrosis factor-alpha production in monocytes and activated platelet-induced generation of reactive oxygen species in neutrophils. In conclusion, NMSO3 acts as a specific inhibitor for P-selectin-mediated cell adhesion and for adhesion-dependent leukocyte activation.

Animals↗
PubMed2003

En Bloc removal of soft tumors within the brain parenchyma using spoon retractors: traction-dissection method. Technical note.

BACKGROUND: En bloc removal of soft tumors within the brain parenchyma has rarely been performed. We describe a safe technique for en bloc removal of the tumors using a spoon retractor, which enables retraction while holding the soft mass upward. METHODS: Dissection of a tumor mass is performed under traction of the surrounding brain tissue by retracting the mass using a spoon retractor. A dissection plane is first established in a relatively safe area, not opposite the critical area, after a corticotomy. The dissection plane is then extended toward the critical side. After circumferential dissection of the tumor mass, the dissection is continued spirally into deeper regions by retracting and holding the mass upward using a spoon retractor. In this way, the tumor is removed en bloc. CONCLUSIONS: The traction-dissection method using spoon retractors is useful in performing en bloc removal of soft tumors within the brain parenchyma less invasively and provides an appropriate operating field even at depth, reducing intraoperative bleeding, and in vascular rich tumors, possibly preventing tumor seeding in cases of malignant tumors.

Brain Neoplasms↗
PubMed2003

Lung-heart weight ratio as a possible index of cardiopulmonary pathophysiology in drowning.

The aim of the present study was to investigate the lung-heart weight ratio in fresh- and saltwater drowning (n=67 and n=75, respectively) as a possible index of cardiopulmonary pathophysiology, in comparison with acute myocardial infarction/ischemia (AMI, n=75) and asphyxiation (n=85). In drowning cases, the total value of the combined lung weight and the amount of pleural effusion was regarded as a possible total lung weight. The median value of the combined/total lung weight was the highest in saltwater drowning, which was followed by freshwater drowning, AMI and asphyxiation, showing a tendency to be mildly increased depending on the heart weight. The lung-heart weight ratio was significantly higher in fresh-/saltwater drownings (3.944+/-1.538 and 4.825+/-2.242, respectively) than in asphyxiation (2.846+/-1.042) and AMI (2.641+/-0.916) (P<0.0001), showing a tendency to be higher in saltwater than freshwater drowning. However, the value depended on the gender and age of the subjects, and the difference between freshwater drowning and asphyxiation was insignificant in females. These results suggested that the lung-heart weight ratio may be an index for investigating the influence of aspirated immersion medium in drownings.

Acute Disease↗
PubMed2003

Postmortem lung weight in drownings: a comparison with acute asphyxiation and cardiac death.

There are several controversial findings and arguments about the lung weight as a marker of drowning. The aim of the present study was to examine the difference in the lung weight and the amount of pleural effusion between freshwater and saltwater drownings (n=70 and n=75, respectively), in comparison with asphyxiation (n=85) and acute cardiac death (n=82), for the diagnosis of drowning. In drowning cases, a gradual postmortem time-dependent decrease in the lung weight and a reciprocal increase in the pleural effusion suggested postmortem transudation from the lungs. The decrease in the total value of the combined lung weight and the amount of pleural effusion was marked in saltwater immersion after 3 days postmortem, suggesting a leakage of the effusion out of the thoracic cavity under an osmotic effect of the immersion medium. In cases within 3 days postmortem, when the combined lung weight and amount of pleural effusion were added to estimate possible combined lung weight at the time of death, there was a gross difference among the causes of death: the value was the largest in saltwater drowning, followed by freshwater drowning, acute cardiac death and asphyxiation. However, the value depended on the gender and age of the subjects, suggesting a relation to the individual physical constitution and survival time or vital activity. These factors should be taken into consideration in evaluation of the lung weight in the diagnosis of drownings.

Adult↗
PubMed2003

Two regions responsible for the actin binding of p57, a mammalian coronin family actin-binding protein.

The actin-binding protein p57, a member of the coronin protein family, is expressed in a variety of immune cells. It has five WD repeats and a coiled-coil motif containing a leucine zipper, both of which are known to mediate protein-protein interactions. In order to identify the precise actin-binding regions in p57, and to assess the contribution of these structural motifs, we prepared various truncated p57 as fusion proteins with glutathione S-transferase (GST) and examined their actin-binding activity. A co-sedimentation assay demonstrated that p57(1-371) (C-terminal truncated p57) had the ability to bind F-actin, but p57(372-461) (a fragment containing the coiled-coil motif) did not. A segment consisting of the N-terminal 34 amino acids of p57 (p57(1-34)) was found to bind to F-actin in the co-sedimentation assay. Furthermore, fluorescence microscopic observation showed that p57(1-34) was co-localized with F-actin in COS-1 cells after the transfection with the p57(1-34) construct. Deletion of (10)KFRHVF(15), a sequence conserved among coronin-related proteins, from p57(1-34) abolished its actin-binding activity, suggesting that this sequence with basic and hydrophobic amino acids is crucial for p57 to bind to F-actin. However, the N-terminal deletion mutant p57(63-461) retained the binding ability to F-actin. This result suggests the presence of a second actin-binding region. Further deletion analysis revealed that p57(111-204), which includes the second and third WD repeats, also exhibited weak actin-binding activity in the co-sedimentation assay. Taken together, these data strongly suggest that at least two regions within Met-1 to Asp-34 and Ile-111 to Glu-204 of p57 are responsible for its binding to the actin cytoskeleton.

Actins↗
PubMed2002

Type III intestinal metaplasia and carcinoma express an identical carbohydrate-epitope revealed by a novel monoclonal antibody (2D11).

A monoclonal antibody (2D11, IgG2b) obtained by immunizing mice with a mucin fraction of the human gastric mucosa reacted specifically to intestinal metaplasia of human gastric mucosa and fetal intestinal mucosa but not to normal adult gastric, small intestinal or colonic mucosa in immunohistochemical staining. The results of Western blotting indicated that 2D11 recognized the high molecular weight glycoprotein(s) (mucin) of the stomach. Treatment of the antigens with sodium periodate abolished their reactivity to 2D11, and digestion of the antigens with beta-galactosidase reduced their reactivity to 2D11. Digestion of the antigens with pronase had no effect, however, suggesting that 2D11 recognizes the oligosugar moiety but not the peptide moiety of the antigens. Further immunohistochemical investigation showed that the reactivity of 2D11 was restricted to the Type IotaIotaIota intestinal metaplasia that is identified by a characteristic staining pattern with the high iron diamine-Alcian blue stain. 2D11 also reacted in high frequency to adenocarcinomas of the stomach (66.7%), pancreas (66.7%) and gallbladder (50.0%), but in low frequency to those in lung (8.3%) and colon (11.1%). It is of interest that 2D11 reacted to very restricted regions of the gastric adenocarcinomas. All monoclonal antibodies to mucin polypeptides (MUC1, 2, 3, 5AC and 6) examined stained intestinal metaplasia and carcinomas in a different pattern from 2D11 in immunohistochemistry. These facts indicate that Type IotaIotaIota intestinal metaplasia and carcinomas express carbohydrate chains identical to those expressed in the fetal intestinal mucosa, suggesting that both of them are closely related to fetal intestinal mucosa.

Animals↗