Biomedical subjects

Tsuyoshi Sato

Publications and source records attributed to Tsuyoshi Sato.

13 recordsLinked to original sources

PubMed2006

Effect of combined antisense oligonucleotides against high-glucose- and diabetes-induced overexpression of extracellular matrix components and increased vascular permeability.

The effect of combined antisense oligonucleotides (AS-oligos) against overexpression of extracellular matrix (ECM) components, fibronectin, laminin, and collagen IV and on cell monolayer permeability was examined in rat microvascular endothelial cells (RMECs) grown in high glucose medium and on retinal vascular permeability in diabetic rats. RMECs grown in high glucose for 10 days and transfected with combined AS-oligos showed a significantly reduced fibronectin, laminin, and collagen IV protein level. In parallel studies, high-glucose-induced excess monolayer permeability was also reduced in RMECs transfected with the combined AS-oligos. Similarly, diabetic rats intravitreally injected with the combined AS-oligos and examined after 2 months of diabetes showed significant reduction in retinal fibronectin, laminin, and collagen IV expression. In addition, vascular permeability, as determined from extravasation of fluorescein isothiocyanate-BSA in the surrounding areas of the retinal capillaries, was partially reduced in the combined AS-oligos-treated diabetic retinas. Our results indicate that the combined AS-oligos strategy is effective in simultaneously reducing fibronectin, collagen IV, and laminin overexpression and reducing vascular leakage in the retinal capillaries of diabetic rat retinas. The findings suggest that abnormal synthesis of ECM components may contribute to vascular leakage in the diabetic retina.

Animals↗

PubMed2006

[The first graduates].

The Hirosaki University School of Health Sciences, started in April, 2001 graduated its first student in March, 2005 and we introduced a health studies specialty master's course at the same time. We report medical technologist training in a principal school and the situation for graduate school master's courses. The course of study includes physics, chemistry, biology and statistics with a compulsory subject by basic seminar that is an introduction subject, a basic education subject, serving as a supplementary lesson and considering the professional training to be connected. We mainly receive practical training by attending the university hospital clinical central laboratory, pathology, and blood transfusion area for three years and can continue for one year to carry out graduation study. As for the course situation, a person of entrance into a school of higher grade to a graduate school compared 11 students out of 41, company relation 4 with the school of allied medical sciences other than finding employment in a hospital and clinical laboratory and became various. 19 students out of 30 are members of society, and we consider this, and the new student situation to Hirosaki University graduate school master's course does a class on after half past 5 and Saturday.

Curriculum↗

PubMed2005

Electrochemical synthesis of a polypyrrole thin film with supercritical carbon dioxide as a solvent.

A conductive polypyrrole (PPy) film was successfully synthesized in a homogeneous supercritical carbon dioxide (scCO2)/acetonitrile (AN) system. The occurrence of a homogeneous supercritical state was confirmed by observations of the phase behavior of the system through a high-pressure cell with a viewing window. The concentration of a supporting electrolyte, tetrabutylammonium hexafluorophosphate (TBAPF6), significantly changed the phase behavior of the scCO2/AN system. The polymerization rate of the film in that system decreased with further addition of CO2. This result suggested that the low viscosity of scCO2 did not play an important role in improving the growth rate of the PPy film. The low polymerization rate might have been due to the electron-transfer resistance arising from the low dielectric constant of scCO2/AN mixture. The roughness of the film prepared in the homogeneous scCO2/AN system was 1/10 that synthesized in AN itself as a solvent. The slow growth of film and the high diffusion rate of the monomer seemed to account for the smooth flat film formation.

Journal Article↗

PubMed2003

A novel optical assay system for the quantitative measurement of chemotaxis.

We have developed an optically accessible, horizontal chemotaxis apparatus consisting of an etched silicon substrate and a flat glass plate, both of which form two compartments with a 5-microm-deep microchannel in between. The device is held together with a stainless steel holder with holes for injecting cells and a chemoattractant to the different compartments. Migration of cells in the channel is traced with time-lapse intervals using a CCD camera. By developing a method for aligning cells at the edge of the channel, we could successfully reduce the number of cells required for a chemotactic assay, depending on the experiment, to 100 or less. To prevent ceaseless flow of contents between the adjacent compartments via the communicating microchannel, a space at the top end of the holder was filled with medium after aligning the cells. By using a fluorescent probe, we demonstrated experimentally that a stable concentration gradient could be maintained. Furthermore, we determined theoretical details of the gradient established using a model chemokine and a computational fluid dynamics code. Reproducible kinetic results of cell migration were obtained using human neutrophils and IL-8 as a model. Migration of other cells such as eosinophils, basophils and Jurkat lymphocytes toward the appropriate chemokines were also demonstrated.

Basophils↗

PubMed2003

High glucose alters connexin 43 expression and gap junction intercellular communication activity in retinal pericytes.

PURPOSE: To investigate the role of the gap junction protein, connexin-43 (Cx43) in the maintenance of retinal vascular homeostasis in diabetic retinopathy. METHODS: In human retinal pericytes (HRPs) and bovine retinal pericytes (BRPs) grown for 7 days in normal (5 mM) or high (30 mM)-glucose medium, the Cx43 protein level was determined by Western blot analysis. Parallel experiments were performed in HRPs to determine the Cx43 mRNA level by RT-PCR, the distribution and localization of Cx43 protein by immunostaining, and gap junction intercellular communication (GJIC) activity by a scrape-loading dye transfer technique. Distribution and localization of Cx43 protein was also determined in pericyte-endothelial cell cocultures. RESULTS: Western blot analysis of the Cx43 protein level in HRPs and BRPs indicated reduced Cx43 expression in the high-glucose condition (69.1% +/- 17% of control, P = 0.004; 62.3% +/- 19% of control, P = 0.001, respectively). The Cx43 mRNA level in HRPs grown in high-glucose medium also showed significant reduction (71.4% +/- 16.8% of control, P = 0.02). The relative number of Cx43 plaques indicative of Cx43 localization at specific sites of contact between adjacent cells showed significant reduction in the high-glucose condition (61% +/- 10% of control, P = 0.002); similarly, a significant reduction in the number of plaques was observed in cocultures grown in high-glucose medium compared with those in normal medium (59.4% +/- 29% of control, P = 0.001). Cells with reduced Cx43 expression showed significantly reduced transfer of lucifer yellow (61% +/- 13% of control, P = 0.001; r = 0.9). CONCLUSIONS: High-glucose-induced downregulation of Cx43 expression and inhibition of GJIC in retinal pericytes may play a role in the disruption of vascular homeostasis in diabetic retinopathy.

Animals↗

PubMed2003

Downregulation of fibronectin overexpression reduces basement membrane thickening and vascular lesions in retinas of galactose-fed rats.

Overexpression of extracellular matrix (ECM) components is closely associated with the development of vascular basement membrane (BM) thickening, a histological hallmark of diabetic microangiopathy. To determine whether BM thickening of retinal capillaries could be prevented by down regulating synthesis of fibronectin, an ECM component, we used antisense oligos targeted against translation initiation site of the fibronectin transcript in galactose-fed rat, an animal model of diabetic retinopathy. After 2 months of galactose-feeding, intravitreal administration of 3 micro mol/l antisense fibronectin oligos was initiated at monthly intervals for 3 months. The antisense strategy significantly reduced fibronectin mRNA and protein level in the retinas of treated eyes compared with untreated eyes of galactose-fed rats (130 +/- 16 vs. 179 +/- 18% of control, P < 0.01, and 144 +/- 28 vs. 204 +/- 22% of control, respectively, r = 0.9) and resulted in partial reduction of retinal capillary BM width (123 +/- 16 vs. 201 +/- 12 nm, P < 0.03). In eyes treated with antisense fibronectin oligos, approximately 35% reduction in both pericyte loss and acellular retinal capillaries was observed (P < 0.04 and P < 0.03, respectively). Glycohemoglobin level was consistently elevated in the treated (6.9 +/- 0.6%) and untreated (6.5 +/- 0.7%) galactose-fed rats compared with control rats (4.5 +/- 0.8%). Overall, these results indicate that downregulation of fibronectin synthesis reduces BM thickening in retinal capillaries with beneficial effect to retinal lesions. The antisense fibronectin oligos may provide a useful approach for reducing vascular lesions in diabetic retinopathy. The thickened vascular BM may be a potential therapeutic target for preventing retinal lesions in diabetic retinopathy.

Animals↗

PubMed2002

Copper(II)- cis, cis-1,3,5-triaminocyclohexane complex-promoted hydrolysis of dipeptides: kinetic, speciation and structural studies.

The hydrolysis of glycylglycine (GylGly), glycyl-L-leucine (GlyLeu), L-leucylglycine (LeuGly) and glycyl-DL-serine (GlySer) promoted by a copper(II)- cis, cis-1,3,5-triaminocyclohexane complex [Cu(II)TACH] was investigated at 70 degrees C and pH 7-10, using HPLC. The observed pseudo-first-order rate constants (k(obs)) and rate enhancing factors (REF) were as follows: 4.1x10(-3 )h(-1)(REF=23) for GylGly, 1.6x10(-3 )h(-1)(REF=21) for GlyLeu, 5.1x10(-3 )h(-1)(REF=64) for LeuGly and 9.2x10(-2 )h(-1)(REF=47) for GlySer [pH 8.1, dipeptide 2 mM, copper(II) 2 mM and TACH 2 mM]. Based on the pH dependence and dipeptide concentration dependence of the initial rates and speciation of the Cu(II)-TACH-dipeptide system at 25 degrees C and I=0.1, the reactions proceed via the formation of a ternary complex [Cu(TACH)(dipeptide)](+) as an intermediate followed by OH(-)-dependent and OH(-)-independent paths to give amino acid(s). GylGly, GlyLeu and LeuGly preferred the OH(-)-dependent path, while GlySer preferred the OH(-)-independent path. The latter can be explained by the intramolecular attack of the amide carbonyl group coordinated with its oxygen atom by the OH group in the serine residue. The X-ray crystal structure of [Cu(TACH)(GlyGly)]BPh(4).MeOH confirmed that GlyGly coordinates to copper(II) ion with its terminal amino N and amide O atoms. The crystal structures of [Cu(TACH)(Gly)]BPh(4) and [Cu(2)(TACH)(2)(OH)(2)](ClO(4))(2).NaClO(4).H(2)O are also reported.

Chromatography, High Pressure Liquid↗

PubMed2002

Role of TGF-beta family in osteoclastogenesis induced by RANKL.

Recent studies have revealed that both transforming growth factor-beta (TGF-beta) and activin A play pivotal roles in osteoclastogenesis. In this report, we show that the effect of TGF-beta family members, TGF-beta1 and activin A, but not BMP-2, enhance multinucleated osteoclast-like cell (OCL) formation induced by receptor activator of NF-kappaB ligand (RANKL) in isolated bone marrow macrophages and monocytic cell line, RAW264.7. TGF-beta1 and activin A caused the growth suppression and concomitant expression of tartrate-resistant acid phosphatase (TRAP) and c-Src, without inducing syncytium formation or increasing the survival rate in RAW264.7 cells. Although TGF-beta1 and activin A had no effect on NF-kappaB and JNK activities, these factors enhanced the expression of JunB, a component of the AP-1 transcriptional complex. These results suggest that TGF-beta1 and activin A may function as commitment factors in osteoclastic differentiation, not as a crucial component for terminal differentiation to form multinucleated OCLs nor in OCL survival.

Acid Phosphatase↗

PubMed2002

Nrf2 transactivator-independent GSTP1-1 expression in "GSTP1-1 positive" single cells inducible in female mouse liver by DEN: a preneoplastic character of possible initiated cells.

Whether single cells immunohistochemically positive for glutathione S-transferase P1-1 (GSTP1-1) induced in the female mouse liver by DEN (Hatayama et al., Carcinogenesis, 14, 537-538, 1993) are precursor initiated cells of preneoplastic foci, is of importance in chemical hepatocarcinogenesis. Nrf2 transactivates a wide variety of ARE (anti-oxidant response element)-mediated enzymes including GSTP1-1. Quantitative examination revealed that the basal expression of hepatic GSTP1-1 was 60% lower in Nrf2 gene knock-out female mice(-/-) than in wild type females, and that treatment with butyrated hydroxyanisole (BHA) increased by 10-fold GSTP1-1 expression in the liver of wild type female mice but not in knockout female mice(-/-). Despite the lack of Nrf2, GSTP1-1-positive single cells were detected in livers of DEN-treated female(-/-) 3 months after treatment. Subsequent BHA feeding to the positive cell-bearing females for one more week clearly showed that the single cells were detectable with females(-/-) but not with females(+/+,+/-) due to the strong induction of GSTP1-1 in the surrounding hepatocytes. The sensitivity to DEN hepatocarcinogenesis was not significantly different among genotypes. These results demonstrate that Nrf2 is regulatory in normal hepatocytes but not in the single cells positive for GSTP1-1 inducible in the female mouse liver by DEN. The transcriptional distinction observed for the DEN-transformants is suggestive of a preneoplastic character of precursor initiated cells.

Animals↗

PubMed2002

Enrichment of fetal cells from maternal blood by magnetic activated cell sorting (MACS) with fetal cell specific antibodies: one-step versus two-step MACS.

We report here the results of fetal cell enrichment from maternal blood in 58 pregnant women by the use of magnetic activated cell sorting (MACS) with erythroblast-specific and/or maternal cell specific antibodies. Two approaches were compared; one-step MACS to enrich CD71+ (a membrane-bound marker) or GPA+ (another marker, glycophorin A) fetal cells versus two-step MACS to deplete CD14+ maternal cells and subsequently to enrich fetal (CD71+ or GPA+) cells. The existence of fetal cells was ensured by both FISH with Y-specific probes and karyotyping of respective amniotic and/or chorionic vullus cells, the results being applied for comparison of detection rate for XY fetuses between the two MACS procedures. In 24 (38.8%) of the 58 blood samples examined, Y-positive cells were observed by FISH, whereas there were 38 true XY fetuses later confirmed by karyotyping, including two cases of 47,XY,+21. On the other hand, in Y-negative cells by FISH, there were two cases of 47,XX,+18. The average number of cells sorted did not differ among one-step MACS procedures with anti-CD14, anti-CD71 and anti-GPA antibodies. With the latter, 12 (75%) of 16 Y-positive fetuses were detected, while only one (20%) of 5 Y-positive fetuses was detected by two-step MACS with anti-CD14/anti-GPA antibodies. The detection rate significantly varied (p = 0.0024) between the two procedures, although the numbers of cases examined were small. There was no statistical difference (p > 0.05) between one-step and two-step MACS with other combinations of antibodies. These findings indicate that one-step MACS using the anti-GPA antibody is more effective than two step MACS for enrichment of fetal cells from maternal blood.

Adult↗

PubMed2002

p53-independent expression of p21(CIP1/WAF1) in plasmacytic cells during G(2) cell cycle arrest induced by Actinobacillus actinomycetemcomitans cytolethal distending toxin.

The cytolethal distending toxin (CDT) from Actinobacillus actinomycetemcomitans has been shown to induce cell cycle arrest in the G(2)/M phase in HeLa cells. In the present study, the mechanism of CDT-induced cell cycle arrest was investigated by using HS-72 cells, a murine B-cell hybridoma cell line. Using flow cytometric analysis, we found that the recombinant CDT (rCDT) from A. actinomycetemcomitans induced G(2) cell cycle arrest in HS-72 cells and that rCDT upregulated expression of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) and the tumor suppressor protein p53. HS-72 cells transfected with the E6/E7 gene of human papillomavirus type 16, which lacked rCDT-induced accumulation of p53, exhibited expression of p21(CIP1/WAF1) or G(2) cell cycle arrest upon exposure to rCDT. Furthermore, ectopic expression of a dominant negative p53 mutant did not inhibit rCDT-mediated p21(CIP1/WAF1) expression or G(2) cell cycle arrest in HS-72 cells. These results suggest that the CDT from A. actinomycetemcomitans induces p21(CIP1/WAF1) expression and G(2) cell cycle arrest in B-lineage cells by p53-independent pathways. Together with additional observations made with HeLa cells and COS-1 cells cultured with the rCDT from A. actinomycetemcomitans, the results of this study indicate that CDT-induced p53 accumulation may not be required for G(2) cell cycle arrest and that an increased level of p21(CIP1/WAF1) may be important for sustaining G(2) cell cycle arrest in several mammalian cells.

Aggregatibacter actinomycetemcomitans↗

PubMed2002

Downregulation of connexin 43 expression by high glucose reduces gap junction activity in microvascular endothelial cells.

Impairment of retinal vascular homeostasis is associated with the development and progression of diabetic retinopathy involving gap junction intercellular communication (GJIC) activity. The principal gap junction protein of intercellular communication, connexin, was investigated to determine the effects of high glucose concentrations on the expression of endothelial-specific connexins (Cx37, Cx40, and Cx43), connexin phosphorylation pattern, and GJIC activity. Rat microvascular endothelial (RME) cells grown in high (30 mmol/l)-glucose medium for 9 days had reduced Cx43 expression: Cx43 mRNA (68 +/- 13% of control; P = 0.019, n = 5) and protein (55.6 +/- 16% of control; P = 0.003, n = 5) levels were reduced; however, Cx37 and Cx40 expression was not affected. Using alkaline phosphatase and Western blot analyses, we identified three forms of Cx43: a nonphosphorylated form (P0) and two phosphorylated forms (P1 and P2). Expression of all three forms was decreased in cells grown in high-glucose medium: PO, 73 +/- 15% of control (P = 0.04); P1, 57 +/- 16% of control (P = 0.01); and P2, 42 +/- 22% of control (P = 0.006). Using immunofluorescence microscopy, we observed Cx43 localization at specific sites of contact (plaques) between adjacent cells. In cells grown in high-glucose medium, we observed reduced plaque counts (63 +/- 6% of control; P = 0.009) and decreased intensity of Cx43 immunofluorescence compared with cells grown in normal medium. Furthermore, using scrape load dye transfer (SLDT) technique, we found that these cells exhibited reduced GJIC activity (60% of control; P = 0.01, n = 5). The reduction in GJIC activity correlated with the decreased Cx43 protein levels (r = 0.9). These results indicate that high glucose concentrations inhibited GJIC activity by reducing Cx43 synthesis in RME cells. Impaired intercellular communication may contribute to breakdown of homeostatic balance in diabetic microangiopathy.

Adipose Tissue↗

PubMed2002

Effect of high glucose on fibronectin expression and cell proliferation in trabecular meshwork cells.

PURPOSE: Increased fibronectin accumulation in the trabecular meshwork of glaucomatous eyes may contribute to the resistance of aqueous outflow and the development of primary open-angle glaucoma (POAG). Because the glucose level is increased in the aqueous humor of patients with diabetes, this study was conducted to determine whether a high-glucose condition alters fibronectin expression and contributes to cell loss in trabecular meshwork. METHODS: The fibronectin mRNA level was determined using RT-PCR in bovine trabecular meshwork cells grown in normal (5 mM) or high (30 mM)-glucose medium for 7 days, and cell counts were measured during this period. Distribution and the relative amount of fibronectin protein were determined in these cells by immunofluorescence microscopy and Western blot analysis. RESULTS: Fibronectin mRNA level in cells grown in high-glucose medium was significantly upregulated two- to threefold compared with cells grown in normal medium (P < 0.05). In cells grown in high-glucose medium, fibronectin immunofluorescence was more intense, and the relative amount of fibronectin protein was significantly increased (131% +/- 15% of control, P < 0.05) compared with the amount in cells grown in normal medium. A moderate decrease in cell number was observed in cells grown in high-glucose medium (78% +/- 7% of control, P < 0.05) CONCLUSIONS: These findings indicate that a high glucose level in aqueous humor of patients with diabetes may increase fibronectin syntheses and accumulation in trabecular meshwork and accelerate the depletion of trabecular meshwork cells, a characteristic feature of the outflow system in POAG. The striking similarity between high glucose-induced alterations in trabecular meshwork cells and those of vascular endothelial cells may represent a common biochemical link in the pathogenesis of POAG and diabetic microangiopathy.

Animals↗