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Biomedical subjects

Y Asada

Publications and source records attributed to Y Asada.

At least 109 records · Page 6Linked to original sources

The role of tissue factor in the pathogenesis of thrombosis and atherosclerosis.

TF is a major regulator of coagulation and hemostasis. High levels of TF antigen and activity are detected in atherosclerotic lesions, particularly in the advanced lesions. When the plaques are ruptured or eroded, exposure of cellular and extracellular TF to circulating blood play a pivotal role in mediating fibrin-rich thrombus formation leading to acute coronary syndromes. On the other hand, activation of blood coagulation and deficiency of coagulation inhibitors, without endothelial cell denudation, are considered to be an important factor of thrombogenesis in the microcirculation. The imbalance between TF and TFPI seems to be important in promoting fibrin thrombus formation in the lung of endotoxin induced DIC condition.

Animals↗

Fibrin-rich and platelet-rich thrombus formation on neointima: recombinant tissue factor pathway inhibitor prevents fibrin formation and neointimal development following repeated balloon injury of rabbit aorta.

Thrombus formation and neointimal growth are the critical events in restenosis after balloon angioplasty. However, the responses of diseased vessels to injuries caused by balloon angioplasty have not been well examined. We investigated the thrombus formation and neointimal development following the balloon injury to the previously induced neointima in the rabbit aorta and the effects of recombinant tissue factor pathway inhibitor (rTFPI) on these responses. Rabbit thoracic aortas were subjected to injury with a Fogarty 4F balloon catheter at 1.75 atm (first injury), and 4 weeks later the same vessels were subjected to the second injury with a Swan-Ganz 5F balloon catheter at 1.4 atm (mild-injury group) or 1.8 atm (severe-injury group), and immediately after that a retrograde bolus injection of rTFPI (100 microg/kg body weight) or saline was performed into the injured segments via the central tube of the Swan-Ganz catheter. Twenty minutes after the second injury, the injured surfaces were covered with platelet-rich thrombi in the mild-injury group and with fibrin-rich thrombi in the severe-injury group. Damaged intimal smooth muscle cells, which were immunohistochemically positive for tissue factor (TF), were observed beneath the fibrin-rich thrombi. The neointima 4 weeks after the second injury was significantly thicker in the severe-injury group than in the mild-injury group. The bolus infusion of rTFPI markedly inhibited fibrin formation on the injured surfaces, and significantly reduced the neointimal development in the severe-injury group at 4 weeks after the second injury. These results indicate that TF-dependent coagulation pathway is primarily responsible for fibrin-rich thrombus formation and may play an important role in neointimal development following the balloon injury to the rabbit aortic neointima. Additionally the bolus administration of rTFPI to the injured vessels could prevent mural thrombus formation and neointimal growth after balloon angioplasty.

Animals↗

[High-dose steroid therapy for sudden deafness--efficacy in severe cases].

We studied the efficacy of high-dose steroid therapy for severe sudden deafness, and evaluated various factors that affect hearing recovery; i.e., severity of hearing loss, age, presence of vertigo and time to the beginning treatment. Twenty-eight patients with idiopathic sudden sensorineural hearing loss whose hearing levels were 60 dB or more were analyzed. They were given 200 mg of prednisolone per day intravenously according to Stennert's method. Hearing recovery was assessed by the criteria of the Sudden Deafness Research Group of the Japanese Ministry of Health and Welfare. The overall recovery rate and cure rate were 50% and 14%, respectively. Patients whose initial hearing levels were within 90 dB showed a significantly higher cure rate than those with above 90 dB (36% (n = 11) vs 0% (n = 17), p < 0.05). The cure rate was significantly higher in the vertigo (-)-group than in the vertigo (+)-group (40% (n = 10) vs 0% (n = 18), p < 0.02). The cure rate was also higher in patients under 50 years of age who were treated within 7 days after onset than in the others (31% (n = 13) vs 0% (n = 15), p < 0.05). The recovery rate was higher when patients were under 50 years of age (63% vs 33%), treated within 7 days after onset (56% vs 42%), or had an initial hearing level within 90 dB (55% vs 47%); however, the differences were not statistically significant. We conclude that these prognostic factors are important for predicting the hearing recovery of patients with severe sudden deafness who undergo high-dose steroid therapy and, therefore, for determining whether this therapy is indicated.

Adult↗

Analysis of tissue factor and tissue factor pathway inhibitor expression in human colorectal carcinoma cell lines and metastatic sublines to the liver.

To investigate the expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in human colorectal carcinomas, Northern blot analysis was performed in a series of human colorectal carcinoma cell lines and in normal or tumoral colorectal tissues. Of 16 human colorectal carcinoma cell lines examined, most expressed TF mRNA, though the levels of expression varied significantly. Considerably higher expression was observed in the cell line CaR-1, while lines established from metastatic lesions tended to express abundant TF mRNA. By contrast, TFPI mRNA levels were low in these high TF-expressing cell lines. TFPI was expressed abundantly in WiDr and in a few other cell lines which expressed a very low level of TF mRNA. Immunocytochemically, both proteins were stained predominantly on the cell surface; however, diffuse cytoplasmic staining for TF also was observed in CaR-1 cells. In addition, the cell surface TF activity was significantly higher in CaR-1 cells than in WiDr cells, confirming the results of mRNA analysis. The level of TF mRNA in colorectal carcinoma tissue in vivo and its ratio to the normal counterpart also varied significantly among the cases. To search for a possible role of TF/TFPI in metastasis of colorectal carcinoma cells, the expression of these genes was compared between a rectal adenocarcinoma cell line, RCM-1, and its highly metastatic subline, RCM-1 L-10. Compared with the parent line, RCM-1 L-10 expressed 7.5-fold higher levels of TF mRNA, whereas TFPI expression was not altered significantly or even decreased slightly. The higher cellular TF activity was confirmed in the metastatic subline in comparison with the parent line.

Adenocarcinoma↗

Cloning, sequencing, and expression of Arthrobacter protophormiae endo-beta-N-acetylglucosaminidase in Escherichia coli.

The gene encoding endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was cloned, and its nucleotide sequence was determined. A single open reading frame consisting of 1935 base pairs and encoding a polypeptide composed of signal peptides of 24 amino acids and a mature protein of 621 amino acids was found. The primary structure of Endo-A exhibited significant homology with FO1F.10 gene product from Caenorhabditis elegans and weak homology with peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum and chitinase from Streptomyces olivaceoviridis. However, the enzyme had no significant homology with any previously reported endo-beta-N-acetylglucosaminidases. Transformed Escherichia coli cells carrying the 4.5-kb fragment expressed Endo-A activity. This enzyme activity was detected in the medium as well as in the periplasmic space of cells under the control of the Endo-A gene promoter. The recombinant Endo-A was efficiently isolated from the periplasmic space of the cells. N-terminal sequence analysis revealed that native and recombinant Endo-A have the same N-terminus. Recombinant and native Endo-A also showed very similar optimum pH profiles and transglycosylation activity.

Amino Acid Sequence↗

Intravenous injection of sonicated blood induces pulmonary microthromboembolism in rabbits with ligation of the splenic artery.

Pulmonary thromboembolism (PTE) is found in long hospitalized patients. Chronic PTE has been reported to play an important role in cardiac failure in thalassemic patients after splenectomy. However, the mechanism of PTE in these patients remains unclear. In this study, we attempted to establish an animal model of PTE. We divided New Zealand white rabbits into three groups: Group I was injected sonicated blood, II was injected non-sonicated blood after ligation of the splenic artery, and III was injected sonicated blood after ligation of the splenic artery. After injection of the sonicated blood, we examined the platelet counts every 10 minutes until 1 hour and the rabbits were sacrificed for histological examination. Platelets significantly decreased in number immediately after the injection of sonicated blood in Groups I and III. Many pulmonary thromboemboli composed mainly of platelets were found in Group III but not in other groups. These pathological changes seem to be partly similar to those of thalassemic patients after splenectomy. This animal model is thought to be useful to study the pathogenesis of pulmonary thromboembolism, especially in thalassemic patients after splenectomy.

Animals↗

Effects of 17 beta-estradiol and progesterone on migration of human monocytic THP-1 cells stimulated by minimally oxidized low-density lipoprotein in vitro.

OBJECTIVE: Many epidemiological studies have shown that postmenopausal hormone replacement therapy (HRT) has a beneficial effect on atherosclerotic cardiovascular disease. The aim of this study was to investigate the effects of estrogen and progestin on the migration of monocytes induced by minimally oxidized low-density lipoprotein (m-ox-LDL) in vitro. METHODS: Human monocytic THP-1 cells were used for the study. Migration assay was performed using a modified Boyden chamber. RESULTS: The presence of estrogen receptors was determined in THP-1 cells by Western and Northern blot analysis. Although native LDL had no significant effects on the migration of THP-1 cells, m-ox-LDL increased the migration of THP-1 cells in a dose-dependent manner. Although 17 beta-estradiol (E2, 10(-9)-10(-6) M) inhibited the 10 micrograms/ml-induced migration of THP-1 cells in a dose-dependent manner, estrone (E1), estriol (E3) and progesterone (P) had no significant effects. The combination of P (10(-9)-10(-6) M) did not show any effect on the inhibitory effect of 10(-7) M E2. Preincubation of THP-1 cells with the anti-estrogenic agent, tamoxifen (10(-6) M), significantly antagonized the inhibitory effect of 10(-7) M E2. m-ox-LDL stimulated MCP-1 secretion from THP-1 cells, which was reduced by E2. Anti-human MCP-1 neutralizing antibody inhibited the migration of THP-1 cells stimulated by m-ox-LDL. E2 also inhibited the 10 ng/ml MCP-1-induced migration of THP-1 cells in a dose-dependent manner. CONCLUSIONS: These findings suggest that the inhibitory effect of E2 on the migration of monocytes might be one of the factors involved in the decreased incidence of atherosclerotic cardiovascular disease in premenopausal women and postmenopausal HRT.

Adult↗

Localization and activity of tissue factor in human aortic atherosclerotic lesions.

Tissue factor (TF) is a transmembrane protein that serves as the major initiator of the blood coagulation cascade. The overexpression of TF antigen and mRNA has previously been reported in advanced atherosclerotic lesions. Recently TF procoagulant activity has also been identified in these lesions. However, localization and activity of TF in various stages of atherosclerosis have not yet been reported. We studied TF localization and its activity in three stages of the human atherosclerotic lesions (diffuse intimal thickening, fatty streak, and atheromatous plaque). The thoracic aortas were obtained from 23 autopsy cases and were examined immunohistochemically using an anti-human TF polyclonal antibody and biotinylated factor VIIa (FVIIa) as a probe to test the FVIIa-binding ability of TF. In addition, the TF-mediated activation of factor X (FX) was quantitatively assessed using a chromogenic assay. In lesions of the diffuse intimal thickening and the fatty streak, almost all of intimal smooth muscle cells (SMCs), macrophages, and endothelial cells were positive for TF. In the atheromatous plaques, TF antigen was detected extensively in the extracellular matrix as well as in the intimal cells. TF in all stages of atherosclerotic lesions had the ability to bind biotinylated FVIIa. TF activity was detected in each lesion and was more prominent in fatty streaks and atheromatous plaques than in the diffuse intimal thickening. These results indicate that active TF is expressed in the early stage of atherosclerotic lesions as well as in the advanced stage, and it contributes to the thrombotic property of human atherosclerotic lesions.

Adolescent↗

Schizophyllum commune A alpha mating-type proteins, Y and Z, form complexes in all combinations in vitro.

Tha A alpha locus of the basidiomycete fungus, Schizophyllum commune, regulates sexual development via proteins Y and Z. Each A alpha mating type encodes unique Y and Z isoforms. We used two isoforms of Y (Y4 and Y5) and two isoforms of Z (Z4 and Z5) in affinity assays of protein binding. These assays identified two types of protein interactions. Each full-length Y or Z protein binds to itself and other Y or Z proteins regardless of the A alpha mating type from which they are encoded (i.e., mating-type independent binding). A second type of binding, detected with partial-length polypeptides, occurs only between N-terminal regions of Y and Z proteins encoded from different A alpha mating types (e.g., Y4Z5 or Y5Z4); we refer to this binding as mating-type dependent binding. Deletion analysis shows that the Y4 specificity domain (an N-terminal region conferring recognition uniqueness to the Y4 isoform) is essential for mating-type dependent binding. Other regions of Y and Z are involved in mating-type independent binding. These results, obtained in vitro, raise the possibility that either of several protein complexes composed of Y and/or Z proteins may occur in vivo.

Fungal Proteins↗

Control of poly-beta-hydroxybutyrate synthase mediated by acetyl phosphate in cyanobacteria.

Poly-beta-hydroxybutyrate (PHB) synthesis in a cyanobacterium, Synechococcus sp. strain MA19, is controlled at the enzyme level and is dependent on the C/N balance in the culture medium. The control involves at least two enzymes. The first enzyme is PHB synthase. Little PHB synthase activity was detected in crude extracts from cells grown under nitrogen-sufficient conditions (MA19(+N)). The activity was detected exclusively in membrane fractions from nitrogen-deprived cells (MA19(-N)) under light but not dark conditions. The shift in the enzyme activity was insensitive to chloramphenicol, which suggests posttranslational activation. Acetyl phosphate activated PHB synthase in membrane fractions from MA19(+N). In vitro, the activation level of PHB synthase changed, depending on the concentration of acetyl phosphate. The second enzyme was phosphotransacetylase (EC 2.3.1.8), which catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate. The activity was detected in crude extracts from MA19(-N) but not in those from MA19(+N). The results suggested that intracellular acetyl phosphate concentration could be controlled, depending on C/N balance and intracellular acetyl-CoA concentration. Acetyl phosphate probably acts as a signal of C/N balance affecting PHB metabolism in MA19.

Acetyl Coenzyme A↗

Nitric oxide-dependent and -independent norepinephrine release in rat mesenteric arteries.

The role of nitric oxide (NO) in endogenous norepinephrine (NE) release in the perfused isolated rat mesenteric vasculature was examined. NE overflow elicited by electrical field stimulation (EFS) at various frequencies was significantly smaller at 24 than at 37 degrees C. The pressor response upon EFS at 8 and 10 Hz, however, was higher at 24 than at 37 degrees C. When production of NO was blocked by N omega-nitro-L-arginine (L-NNA), NE overflow upon EFS at each frequency of stimulation was diminished by 50% at 37 degrees C but remained unchanged at 24 degrees C, whereas the pressor response elicited by EFS became greater at 37 than at 24 degrees C. These effects of L-NNA were reversed by L-arginine, but not by its D-enantiomer. Sodium nitroprusside, an NO donor, increased EFS-elicited NE overflow at 24 degrees C but had no effect at 37 degrees C. These results demonstrate that NE release is NO dependent and NO independent. The NO-dependent mechanism is more sensitive to cooling than the NO-independent mechanism. The increase in EFS-elicited perfusion pressure at 24 degrees C may be due to reduction in synthesis of NO (a potent vasodilator), thus unmasking the effect of NE and other noncatecholamine vasoconstrictors.

Animals↗

Role of nitric oxide in mucosal blood flow response and the healing of HCl-induced lesions in the rat stomach.

The role of nitric oxide (NO) in the gastric mucosal blood flow response and the healing of HCl-induced gastric lesions was investigated in rats. After 18 h fasting rats were given 0.6 N HCl p.o. for the induction of gastric lesions, and 1 h later they were fed normally. After induction of gastric lesions, they were repeatedly administered the NO synthase inhibitors NG-nitro-L-arginine methyl ester (L-NAME 5-20 mg/kg p.o. twice daily) or aminoguanidine (20 mg/kg s.c. once daily) for 7 days. Gastric lesions caused by HCl healed almost completely within 5 days with granulation and to an extent with re-epithelialization. Repeated administration of L-NAME but not aminoguanidine significantly delayed the healing of gastric lesions in a dose-dependent manner. The damaged mucosa secreted less acid, but showed a marked rise in H+ permeability, resulting in luminal acid loss accompanied by an increase of mucosal blood flow. Aminoguanidine did not significantly affect any of these functional changes observed in the stomach after damage by HCl, whereas L-NAME treatment slightly reversed the decreased acid response, increased the luminal H+ loss, and totally inhibited the mucosal hyperemic response associated with luminal acid loss in the damaged mucosa. In addition, the deleterious influences of L-NAME on the mucosal blood flow response and the healing of gastric lesions were significantly antagonized by co-administration of L-arginine but not of D-arginine (500 mg/kg x 2, i.p.). Luminal output of NO2-/NO3- was significantly increased in pylorus-ligated stomachs in control rats on days 3 and 5 after damage, and such increases in gastric NO output were completely attenuated by L-NAME treatment. These results suggest that endogenous NO may contribute to the healing of acute gastric injury by mediating the mucosal hyperemic responses associated with acid back-diffusion and by facilitating acid disposal in the damaged mucosa. NO mediating such responses and participating in the healing aspect of gastric lesions may be produced by the constitutive type of NO synthase.

Animals↗

Epididymal sperm retrieval by epididymal micropuncture combined with intracytoplasmic sperm injection: difference between acquired and congenital irreparable obstructive azoospermia.

To evaluate the differences in fertilization and pregnancy rates following intracytoplasmic sperm injection (ICSI) of retrieved epididymal sperm between congenital (group 1) and acquired (group 2) unreconstructable obstructive azoospermic patients, we compared the outcome of the ICSI procedure between these two groups. Thirty-six patients with obstructive azoospermia received epididymal sperm retrieval by the micropuncture method for the ICSI procedure. Main parameters evaluated were epididymal fluid volume, sperm concentration, sperm-motility; fertilization rate and clinical pregnancy rate. There were no significant differences in epididymal fluid and sperm concentration between the two groups. However, the sperm motility in group 1 was significantly higher than that in group 2. The fertilization rates per couple in groups 1 and 2 were 78.2 and 82% (nonsignificant). The pregnancy rate per couple in group 1 was 37.5% (6/16), while that in group 2 was only 5% (1/20); this difference was statistically significant (p < 0.05). Using epididymal sperm for the ICSI procedure, the chances of pregnancy for couples with congenital absence of the vas deferens were significantly higher compared with couples with acquired unreconstructable obstructive male infertility.

Adult↗

Effects of 17beta-estradiol and progesterone on the adhesion of human monocytic THP-1 cells to human female endothelial cells exposed to minimally oxidized LDL.

It is known that hormone replacement therapy inhibits the progression of atherosclerosis in postmenopausal women. Focal attachment of monocytes to endothelial cells is observed in early atherosclerotic lesions. The aim of this study was to investigate the effects of 17beta-estradiol (E2) and progesterone on the adhesion of human monocytic THP-1 cells to human female aortic endothelial cells (HAECs) in vitro. Minimally oxidized low-density lipoprotein (LDL) significantly increased THP-1 cell adhesion to HAECs as compared with native LDL at the same concentration. Though E2 inhibited minimally oxidized LDL-induced THP-1 cell adhesion in a dose-dependent manner, progesterone had no significant effects. Preincubation of HAECs with tamoxifen significantly antagonized the inhibitory effect of E2. These findings suggest a beneficial effect of hormone replacement therapy on atherosclerosis.

Adult↗

Orientation of photosynthetic reaction center reconstituted in neutral and charged liposomes.

The photosynthetic reaction center from the photosynthetic bacterium Rhodobacter sphaeroides was reconstituted into neutral, positively charged, or negatively charged liposomes. About 70% of photosynthetic reaction centers were reconstituted in the proteoliposomes exposing their H-subunit outside with positively charged lipids while only 30-40% of them were in the same topological orientation with neutral or negatively charged lipids.

Cytochrome c Group↗

Purification and properties of two isozymes of gamma-glutamyltranspeptidase from Bacillus subtilis TAM-4.

Two isozymes of gamma-glutamyltranspeptidase, GGT-A and GGT-B, were purified to electrophoretic homogeneity from a culture broth of Bacillus subtilis TAM-4, which produces poly(gamma-glutamic acid) (PGA) de novo. GGT-A was composed of three subunits with molecular weights of 23,000 (I), 39,000 (II), and 40,000 (III). GGT-B was composed of two subunits with molecular weight of 22,000 (I) and 39,000 (II). The N-terminal amino acid sequences of GGT-A subunit I and GGT-B subunit I were very similar. GGT-A subunit II and GGT-B subunit II had an identical N-terminal amino acid sequence. That of GGT-A subunit III showed no similarity to the other subunits. Both GGTs had similar enzymatic properties (optimum pH and temperature: pH 8.8 and 55 degrees C) but showed a significantly different thermal stability at 55 degrees C. Both GGT-A and -B used D-gamma-glutamyl-p-nitroanilide as well as the L-isomer as the gamma-glutamyl donor and used various amino acids and peptides as the acceptor. It was also found that the PGA produced by the strain was hydrolyzed to glutamic acid by its own GGTs.

Amino Acid Sequence↗

Modulation of E-cadherin expression in TPA-induced cell motility: well-differentiated human adenocarcinoma cells move as coherent sheets associated with phosphorylation of E-cadherin-catenin complex.

We previously presented a two-dimensional cell motility assay using L-10, a highly metastatic variant of the human rectal adenocarcinoma cell line RCM-1, as a motility model of tumor cells of epithelial origin. In this model, L-10 cells moved outward from the cell islands mainly as a localized coherent sheet of cells when stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA). Electronmicroscopic study of the migrating cell sheets revealed that wide intercellular gaps had developed at the lower portion of the cells, allowing them to extend leading lamellae, whereas close cell-cell contacts remained at the upper portion of the cells. In the present study, the mechanism involved in this localized modulation of cell-cell adhesion at the lower portion of the cells was investigated with special reference to E-cadherin expression. E-cadherin immunostaining, which was demonstrated using an anti-E-cadherin mAb, HECD-1, was decreased in migrating L-10 cell sheets. Apparently, however, E-cadherin was involved in the sheet formation of migrating cells because simultaneous or sequential treatment with TPA and HECD-1 inhibited sheet formation and caused scattering of migrating cells. With immunoelectron microscopic study, E-cadherin immunoreactivity was confined to the upper portion of migrating cells and lost at the lower portion. The level of E-cadherin and alpha-catenin expression was not altered by TPA treatment, although tyrosine phosphorylation of E-cadherin and catenins increased 1.6- to 1.9-fold. We propose that cells are released from cell-cell adhesion only at the lower portion of the cells via phosphorylation of the E-cadherin-catenin complex when stimulated with TPA. This change allows the cells to extend leading lamella and thus move together as coherent sheets (cohort migration).

Adenocarcinoma↗

[Quantitative evaluation of regional pulmonary functions after lung resection using three-dimensional images of ventilation and perfusion SPECT].

To evaluate changes after Lobectomy in the regional functions of the remaining lung and of the unaffected lung, three-dimensional (3-D) images of lung ventilation and perfusion SPECT were studied. In 60 patients who underwent lung resection, ventilation (99m Tc-phytate aerosol inhalation) and perfusion (99m Tc-MAA) SPECT were performed before and one month after operation. 3-D images were obtained from SPECT by the volume-rendering method setting thresholds at three levels, and the 3-D functional capacity of the right and left lung was determined at a threshold of 25%. Changes were evaluated using the ratio of the value obtained after operation to the one obtained before operation. The remaining rate of perfusion in the remaining lung was 0.58 +/- 0.22 after the right upper lobectomy, 0.32 +/- 0.04 after the right upper & middle bilobectomy, 0.86 +/- 0.11 after the right middle lobectomy, 0.55 +/- 0.17 after the right lower lobectomy, 0.24 +/- 0.18 after the left upper lobectomy, 0.46 +/- 0.05 after the left lower lobectomy, and 0 after the left pneumonectomy. The remaining rate was significantly low in the right upper & middle bilobectomy and the left upper lobectomy. The large decrease in the rate in these operations was considered to have resulted from a shift of the remaining lung towards the head and excessive expansion of the remaining lung. The distribution of the aerosol inhalation decreased more significantly than that of perfusion in almost all patients. The remaining rate of ventilation of the remaining lung after the lower lobectomy was lower than that after the upper lobectomy. This may have been due to the characteristics of the aerosol practices. We found that changes in the regional functions of the lung after lung resection can be topographically and quantitatively evaluated by 3-D image processing of pulmonary ventilation and perfusion SPECT, such analysis is usefulness in evaluation of function preservation operation.

Female↗