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M Sugimoto

Publications and source records attributed to M Sugimoto.

At least 91 records · Page 5Linked to original sources
PubMed1999

Activation of caspase-3 in beta-amyloid-induced apoptosis of cultured rat cortical neurons.

Amyloid beta protein (Abeta) has been thought to participate in the neurodegeneration associated with Alzheimer's disease. We here report on caspase-3 activation by Abeta-treatment of cultured neurons. Treatment of rat primary cortical culture with Abeta 25-35, an active fragment of Abeta, induced neuronal death as determined by a decrease in neuron-specific microtubule-associated protein 2 (MAP2)-like immunoreactivity and by the release of cellular lactate dehydrogenase (LDH). Abeta 25-35 also induced elevation of caspase-3-like Ac-DEVD-MCA cleavage activity in advance of neuronal death with similar concentration-dependency for neuronal death. Inhibitor sensitivity of the Abeta-induced proteolytic activity was similar to that of human recombinant caspase-3. Cleavage of pro-caspase-3 and cleavage of its endogenous substrates, poly (ADP-ribose) polymerase (PARP) and alpha-fodrin, were produced by Abeta-treatment. A caspase-3 inhibitor, Ac-DEVD-CHO, prevented Abeta-induced DNA fragmentation and cleavage of alpha-fodrin, but not of PARP. Caspase inhibitor of broad specificity, Z-VAD-CH(2)-DCB, additionally prevented Abeta-induced cleavage of PARP and some early loss of cell membrane integrity measured by LDH release. However, Abeta-induced condensation of nuclear chromatin and most of the late disintegration of cell membranes were not prevented in the presence of these caspase inhibitors. These results suggest that activation of both caspase-3 and caspase(s) other than caspase-3 play distinct roles in Abeta-induced apoptosis of rat cortical neurons. Furthermore, in the presence of caspase inhibitors, Abeta-induced neuronal death still occurred with different morphological features.

Amyloid beta-Peptides↗
PubMed1999

Cytosolic calcium changes in a process of platelet adhesion and cohesion on a von Willebrand factor-coated surface under flow conditions.

Recent flow studies indicated that platelets are transiently captured onto and then translocated along the surface through interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF). During translocation, platelets are assumed to be activated, thereafter becoming firmly adhered and cohered on the surface. In exploring the mechanisms by which platelets become activated during this process, we observed changes in platelet cytosolic calcium concentrations ([Ca(2+)]i) concomitantly with the real-time platelet adhesive and cohesive process on a vWF-coated surface under flow conditions. Reconstituted blood containing platelets loaded with the Ca(2+) indicators Fura Red and Calcium Green-1 was perfused over a vWF-coated glass surface in a flow chamber, and changes in [Ca(2+)]i were evaluated by fluorescence microscopy based on platelet color changes from red (low [Ca(2+)]i) to green (high [Ca(2+)]i) during the platelet adhesive and cohesive process. Under flow conditions with a shear rate of 1,500 s(-1), no change in [Ca(2+)]i was observed during translocation of platelets, but [Ca(2+)]i became elevated apparently after platelets firmly adhered to the surface. Platelets preincubated with anti-GP IIb-IIIa antibody c7E3 showed no firm adhesion and no [Ca(2+)]i elevation. The intracellular Ca(2+) chelator dimethyl BAPTA did not inhibit firm platelet adhesion but completely abolished platelet cohesion. Although both firm adhesion and cohesion of platelets have been thought to require activation of GP IIb-IIIa, our results indicate that [Ca(2+)]i elevation is a downstream phenomenon and not a prerequisite for firm platelet adhesion to a vWF-coated surface. After platelets firmly adhere to the surface, [Ca(2+)]i elevation might occur through the outside-in signaling from GP IIb-IIIa occupied by an adhesive ligand, thereby leading to platelet cohesion on the surface.

Blood Platelets↗
PubMed1999

Real-time analysis of mural thrombus formation in various platelet aggregation disorders: distinct shear-dependent roles of platelet receptors and adhesive proteins under flow.

We evaluated real-time processes of platelet thrombus formation on a collagen surface in a flow chamber with whole blood from patients with various platelet aggregation disorders, such as Bernard-Soulier syndrome (BSS), Glanzmann's thrombasthenia (GTA), type 3 von Willebrand disease (vWD), and congenital afibrinogenemia (Af), who lack platelet glycoprotein (GP) Ib-IX complex, GP IIb-IIIa, von Willebrand factor (vWF), and fibrinogen, respectively. Blood from GTA patients showed impaired thrombus growth but significant initial platelet-surface interaction under all shear conditions tested (50 to 1,500 s(-1)). By contrast, blood from patients with BSS or type 3 vWD showed no platelet-surface interaction under high shear (>/=1, 210 s(-1)) but normal thrombus formation under low shear (</=340 s(-1)). When shear rate was increased stepwise to 1,500 s(-1) during perfusion, the thrombus growth observed in type 3 vWD or BSS under low shear was arrested, whereas that in control blood was sharply accelerated as a function of shear rate. Overall thrombus formation in Af appeared indistinguishable from that of a control under shear rates between 50 and 1,500 s(-1). However, Af thrombi formed under such conditions collapsed immediately when shear rate was further increased to 4,500 s(-1), whereas thrombi of type 3 vWD or BSS formed under low shear were stable even when shear rate was elevated to 9,000 s(-1) during perfusion. These findings suggest that distinct molecular mechanisms underlie the pathologic bleeding in these diseases and point to the distinct roles of two major adhesive proteins, vWF and fibrinogen. In mural thrombus formation under flow conditions, vWF, perhaps mainly through its interaction with GP Ib-IX, acts as an "initiator and promoter," whereas fibrinogen, via its binding to GP IIb-IIIa, acts as a "stabilizer" against heightened shear forces that could lead to peeling off of platelets from the surface.

Blood Coagulation Disorders↗
PubMed1999

Reconsideration of senescence, immortalization and telomere maintenance of Epstein-Barr virus-transformed human B-lymphoblastoid cell lines.

We review recent data on senescence and immortalization of human B-lymphoblastoid cell lines (LCLs) transformed by the Epstein-Barr virus (EBV). Although EBV-transformed LCLs are generally believed to be immortalized, a series of recent studies, including ours, provided strong evidence that they are mostly mortal and have non-malignant properties, except for a small proportion of LCLs that are immortalized by developing a strong telomerase activity. A large proportion of mortal LCLs have exceptionally long lifespans. Some of them have a lifespan over 150 population-doubling levels, keeping a relatively constant telomere length in spite of the absence of a detectable telomerase activity, suggesting that they maintain telomeres by a pathway other than that using telomerase. Here we propose a model of an alternative pathway to maintain telomeres of such long-lived mortal LCLs by exploiting extra-chromosomal telomere repeat DNA, which was recently found by us.

B-Lymphocytes↗
PubMed1999

Detection by epitope-defined monoclonal antibodies of Werner DNA helicases in the nucleoplasm and their upregulation by cell transformation and immortalization.

We prepared several monoclonal antibodies (mAbs) specific for the NH2- and COOH-terminal regions of the DNA helicase (WRN helicase) responsible for Werner's syndrome known as a premature aging disease. With these antibodies, we detected by immunoblot analysis the endogenous WRN helicase of a relative mass of 180 kD in several lines of cultured cells, but not in patient cells with a defined mutation. Immunocytochemical staining of proliferating fibroblasts and tumor cells showed that the major part of WRN helicase is in the nucleoplasm and not in the nucleolus. Similar experiments with a rat mAb specific to the mouse homologue of human WRN helicase yielded an identical conclusion. Although this nucleoplasmic staining was evident in cells in interphase, the condensed chromatin structure in metaphase was not stained by the same mAbs, suggesting that WRN helicases exist perhaps in a soluble form or bound to the unfolded chromatin structure. From quantitative immunoblot analysis, higher levels of WRN helicase were observed in all transformed cells and tumor cells examined than those of normal cells. The expression of WRN helicase was enhanced consistently in fibroblasts and B-lymphoblastoid cells by transformation with SV-40 and Epstein-Barr virus, respectively, suggesting that rapidly proliferating cells require a high copy numbers of WRN helicase.

Animals↗
PubMed1999

The effect of takusha, a kampo medicine, on renal stone formation and osteopontin expression in a rat urolithiasis model.

Kampo medicine is a traditional Japanese therapeutic system which originated in China and was used to treat various diseases for hundreds of years. Kampo medicine had been also used for the cure and the prevention of urinary calculi for many years, but the effect and the mechanism of this use of kampo medicine are unclear. We examined the inhibitory effect of the kampo medicine takusha on the formation of calcium oxalate renal stones induced by ethylene glycol (EG) and vitamin D3 in rats. We also investigated the effect of takusha on osteopontin (OPN) expression, which we previously identified as an important stone matrix protein. The control group rats were non-treated; the stone group rats were administered EG and vitamin D3, and the takusha group was administered takusha in addition to EG and vitamin D3. The rate of renal stone formation was lower in the takusha group than in the stone group; thus, the OPN expression in the takusha group was smaller than in the stone group. Takusha was effective in preventing oxalate calculi formation and OPN expression in rats. These findings suggest that takusha prevents stone formation including not only calcium oxalate aggregation but also proliferation.

Animals↗
PubMed1999

Expression of bone matrix proteins in urolithiasis model rats.

Urinary calcium stones are a pathological substance, and they show similarities to physiological mineralization and other pathological mineralizations. The expression of messenger (m) RNAs of osteopontin (OPN), matrix Gla protein (MGP), osteonectin (ON) and osteocalcin (OC) in bones and teeth has been described. We previously identified OPN as an important stone matrix protein. In addition, the spontaneous calcification of arteries and cartilage in mice lacking MGP was recently reported, a finding which indicates that MGP has a function as an inhibitor of mineralization. Here, we examined the mRNA expressions of OPN, MGP, ON, and OC in the kidneys of stone-forming model rats administered an oxalate precursor, ethylene glycol (EG) for up to 28 days. The Northern blotting showed that the mRNA expressions of OPN and MGP were markedly increased with the administration of EG, but their expression patterns differed. The OPN mRNA expression reached the maximal level at day 7 after the initiation of the EG treatment and showed no significant difference after 14 and 28 days, whereas the MGP mRNA expression rose gradually to day 28. The in situ hybridization demonstrated that the cell type expressing OPN mRNA was different from that expressing MGP. We suggest that OPN acts on calcification and MGP acts on suppression.

Animals↗
PubMed1999

Immunological diagnosis of Werner syndrome by down-regulated and truncated gene products.

Although immunological methods are widely used to diagnose various infectious diseases, they have rarely been employed to detect genetic diseases. In this study, we have established an immunoblot analysis system for the diagnosis of Werner syndrome (WS), a recessive genetic disorder causing premature aging and an enhanced risk of rare cancers. The method uses an immunoblot technique with specific monoclonal antibodies to WS gene product, and B-lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus; these cell lines express an increased level of normal WS gene product DNA helicase. The method clearly distinguishes normal from patient LCLs containing any of the mutation types found so far in Japan, primarily because of the drastically reduced levels of mutated gene products, and secondarily because of the truncated product sizes. A comparison of this immunological diagnosis with the symptom-based clinical diagnosis has narrowed down the criteria of symptoms essential for WS diagnosis. This procedure is compatible with, and has some advantage over, the genetic method, because WS patients can be diagnosed without determining the mutated gene sequences. The method exemplified in WS may also be applied to detect some other genetic diseases.

Adult↗
PubMed1999

Allosteric effects of four stereoisomers of a fused indole ring system with 3H-N-methylscopolamine and acetylcholine at M1-M4 muscarinic receptors.

We previously demonstrated that brucine and some analogues allosterically enhance the affinity of ACh at muscarinic receptor subtypes M1, M3 or M4. Here we describe allosteric effects at human M1-M4 receptors of four stereoisomers of a pentacyclic structure containing features of the ring structure of brucine. All compounds inhibited 3H-NMS dissociation almost completely at all subtypes with slopes of 1, with similar affinity values at the 3H-NMS-occupied receptor to those estimated from equilibrium assays, consistent with the ternary complex allosteric model. Compound 1a showed positive cooperativity with H-NMS and small negative or neutral cooperativity with ACh at all subtypes. Its stereoisomer, 1b, showed strong negative cooperativity with both 3H-NMS and ACh across the subtypes. Compound 2a was positive with 3H-NMS at M2 and M4 receptors, neutral at M3 and negative at M1 receptors; it was negatively cooperative with ACh at all subtypes. Its stereoisomer, 2b, was neutral with 3H-NMS at M1 receptors and positive at the other subtypes; 2b was negatively cooperative with ACh at M1, M3 and M4 receptors but showed 3-fold positive cooperativity with ACh at M2 receptors. This latter result was confirmed with further 3H-NMS and 3H-ACh radioligand binding assays and with functional assays of ACh-stimulated 35S-GTPgammaS binding. These results provide the first well characterised instance of a positive enhancer of ACh at M2 receptors, and illustrate the difficulty of predicting such an effect.

Acetylcholine↗
PubMed1999

Differential effects of troglitazone and D-chiroinositol on glucosamine-induced insulin resistance in vivo in rats.

Troglitazone and D-chiroinositol have been shown to exert antidiabetic effects by either potentiating or mimicking insulin action. We studied whether pretreatment with these compounds can prevent the deleterious effects of glucosamine on insulin action that may play an important role in hyperglycemia-induced insulin resistance. Normal Wistar rats were pretreated with troglitazone (100 mg/kg/d), D-chiroinositol (100 mg/kg/d), or placebo (saline) for 7 days. Glucosamine (50 micromol/kg/min) was then infused for 210 minutes, and a euglycemic glucose clamp was performed during the last 120 minutes. Pretreatment with troglitazone or D-chiroinositol had no effect on fasting plasma glucose or insulin or basal hepatic glucose output (HGO). Under the euglycemic-hyperinsulinemic (956+/-93 pmol/L) clamp condition, HGO in glucosamine-infused placebo-treated rats was not suppressed, but instead was increased over the basal level, indicative of hepatic insulin resistance. In contrast, HGO failed to increase during glucosamine infusion in rats pretreated with troglitazone but was not normally suppressed. This may indicate a partial improvement in the hepatic insulin resistance. D-Chiroinositol pretreatment had no effect on the glucosamine-induced increase in HGO. The glucose disposal rate (GDR) was 25% lower in rats infused with glucosamine versus saline-infused rats (25.5+/-2.5 v 34.1+/-2.0 mg/kg/min), indicative of peripheral insulin resistance. Pretreatment with D-chiroinositol (34.5+/-2.3 mg/kg/min) prevented the glucosamine-induced decrease in the GDR, indicating an improvement in peripheral insulin resistance. Troglitazone (25.2+/-3.3 mg/kg/min) was without effect. In conclusion, (1) in normal control rats, glucosamine infusion induced hepatic and peripheral insulin resistance; (2) D-chiroinositol, but not troglitazone, pretreatment prevented glucosamine-induced peripheral insulin resistance; and (3) troglitazone, but not D-chiroinositol, partially blocked the glucosamine-induced hepatic insulin resistance. D-Chiroinositol may provide a novel pharmacological approach to hexosamine-induced peripheral insulin resistance.

Animals↗
PubMed1999

Effects of ammonia solution on the gastric mucosa in cirrhotic rats and therapeutic effects of geranylgeranylacetone.

BACKGROUND: We designed an animal model in order to clarify whether Helicobacter pylori infection causes the gastric mucosal lesion frequently seen in cirrhotic patients. METHODS: Ammonia (NH3) solution was given to rats with carbon tetrachloride-induced cirrhosis. The gastric mucosal hexosamine (Hx) content and histopathological findings in the cirrhotic rats were analysed and compared with those of the intact liver rats. Moreover, the usefulness of geranylgeranylacetone (GGA) was investigated in both rat groups. Both rat groups were subdivided according to the treatment as follows: a control group, an NH3 (0.02% solution) group, and an NH3 + GGA (400 mg/kg per day) group; and fed for 4 weeks. RESULTS: The gastric mucosal Hx content of the control group of the cirrhotic rats (16.7 +/- 5.2 microg/mg) was significantly lower than that of the control group of the intact liver rats (26.6 +/- 4.5 microg/mg, P < 0.05). In the cirrhotic rats, the Hx content of both the NH3 (31.9 +/- 13.1 microg/mg) and the NH3 + GGA group (31.9 +/- 9.8 microg/mg) was significantly higher than the Hx content of the control group (P < 0.05). Microscopically, in the cirrhotic rats, while scattered mucosal erosions were recognized in three of five rats of the NH3 group, there were no erosions in any rats of the NH3 + GGA group. CONCLUSIONS: These data suggest that the gastric mucosal defence mechanism is defective in liver cirrhosis and that NH3 enhances this defensive mechanism by acting as mild irritant; however, in some cirrhotics this results in gastric erosion due to excessive irritation. Geranylgeranylacetone protects the gastric mucosa against NH3 irritation in cirrhotics without enhancing the Hx content. Thus, H. pylori infection may be a possible cause of the gastric mucosal lesion in patients with liver cirrhosis. The mechanism of the therapeutic effect of GGA is not due to an enhancement of the gastric mucosal Hx content.

Ammonia↗
PubMed1999

Biological roles of angiotensin II via its type 2 receptor during rat follicle atresia.

Type 1 angiotensin II (ANG II) receptors play crucial roles in the regulation of blood pressure and fluid osmolarity, whereas the physiological roles of type 2 ANG II receptors (AT2) remain unclear. Because AT2 is expressed in atretic follicles where granulosa cells undergo apoptosis, we examined the space and time relationship between AT2 expression and follicle atresia in vivo and the effect of AT2 on follicle-stimulating hormone (FSH) actions in vitro. Binding studies, autoradiography, and RT-PCR of AT2 revealed that the AT2 content in granulosa cells was time dependently increased at both protein and mRNA levels in equine chorionic gonadotropin-treated immature female rats. This increase paralleled the progression of atresia. ANG II suppressed FSH-caused prevention of DNA fragmentation, increases in luteinizing hormone receptor content, and estrogen production through AT2 in cultured granulosa cells. Moreover, FSH-induced stimulation of extracellular signal-regulated kinase activity, critical for cell survival, was inhibited by AT2 stimulation. These results suggest that AT2 mediates the progression of follicle atresia through granulosa cell apoptosis by inhibiting FSH actions.

Angiotensin II↗
PubMed1999

An inhibitor of p38 and JNK MAP kinases prevents activation of caspase and apoptosis of cultured cerebellar granule neurons.

Both p38 mitogen-activated protein kinase (p38) and c-Jun N-terminal kinase (JNK) are known to play important roles in neuronal apoptosis. However, the relationship between these kinases and caspases, another key mediator of apoptosis, is unclear. In the present study, we investigated the possible effects of SB203580 [(4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-i mid azole], an inhibitor of p38, on caspase activation and apoptosis of cultured rat cerebellar granule neurons. In granule neurons, SB203580 prevented apoptosis that was induced by lowering the concentration of KCl in the culture medium for 24 hr. SB203580 also prevented augmentation of caspase-3-like protease activity at 8 hr after the low KCl treatment. The IC50 values of SB203580 for both events were between 3 microM and 10 microM. Expression and phosphorylation of c-Jun, potently induced by low KCl treatment, were prevented by SB203580 at 10 microM. Z-Asp-CH2-DCB, a caspase inhibitor with anti-apoptotic activity, did not inhibit the induction and phosphorylation of c-Jun. Granule neurons displayed high levels of p38 and JNK activities. SB203580 inhibited not only p38 but also JNK activities extracted from granule neurons. These results suggest that activation of c-Jun by p38 and/or JNK mediates the activation of caspase in the low KCl-induced apoptosis in cerebellar granule neurons.

Animals↗
PubMed1999

Further characterization of earthworm serine proteases: cleavage specificity against peptide substrates and on autolysis.

Cleavage specificity of two fibrinolytic enzymes from Lumbricus rubellus [Nakajima, N., et al., Biosci. Biotechnol. Biochem., 57, 1726-1730 (1993) and 60, 293-300 (1996)] was investigated using beta-amyloid 1-40 and oxidized insulin B-chain as peptide substrates. The serine protease, F-III-2, cleaved the former substrate at six sites, and the latter at five sites. F-II digested them at six and ten, respectively. The cleavage specificity of F-III-2 resembled those of both trypsin and chymotrypsin. F-II had a broader specificity than F-III-2 and preferred also the bonds consisting neutral or hydrophobic amino acids. Furthermore, F-III-2 itself was digested initially on the site of Arg(144)-Tyr(145) to produce two peptide fragments, when it was autolyzed regularly by heating.

Amino Acid Sequence↗
PubMed1999

Role of osteopontin in bone remodeling caused by mechanical stress.

Changes in the number and proportion of osteopontin mRNA (Opn) expressing osteocytes and osteoclasts caused by the mechanical stress applied during experimental tooth movement were examined in the present study. Opn expression was detected in the osteocytes on the pressure side at the early stage, and gradually spread to those on the tension side and also to the osteoblasts and bone-lining cells in the alveolar bone. Only 3.3% of the osteocytes located on the pressure side expressed Opn in the interradicular septum of control rats; in contrast, the value was increased to 87.5% at 48 h after the initiation of tooth movement. These results indicate that these cells responded to mechanical stress loaded on the bone with expression of the osteopontin gene. Following the increased expression of Opn in these cells, a 17-fold greater number of osteoclasts compared with the control and numerous resorption pits were observed on the pressure side of the alveolar bone. Injection of arginine-glycine-aspartic acid-serine peptide but not that of arginine-glycine-glutamic acid-serine peptide strongly inhibited the increase in the number of osteoclasts. Furthermore, an in vitro migration assay demonstrated the chemotactic activity of osteopontin (OPN) on the precursor of osteoclasts. Our study strongly suggests that OPN is an important factor triggering bone remodeling caused by mechanical stress.

Animals↗