Biomedical subjects

K Fujitani

Publications and source records attributed to K Fujitani.

At least 37 records · Page 2Linked to original sources

PubMed1997

Identification of mu-, m-calpains and calpastatin and capture of mu-calpain activation in endothelial cells.

The presence of the calpain-calpastatin system in human umbilical vein endothelial cells (HUVEC) was investigated by means of ion exchange chromatography, Western blot analysis, and Northern blot analysis. On DEAE anion exchange chromatography, calpain and calpastatin activities were eluted at approximately 0.30 M and 0.15-0.25 M NaCl, respectively. For half-maximal activity, the protease required 800 microM Ca2+, comparable to the Ca2+ requirement of m-calpain. By Western blot analysis, the large subunit of mu-calpain (80 kDa) was found to be eluted with calpastatin (110 kDa). Both the large subunit of m-calpain (80 kDa) and calpastatin were detected in the respective active fractions. By Northern blot analysis, mRNAs for large subunits of mu- and m-calpains were detected in single bands, each corresponding to approximately 3.5 Kb. Calpastatin mRNA was observed in two bands corresponding to approximately 3.8 and 2.6 Kb. Furthermore, the activation of mu-calpain in HUVEC by a calcium ionophore was examined, using an antibody specifically recognizing an autolytic intermediate form of mu-calpain large subunit (78 kDa). Both talin and filamin of HUVEC were proteolyzed in a calcium-dependent manner, and the reactions were inhibited by calpeptin, a cell-permeable calpain specific inhibitor. Proteolysis of the cytoskeleton was preceded by the appearance of the autolytic intermediate form of mu-calpain, while the fully autolyzed postautolysis form of mu-calpain (76 kDa) remained below detectable levels at all time points examined. These results indicate that the calpain-calpastatin system is present in human endothelial cells and that mu-calpain may be involved in endothelial cell function mediated by Ca2+ via the limited proteolysis of various proteins.

Antibody Specificity↗

PubMed1997

Calpain activation in shear-induced platelet aggregation.

Fluid shear stress has been known to activate platelet reaction such as aggregation, but the exact mechanism of shear-induced platelet aggregation (SIPA) has not been fully understood. Calpain, an intracellular calcium-activated cysteine protease, is abundant in platelets and is considered to be activated and involved in the proteolytic processes during platelet activation. A possible activation of calpain in SIPA was investigated, employing a newly developed aggregometer and specific monoclonal antibodies to detect activation of calpain. When a shear stress gradient varying between 6 and 108 dyn/cm2 was applied to platelets, activation of mu-calpain was observed only in high-shear-stressed platelets, resulting in the proteolysis of talin. At 1 min after the onset of constant high shear stress of 108 dyn/cm2, mu-calpain activation and proteolysis of talin were detected and increased in a time-dependent manner. Constant shear stress more than 50 dyn/cm2, applied for 5 min, caused mu-calpain activation and proteolysis of talin, which were increased in a shear-force-dependent manner. Calpeptin, a calpain-specific peptide antagonist, caused the complete inhibition of both mu-calpain activation and proteolysis of talin, while SIPA profiles with calpeptin showed almost no change compared to those without calpeptin. These results suggest the possibility of calpain involvement in late phases of shear-induced platelet activation such as cytoskeletal reorganization.

Calpain↗

PubMed1997

Direct extracranial-intracranial bypass for children with moyamoya disease.

To improve cerebral hypoperfusion in the ischemic type of Moyamoya disease, we have applied superficial temporal artery-middle cerebral artery (STA-MCA) double anastomoses in combination with encephalo-myo-synangiosis (EMS) for 19 hemispheres of 10 children (age from 5 to 11 years at surgery). Two branches of the STA were anastomosed to the two cortical arteries which were selected in the watershed area of the cerebral hemisphere estimated as a hypoperfusion area on the preoperative angiograms. Before surgery transient ischemic attacks (TIAs) developed from every month to every 6 months in association with hyperventilation or sobbing. No perioperative completed stroke or wound complications was observed, although single TIA developed in four patients within 1 month after surgery. Postoperative angiogram demonstrated that, not only the preoperative watershed area, but also the most of the middle cerebral artery territory was oppacified via the 2 branches of the STA in all 19 hemispheres. In a mean follow-up period of 4 years, no ischemic episode was induced by hyperventilation, and there was no mental or neurological deterioration. STA-MCA double anastomoses, to the cerebral watershed area, in combination with EMS are safe and effective even for younger children with Moyamoya disease.

Anastomosis, Surgical↗

PubMed1996

Sexual orientation in Drosophila is altered by the satori mutation in the sex-determination gene fruitless that encodes a zinc finger protein with a BTB domain.

We have isolated a new Drosophila mutant, satori (sat), the males of which do not court or copulate with female flies. The sat mutation comaps with fruitless (fru) at 91B and does not rescue the bisexual phenotype of fru, indicating that sat is allelic to fru (fru(sat)). The fru(sat) adult males lack a male-specific muscle, the muscle of Lawrence, as do adult males with other fru alleles. Molecular cloning and analyses of the genomic and complementary DNAs indicated that transcription of the fru locus yields several different transcripts. The sequence of fru cDNA clones revealed a long open reading frame that potentially encodes a putative transcription regulator with a BTB domain and two zinc finger motifs. In the 5' noncoding region, three putative transformer binding sites were identified in the female transcript but not in male transcripts. The fru gene is expressed in a population of brain cells, including those in the antennal lobe, that have been suggested to be involved in determination of male sexual orientation. We suggest that fru functions downstream of tra in the sex-determination cascade in some neural cells and that inappropriate sexual development of these cells in the fru mutants results in altered sexual orientation of the fly.

Alleles↗

PubMed1996

Hemostasis activation during sclerotherapy of lower extremity varices.

The influence of compression sclerotherapy upon hemostasis activation was investigated in 41 consecutive patients with lower extremity varices by serial measurement of thrombin-antithrombin III complexes (TAT), D-dimer, fibrinogen and C-reactive protein (CRP). Blood sampling was carried out before operation and on the 7th and 28th post-operative day in patients randomly assigned to either the control group (n = 18), in which high ligation of sapheno-femoral junction and local excision of varices were performed, or the sclerotherapy group (n = 23) in which the comparable surgical intervention and compression sclerotherapy using hypertonic saline were performed simultaneously. In both groups, the TAT, D-dimer and fibrinogen concentrations at day 7 were significantly elevated compared to the value before operation while CRP showed no significant change during the observation period. In the sclerotherapy group, higher incidence of superficial thrombosis was observed and the TAT concentration at day 7 was significantly higher than that in the control group (p < 0.01), and the TAT at day 28 was still significantly elevated compared to the pre-operative level (p < 0.05). However, no relationship between TAT and D-dimer concentrations and the extent of superficial thrombosis was observed. We conclude that compression sclerotherapy for lower extremity varices causes latent activation of coagulation system and can be a risk factor for venous thromboembolism.

Antithrombin III↗

PubMed1996

Separate analysis of nuclear and cytosolic Ca2+ concentrations in human umbilical vein endothelial cells.

Ca2+ concentration inside human umbilical vein endothelial cells was studied separately in cytosol and nucleus by a confocal laser scanning microscopy using fluo-3. The in vivo calibration curve for cytosol and nucleus showed good linearity between fluorescence intensity and Ca2+ concentration in cytosol ([Ca2+]i) and nuclei ([Ca2+]n). After calibration, [Ca2+]n was constantly higher than [Ca2+]i before and after the chelation of extracellular Ca2+ suggesting an active Ca2+ accumulation system on nuclear membrane. [Ca2+]n was also constantly higher than [Ca2+]i after the stimulation of thrombin (0.05 U/ml), FCS (10%), and thapsigargin (Tsg, 1 microM). The temporal change of [Ca2+]n and [Ca2+]i was identical, and [Ca2+]i gradient towards the nucleus and peripheral or central [Ca2+]n rise was observed after these stimulations. From these results, [Ca2+]n is not only regulated by the active Ca2+ accumulation system on nuclear membrane at rest but also the generation of inositol-triphosphate. FCS caused heterogeneous [Ca2+]n or [Ca2+]i rise from cell to cell; single spike or oscillatory change of [Ca2+]n and [Ca2+]i was observed in about 56% of cells, which were completely abolished by the chelation of extracellular Ca2+, suggesting that FCS stimulated [Ca2+]n and [Ca2+]i rise solely depending on Ca2+ influx from extracellular medium. The higher concentration of [Ca2+]n and heterogeneous [Ca2+]n rise may have important roles in nuclear-specific cellular responses.

Aniline Compounds↗

PubMed1996

A new mutation of the L1CAM gene in an X-linked hydrocephalus family.

X-linked hydrocephalus is a genetic form of hydrocephalus that frequently occurs in females. It is characterized by ventricular dilatation, mental retardation, deformity of the thumb and spastic paraparesis. Recently, 23 different mutations of the gene for the neural cell adhesion molecule, L1CAM, located at chromosome region Xq28, have been reported, 16 of which were detected in families with X-linked hydrocephalus. We sequenced the coding region of the L1CAM gene of patients from two different families with X-linked hydrocephalus and found a novel mutation at nucleotide residue 1963 in one family. This mutation from adenine to guanine results in an amino acid change from lysine to glutamic acid at residue 655 of the L1CAM protein, which belongs to the fibronectin type III domain. We report another method of the rapid identification of the mutation based on the polymerase chain reaction. This mutation was not detected among 70 X chromosomes from a healthy population. Ours is the first report demonstrating this gene mutation in X-linked hydrocephalus in an Asian population. Our findings further emphasize the evolving genotypic heterogeneity in X-linked hydrocephalus.

Adolescent↗

PubMed1996

Genetic dissection of sexual orientation: behavioral, cellular, and molecular approaches in Drosophila melanogaster.

Insertional mutagenesis using P-element vectors yielded several independent mutations that cause male homosexuality in Drosophila melanogaster. Subsequent analyses revealed that all of these insertions were located at the same chromosomal division, 91B, where one of the inversion breakpoints responsible for the bisexual phenotype of the fruitless (fru) mutant has been mapped. In addition to the altered sexual orientation, the fru mutants displayed a range of defects in the formation of a male-specific muscle, the muscle of Lawrence. Since the male-specific formation of this muscle was dependent solely on the sex of the innervating nerve and not on the sex of the muscle itself, the primary site of action of the fru gene should be in the neural cells. satori, one of the P-insertion alleles of fru which we isolated, carried the lacZ gene of E. coli as a reporter, and beta-galactosidase expression was found in a subset of brain cells including those in the antennal lobe in the satori mutant. Targeted expression of a sex determination gene, transformer (tra), was used to produce chromosomally male flies with certain feminized glomeruli in the antennal lobe. Such sexually mosaic flies courted not only females but also males when the DM2, DA3 and DA4 glomeruli were feminized, indicating that these substructures in the antennal lobe may be involved in the determination of the sexual orientation of flies. Molecular cloning and analyses of the genomic and complementary DNAs indicated that transcription of the fru locus yields several different transcripts, one of which encodes a putative transcription regulator with a BTB domain and two zinc finger motifs. In the 5' non-coding region, three putative Transformer binding sites were identified. It appears plausible therefore that the fru gene is one of the elements in the sex determination cascade that controls sexual fates of certain neuronal cells. Improper sex determination in these neural cells may lead to altered sexual orientation and malformation of the male-specific muscle. Some implications of the results of our study on sexual orientation in other organisms will be discussed based on the Drosophila research.

Amino Acid Sequence↗

PubMed1996

Calpain activation in plasma membrane bleb formation during tert-butyl hydroperoxide-induced rat hepatocyte injury.

BACKGROUND & AIMS: The mechanism of plasma membrane blebbing (dissociation of the lipid bilayer from the membrane cytoskeleton) in hepatocyte injury is not known. The aim of this study was to investigate the role of calpain, a calcium-dependent cytosolic protease, in bleb formation induced by oxidative stress. METHODS: Hepatocytes from Wistar rats were injured with tertbutyl hydroperoxide in the presence or absence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or a specific calpain inhibitor, calpeptin (Z-Leu-nLeu-H). Bleb formation was examined by phase-contrast and transmission electron microscopies. Intracellular calcium concentration was measured using Fura-2. Western blot analyses were performed for cytoskeletal proteins (talin, alpha-actinin, and vinculin) and the intermediate (activated) and proactivated forms of calpain mu. RESULTS: tert-Butyl hydroperoxide induced a sustained increase in intracellular calciu, bleb formation, and, ultimately, hepatocyte death. Talin and alpha-actinin were degraded in a time-dependent manner, although no apparent changes of actin filament were observed. Before the cytoskeletal protein degradation, the intermediate form of calpain mu appeared as its proactivated form decreased. In addition, calpeptin or EGTA inhibited not only calpain mu activation but also cytoskeletal protein degradation and bleb formation. CONCLUSIONS: In tert-butyl hydroperoxide-treated hepatocytes, the activation of calpain promotes membrane blebbing via degradation of cytoskeletal proteins.

Animals↗

PubMed1995

Involvement of protein phosphatase-1 in cytoskeletal organization of cultured endothelial cells.

The phosphorylation and dephosphorylation of cytoskeletal proteins regulate the shape of eukaryotic cells. To elucidate the role of serine/threonine protein phosphatases (PP) in this process, we studied the effects of calyculin A (CLA), a potent and specific inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A) on the cytoskeletal structure of cultured human umbilical vein endothelial cells (HUVECs). The addition of CLA (5 min) caused marked alterations in cell morphology, such as cell constriction and bleb formation. Microtubules and F-actin were reorganized, becoming markedly condensed around the nucleus. Although the fluorescence intensity of phosphoamino acids was not significantly different according to immunocytochemistry between cells with and without CLA, polypeptides of 135, 140, 158, and 175 kDa were specifically phosphorylated on serine and/or threonine residues. There was no significant effect on tyrosine residues. The effects of CLA on cytoskeletal changes and protein phosphorylation were almost completely inhibited by the non-selective kinase inhibitor, K-252a. The effect of CLA on cell morphology was at least 100 times more potent than that of okadaic acid, consistent with the inhibitory potency against PP-1. The catalytic subunit of PP-1 was also identified in HUVECs by Western blotting with its monoclonal antibody antibody. These results suggest that PP-1 is closely involved in sustaining the normal structure of the cytoskeleton.

Actins↗

PubMed1995

Clinical evaluation on combined administration of oral prostacyclin analogue beraprost and phosphodiesterase inhibitor cilostazol.

Among various oral antiplatelets, a combination of a novel prostacyclin analogue beraprost (BPT) and a potent phosphodiesterase inhibitor cilostazol (CLZ) may result in untoward clinical effects due to possible synergistic elevation of intracellular cAMP (cyclic adenosine 3',5'-monophosphate). Thereby, a clinical study of the combined administration of the two agents was attempted. Twelve healthy volunteers were assigned to take BPT/CLZ in the following schedule; BPT: 40 micrograms at day 1 and 120 micrograms t.i.d. from day 7 to 14, CLZ: 200 mg t.i.d. from day 3 to 14. At various time intervals, physical examination and blood collection for ex vivo platelet aggregation and determination of intraplatelet cAMP were performed. Throughout the observation period, no significant alteration in vital signs was observed. Seven out of 12 subjects experienced headache of a short duration accompanying facial flush in one and nausea in one, especially after ingestion of CLZ. All of these symptoms, probably caused by the vasodilating effect of the two agents, were of mild degree and no special treatment was required. Intraplatelet cAMP content was gradually but significantly increased to 9.84 +/- 4.59 pmol per 10(9) platelets at day 14 in comparison with the initial value (6.87 +/- 2.25 pmol). The platelet aggregability was significantly suppressed at various time intervals but no additive or synergistic inhibitory effect by the combined administration was noted. In conclusion, the combined administration of BPT/CLZ is safe at doses used in the study, though the beneficial clinical effect of the combined administration has yet to be elucidated.

Adult↗

PubMed1995

A clinical and neuroradiological study of X-linked hydrocephalus in Japan.

To clarify the clinicopathological features of X-linked hydrocephalus, the authors studied 30 affected males from 15 families. In utero ultrasonography, performed at 21 to 40 weeks of gestation, revealed 18 fetuses with hydrocephalus. Computerized tomography (CT) revealed bilateral enlargement of the lateral ventricle with preponderant dilation of the posterior horn. In five patients with complete magnetic resonance (MR) imaging data, the most specific finding was localized atrophy of the anterior vermian lobe. Other MR imaging findings included a large massa intermedia, flat corpora quadrigemina, a small brainstem, and diffuse hypoplasia of the cerebral white matter. In all cases, the corpus callosum was hypoplastic or aplastic. The aqueduct was patent in four of five cases. Asymmetrical reduction of the ventricular size and a rippled ventricular wall were characteristic postshunt CT findings. Progressive macrocephaly and symptoms due to increased intracranial pressure were ameliorated by the shunt; however, the neurological outcome was not improved by shunting. Of 14 patients who lived to be between 2 and 18 years of age, all are retarded. These results indicate that X-linked hydrocephalus is not a disease of simple ventriculomegaly due to aqueduct stenosis alone but involves other complicated central nervous system anomalies.

Abnormalities, Multiple↗

PubMed1994

Cerebrospinal fluid edema associated with shunt obstruction.

The authors report four hydrocephalic children with cerebrospinal fluid (CSF) edema extending along the ventricular catheter of an obstructed CSF shunt. Three of the patients exhibited massive CSF edema along the ventricular catheter, yet they manifested neither ventricular enlargement nor apparent periventricular CSF edema despite increased intraventricular pressure. These findings suggested ventricular tautness. The remaining patient, who had dilated ventricles with periventricular CSF edema, displayed CSF edema in a limited area along the ventricular catheter. Replacement of the obstructed peritoneal catheter of the shunt resulted in rapid improvement of the edema in all patients. In the three patients with massive CSF edema, however, a small lesion remained in the subcortical white matter along the ventricular catheter as demonstrated by computerized tomography and/or magnetic resonance imaging 3 to 5 years after shunt revision. It is concluded that shunt obstruction may result in massive CSF edema along the ventricular catheter in hydrocephalic children who have ventricular tautness after installation of the shunt causing irreversible although usually asymptomatic damage to the affected area of the brain.

Adult↗

PubMed1993

Different target-site specificities of the hairpin ribozyme in cis and trans cleavages.

The hairpin ribozyme cleaves a phosphodiester bond at the 5' side of a 5'GUC3' sequence of an RNA with high efficiency. An RNA having a 5'GUA3' sequence instead of the GUC sequence is a poor substrate for this ribozyme. Here, we show that this is indeed so in a trans-acting ribozyme system, but in a cis-acting ribozyme system this ribozyme cleaves the 5' side of a GUA sequence as efficiently as the wild-type cleaves the GUC sequence. One base substitution in the ribozyme also affected the target-site specificity in the cis-acting system.

Base Sequence↗

PubMed1993

The effects of calpeptin (a calpain specific inhibitor) on agonist induced microparticle formation from the platelet plasma membrane.

Platelets activated by various agonists produce formation of vesicles shed from the plasma membrane (microparticles). However, the mechanism of microparticle (MP) formation has not been clarified yet. The aim of the present study was to determine the possibility of involvement of calpain (a Ca(2+)-dependent thiol protease) in MP formation. Washed platelets preincubated with calpeptin, a cell permeable calpain specific inhibitor, or with a vehicle were activated by thrombin plus collagen or by calcium ionophore A23187. Flow cytometry was used to detect the amount of microparticle formation by using murine monoclonal antibodies against GP IIb-IIIa or GP IIb and fluorescein 5-isothiocyanate labeled goat anti-mouse IgG. MP formation stimulated either by thrombin plus collagen or by A23187 was inhibited by calpeptin in a dose dependent manner. The microparticle formation from platelets activated by A23187 reached a plateau in approximately 5 min after activation, whereas that from platelets activated by thrombin plus collagen reached a plateau at 30 min following the stimulation. These time sequences corresponded well with those of degradation of actin-binding protein (ABP), a well known substrate of calpain, of platelets activated by these two stimulations. However, the inhibition of MP formation by calpeptin was more marked in the early stage (within 10 min) than in the late stage (after 30 min) of platelet activation. At 30 min after platelet activation by either two stimulations, a significant amount of microparticle formation was observed in the presence of 30 microM calpeptin, which inhibited hydrolysis of ABP almost completely. Our data suggest the involvement of calpain in the early stage (especially within 10 min) of microparticle formation.

Antigens, Human Platelet↗

PubMed1993

Calyculin A inhibits the exposure of fibrinogen receptor in thrombin-stimulated platelets.

Calyculin A (CLA) and okadaic acid (OA), specific and potent inhibitors of protein phosphatase 1/2A, inhibit platelet aggregation. However, their inhibitory mechanisms remain unknown. We investigated the effects of CLA on the exposure of fibrinogen receptor in thrombin-stimulated platelets, using flow cytometry with a monoclonal antibody against the fibrinogen receptor of activated glycoprotein(Gp)IIb/IIIa complex (PAC-1). CLA inhibited the exposure of fibrinogen receptor in a dose related manner when added either before or 3 min after thrombin stimulation. In contrast, CLA had no significant effect when the expression of GpIIb/IIIa complex was examined in resting platelets, using a monoclonal antibody recognizing non-activated GpIIb/IIIa complex (NNKY1-32). These results suggest that protein phosphatase 1/2A may be directly involved in the exposure of platelet fibrinogen receptor.

Adult↗

PubMed1993

The possible involvement of protein phosphatase 1 in thrombin-induced Ca2+ influx of human platelets.

Protein phosphatase 1 is considered to be involved in thrombin-induced platelet activation (Murata et al., Biochem Int 26:327-334, 1992). To clarify the mechanism, we examined the effects of protein phosphatase 1 and 2A inhibitors (calyculin A, tautomycin, okadaic acid) on Ca2+ influx. In the presence of 1 mM Ca2+, thrombin- (0.1 U/ml) induced platelet aggregation and ATP release were inhibited by calyculin A, while this inhibitory effect was abolished in the absence of Ca2+ (EGTA 1 mM). Furthermore, thrombin-induced Mn2+ influx but not intracellular Ca2+ mobilization was inhibited by calyculin A in a dose-related manner. Calyculin A also blocked the ongoing Ca2+ influx when added 3 min after thrombin stimulation. Similar inhibitory effects were observed with okadaic acid and tautomycin in the same potency sequence as the reported one for protein phosphatase 1 (calyculin A > tautomycin > okadaic acid). These results suggest that the anti-platelet effects of phosphatase inhibitors are due to the inhibition of Ca2+ influx and that protein phosphatase 1 plays a key role in the regulation of receptor operated Ca2+ channel of human platelets.

Antifungal Agents↗

PubMed1993

[Quantitative evaluation of 111In-antimyosin Fab myocardial imaging in acute myocardial infarction patients].

Quantitative evaluation of 111In-antimyosin Fab myocardial (InAM) imaging was performed in 15 patients with acute myocardial infarction to evaluate which organ is the most appropriate for the control of myocardial accumulation, to compare the quantitative method with the conventional visual method, and to study which clinical indices correlate with the InAM quantitative evaluations. InAM images demonstrated the myocardium with 31 +/- 6 mean counts/pixel, lung with 14 +/- 4, upper mediastinum with 20 +/- 5, middle mediastinum with 26 +/- 5, and liver with 75 +/- 10. We considered the lung to be the most appropriate control organ for quantitative evaluations of InAM imaging, because it could be separated from the myocardium, and the measurement range was narrow. The InAM uptake index [IUI = (myocardial counts-lung counts)/lung counts] was calculated as the index of myocardial accumulation. Visual evaluations of myocardial accumulation on InAM images were classified into three grades. The IUI of grade 1 (slight) was 0.98 +/- 0.19, grade 2 (moderate) was 1.34 +/- 0.38, and grade 3 (severe) was 1.97 +/- 0.19. Visual grading was nearly in accordance with the IUI, although it was difficult to distinguish visually between grades 1 and 2. Measurement of wall motion by left ventriculography showed that reduced wall motion was associated with an IUI of 1.01 +/- 0.18 and dyskinesis with an IUI of 1.92 +/- 0.16, showing IUI can indicate regional myocardial damage. However, IUI was not correlated with indices of the overall left ventricular function, such as ejection fraction, cardiac index, and peak creatine kinase level.

Antibodies, Monoclonal↗