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Biomedical subjects

T Okazaki

Publications and source records attributed to T Okazaki.

At least 145 records · Page 8Linked to original sources
PubMed1997

Involvement of protein tyrosine kinase p72syk and phosphatidylinositol 3-kinase in CD2-mediated granular exocytosis in the natural killer cell line, NK3.3.

The granular exocytosis pathway is one mechanism by which NK cells and CTLs induce cytolysis of target cells. Triggering through adhesion molecules such as CD2 and LFA-1 as well as Fc gammaRIII (CD16) can invoke this pathway. CD2 is a cell surface glycoprotein present on CTLs and NK cells that plays an important role in both cellular adhesion and signal transduction. Here we report that cross-linking of CD2 as well as CD16 by immobilized Abs enhances granular exocytosis in an NK cell line, NK3.3. Herbimycin, a protein tyrosine kinase (PTK) inhibitor, or wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI 3-K), inhibited completely or almost completely CD2- or CD16-mediated granular exocytosis, suggesting the involvement of protein tyrosine kinases and PI 3-K in both CD2- and CD16-mediated granular exocytosis. We also observed that cross-linking of CD2 as well as CD16 enhances p72syk tyrosine kinase activity, and this enhancement correlated well with the increased tyrosine phosphorylation of several cellular proteins, including the adapter protein Shc. Furthermore, we have observed that cross-linking of CD2 as well as CD16 enhances the PI 3-K activity associated with the tyrosine-phosphorylated cellular proteins and Shc. These results provide insight into the signaling pathways by which triggering of CD2 and CD16 on NK cells leads to cytolysis of target cells.

Androstadienes↗
PubMed1997

Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8.

The activation of multiple interleukin-1beta converting enzyme-related proteases (caspases) in apoptotic mammalian cells raises questions as to whether the multiple active caspases have distinct roles in apoptotic execution as well as how these proteases are organized in apoptotic signaling pathways. Here we used an affinity-labeling agent, YV(bio)KD-aomk, to investigate the caspases activated during apoptotic cell death. YV(bio)KD-aomk identified six distinct polypeptides corresponding to active caspases in Fas-stimulated Jurkat T cells. On staurosporine treatment, four polypeptides were detected. Competition experiments showed that the labeled caspases have distinct substrate preferences. Stepwise appearance of the labeled caspases in each cell death event was consistent with the view that the activated caspases are organized into protease cascades. Moreover, we found that stepwise activation of caspases similar to that induced by Fas ligation is triggered by exposing non-apoptotic Jurkat cell extracts to caspase-8 (MACH/FLICE/Mch5). Conversely, CrmA protein, a viral suppressor of Fas-induced apoptosis, inhibited the protease activity of caspase-8. Overall, these findings provide evidence that caspase-8, a CrmA-sensitive protease, is responsible for initiating the stepwise activation of multiple caspases in Fas-stimulated cells.

Affinity Labels↗
PubMed1997

Self-rating Barthel index compatible with the original Barthel index and the Functional Independence Measure motor score.

To confirm concurrent validity of the final revision of the self-rating Barthel index (SB) and its test-retest reliability, we investigated 171 stroke outpatients without severe aphasia or dementia who were seen by a doctor on predetermined days at eight different hospitals. For 41 patients, the differences in scores among the original Barthel index (BI) and Granger's BI and SB were examined by the Friedman two-way analysis of variance and Wilcoxon matched-pairs signed-ranks test, and the difference in scores between Functional Independence Measure motor score (FIM-MS) and three-level scale FIM-MS was determined by the Wilcoxon's matched-pairs signed-ranks test. Concurrent validity of the SB was confirmed by Spearman's correlation coefficients with the original BI and FIM-MS, and internal consistency of the five measuring instruments was examined. For all 171 patients test-retest reliability of the SB was examined with the Spearman's correlation coefficient for total scores and kappa coefficients for each ADL item. Regression analysis was performed to determine what factors were related with test-retest reliability. Total scores of the SB and Granger's BI and the three-level scale FIM-MS were significantly higher than those of the original BI and FIM-MS, respectively. Correlation coefficients of the SB with the original BI and FIM-MS were 0.994 and 0.904, respectively, and its alpha-coefficient was 0.842. Test-retest reliability of the total score was 0.835 by the correlation coefficient, and kappa coefficients of 1 and 12 ADL items were fair and good, respectively. Regression analysis revealed that self-rating by a patient with a high SB total score is more accurate. Therefore, the SB has good concurrent validity and is well-related with the original BI and FIM, and its test-retest reliability is sufficiently high for practical use.

Activities of Daily Living↗
PubMed1997

Analysis of the affinity of each haptoglobin polymer for hemoglobin by two-dimensional affinity electrophoresis.

Using polyacrylamide gel electrophoresis we have often encountered a decrease in the high molecular weight polymers of haptoglobin 2-1 in the serum of patients after transfusion and with hemolytic diseases. Using two-dimensional affinity electrophoresis we have attributed this phenomenon to the high affinity of high molecular weight polymers of the haptoglobin 2-1 for hemoglobin.

Biopolymers↗
PubMed1997

Identification of a predominant replication origin in fission yeast.

We have identified five autonomously replicating sequences (ARSs) in a 100 kbp region of the Schizosaccharomyces pombe chromosome II. Analyses of replicative intermediates of the chromosome DNA by neutral/neutral two-dimensional gel electrophoresis demonstrated that at least three of these ARS loci operate as chromosomal replication origins. One of the loci,ori2004, was utilized in almost every cell cycle, while the others were used less frequently. The frequency of initiation from the respective chromosomal replication origin was found to be roughly proportional to the efficiency of autonomous replication of the corresponding ARS plasmid. Replication from ori2004 was initiated within a distinct region almost the same as that for replication of the ARS plasmid. These results showed that the ori2004 region of approximately 3 kbp contains all the cis elements essential for initiation of chromosome replication.

Chromosomes, Fungal↗
PubMed1997

Ceramide-induced translocation of protein kinase C-delta and -epsilon to the cytosol. Implications in apoptosis.

Ceramide is now recognized as an intracellular lipid signal mediator, which induces various kinds of cell functions including apoptosis. Ceramide-induced apoptosis was reported to be blocked by 12-O-tetradecanoylphorbol 13-acetate, a protein kinase C (PKC) activator, but its mechanism remained unclear. Therefore, we investigated whether ceramide has any effects on PKC in the induction of apoptosis. We here report that N-acetylsphingosine (synthetic membrane-permeable ceramide) induced translocation of PKC-delta and -epsilon isozymes from the membrane to the cytosol within 5 min in human leukemia cell lines. Treatment with sphingomyelinase, tumor necrosis factor-alpha, or anti-Fas antibody, all of which can induce apoptosis by generating natural ceramide, similarly induced cytosolic translocation of PKC-delta and -epsilon. In Fas-resistant cells anti-Fas antibody did not induce cytosolic translocation of PKC-delta and -epsilon because of no generation of ceramide, whereas N-acetylsphingosine induced apoptosis with cytosolic translocation of PKC-delta and -epsilon. Furthermore, both 12-O-tetradecanoylphorbol 13-acetate and a nonspecific kinase inhibitor, staurosporine, prevented ceramide-induced apoptosis by inhibiting cytosolic translocation of PKC-delta and -epsilon. These data suggest that cytosolic translocation of PKC-delta and -epsilon plays an important role in ceramide-mediated apoptosis.

Antibodies, Monoclonal↗
PubMed1997

Mitochondrial DNA sequence analysis of the masu salmon--phylogeny in the genus Oncorhynchus.

We determined 2162-bp sequences from the mitochondrial genome of the masu salmon Oncorhynchus masou masou, chum salmon Oncorhynchus keta, and Atlantic salmon Salmo salar by using polymerase chain reactions. These DNA sequences span from the 3' region of the gene for ATPase subunit 6 to the 5' region of the gene for NADH dehydrogenase subunit 4L. With the aid of the sequence data from other Pacific salmonid species (Thomas and Beckenbach, 1989, J. Mol. Evol. 29: 233-245), the evolutionary distances among the masu salmon and other species in the genus Oncorhynchus were calculated. These evolutionary distances were then used to construct a neighbor-joining tree. Further, a maximum parsimony tree was constructed. The evolutionary trees that were obtained suggest that masu salmon first diverged from the common ancestor of the genus Oncorhynchus.

Animals↗
PubMed1997

Pulmonary expression of vascular endothelial growth factor and myosin isoforms in rats with congenital diaphragmatic hernia.

Abnormalities of the pulmonary vasculature are well documented in cases of congenital diaphragmatic hernia (CDH). Vascular endothelial growth factor (VEGF), an angiogenic factor, is a recently described endothelial cell-specific growth factor. Myosin heavy chain (MHC) isoforms such as SMemb, SM1 and SM2 are important molecular markers used to study vascular smooth muscle cell differentiation. SMemb is predominantly expressed in immature smooth muscle cells (SMC), and SM2 is expressed in mature SMCs. The authors investigated the expression of VEGF and SMC differentiation in pulmonary vessels in CDH rat lungs and in controls. The lungs of nitrofen-induced CDH rat fetuses (n = 16, gestational age 16, 18, 20, and 22 days) were stained immunohistochemically using antibodies against VEGF, SMemb and SM2, while alpha-actin was used as a general marker of vascular smooth muscle cells. In the CDH group VEGF expression was negative in pulmonary vessels before birth, and in the control group VEGF was positive in smooth muscle cells in vessel walls from 20 days both in vessels at the hilum and in pulmonary parenchyma. In both control and CDH groups, SMemb expression was positive from 16 days' gestation. SM2 expression was negative in vessel walls during the prenatal period in both groups. Alpha-actin was localized in both lungs obtained from control and CDH groups in the lung hilum from 16 days and around peripheral vessels from 18 days. Differences in vascular smooth muscle cell differentiation were not observed between control and CDH lung. These findings suggest that differences in pulmonary vascular development exist between control and CDH rats for VEGF expression, and maturational differences in smooth muscle cell differentiation are not present. This role of altered endothelial cell growth might be related to the different pulmonary vascular reactivity present in CDH lungs.

Animals↗
PubMed1997

Antianginal effects of YM430, a novel calcium entry-blocking and beta-adrenoceptor-blocking agent in several experimental angina models.

1. We evaluated the antianginal effects of YM430 in several experimental models in vitro and in vivo. 2. In isolated dog coronary artery, YM430 (10(-8)-10(-6) M) inhibited 3,4-diaminopyridine-induced rhythmic contractions with an IC50 value of 59.2 nM. 3. In anesthetized rats, YM430 (10-100 mg/kg PO) inhibited arginine vasopressin-induced ST-segment depression with an IC50 value of 36.6 mg/kg PO. 4. In anesthetized dogs, YM430 (0.3 mg/kg IV) significantly inhibited ST-segment elevation induced by coronary artery occlusion. 5. These findings suggest that YM430 may be of value in the treatment of various types of angina pectoris such as variant and stable angina.

4-Aminopyridine↗
PubMed1997

Ligand-induced down-regulation of Trk messenger RNA, protein and tyrosine phosphorylation in rat cortical neurons.

Chronic exposure of brain neurons to nerve growth factor in vitro and in vivo results in increased levels of the nerve growth factor receptor TrkA. In contrast, in the present study, we have found that chronic exposure of rat embryonic cortical neurons to brain-derived neurotrophic factor (BDNF) leads to a pronounced reduction of the levels of protein and messenger RNA for the full-length but not the truncated BDNF receptor TrkB. Similar effects were observed with the other TrkB ligands neurotrophin-3 and neurotrophin-4/5. After pretreatment with BDNF, neurotrophin-3 or neurotrophin-4/5, subsequent tyrosine phosphorylation responses of the remaining Trks to the same factors were greatly reduced. Three days exposure of rat embryonic cortical neurons to BDNF induced an absolute refractory period of several hours, with no subsequent response to the same factor. Similar but less pronounced refractory effects were observed with neurotrophin-3 and neurotrophin-4/5. Our results suggest a negative regulatory effect of BDNF and other TrkB ligands on TrkB receptors. Down-regulation of the TrkB response by its ligands might play a role in the control of BDNF action during early development, when BDNF levels significantly increase. Our findings are also of potential clinical relevance, since the possibility of ligand-induced down-regulation of the receptor response needs to be addressed when considering BDNF or other neurotrophins for the therapy of neurodegeneration.

Animals↗
PubMed1997

Tyrosine kinase dependent expression of TGF-beta induced by stretch in mesangial cells.

Increased glomerular hydraulic pressure has been suggested as a major causative factor in the development of glomerular sclerosis. The elevation of glomerular pressure increases the magnitude of stretch to mesangial cells. The study was, therefore, designed to investigate the effect of mechanical stretch on expression of TGF-beta and extracellular matrix components in cultured rat mesangial cells. The results showed that mechanical stretch stimulated mRNA expression for TGF-beta1 and TGF-beta3 in a time dependent manner, and that mesangial cells secreted substantial amounts of TGF-beta proteins in response to stretch. Stretch was also shown to stimulate mRNA expression for collagen types I and IV, and fibronectin, major components of mesangial extracellular matrix. The stretch-induced mRNA expression for extracellular matrix components was inhibited by neutralizing antibody to TGF-beta. Moreover, stretch-induced mRNA expression of TGF-beta was inhibited by tyrosine kinase inhibitors, genistein or herbimycin A, whereas Ca channel blockers nitrendipine or Gd3+, and inhibitors for protein kinase A or C had no effect. These findings indicate that stretch induced TGF-beta mRNA primarily through tyrosine kinase-dependent mechanisms in cultured rat mesangial cells, and the secreted TGF-beta may play a significant role for the stretch-induced expression of extracellular matrix proteins. Our results suggest that stretch-induced TGF-beta of mesangial cells might be a mediator in the progression of glomerular sclerosis as an autocrine/paracrine factor.

Angiotensin II↗
PubMed1997

Immunoaffinity purification and characterization of CACGTG sequence-binding proteins from cultured mammalian cells using an anti c-Myc monoclonal antibody recognizing the DNA-binding domain.

c-Myc protein contains a basic helix-loop-helix and leucine zipper (bHLHLZ) structure in its carboxyl terminal region, forms heterodimers with Max, and binds to the CACGTG sequence in DNA. A number of bHLHLZ proteins are present in cells, and some of them bind to the same DNA sequence. Using an anti c-Myc monoclonal antibody, MYC5, raised against a bacterially synthesized c-Myc protein, we have carried out immunoaffinity purification of c-Myc proteins from cultured mammalian cells. The immunoaffinity-purified fraction was found to contain not only c-Myc but also other CACGTG sequence-binding proteins, such as Max, Mad, and USF, indicating a wide cross-reaction to CACGTG sequence-binding proteins. The MYC5 antibody recognized the common structural motif in their basic region required for sequence-specific DNA binding and was shown to inhibit their DNA-binding activities. The immunoaffinity-purified N-Myc, Max, Mad, and, presumably, c-Myc were highly phosphorylated, and phosphatase treatment increased the DNA-binding activity of Myc, suggesting that the DNA-binding activity of c-Myc was regulated by phosphorylation in vivo. From these results, we can conclude that the MYC5 antibody constitutes a powerful tool for the purification and characterization of c-Myc and other CACGTG sequence-binding proteins.

Antibodies, Monoclonal↗
PubMed1997

Vulval reconstruction using rectus abdominis musculocutaneous flap transfer with secondary liposuction in extramammary Paget's disease.

Two female patients with vulval skin defects after extramammary Paget's disease were reconstructed using a pedicled, distally based rectus abdominis musculocutaneous flap. After the operation, the bulkiness of the flap, which might be a major problem in the application for a vulval skin defect, was improved successfully by flap volume reduction with secondary liposuction. We conclude that rectus abdominis musculocutaneous flap transfer with secondary liposuction is one of the acceptable methods for reconstructing vulval skin defects.

Biopsy↗
PubMed1997

RBP-L, a transcription factor related to RBP-Jkappa.

RBP-Jkappa is a sequence-specific DNA binding protein which plays a central role in signalling downstream of the Notch receptor by physically interacting with its intracellular region. Although at least four Notch genes exist in mammals, it is unknown whether each Notch requires a specific downstream signalling molecule. Here we report isolation and characterization of a mouse RBP-Jkappa-related gene named RBP-L that is expressed almost exclusively in lung, in contrast to the ubiquitous expression of RBP-Jkappa. For simplicity, we propose to call RBP-Jkappa RBP-J. The RBP-L protein bound to a DNA sequence almost identical to that of RBP-J. Surprisingly, RBP-L did not interact with any of the known four mouse Notch proteins. Although we found that RBP-L and EBNA-2 cooperated in transcriptional activation, they did not show significantly strong protein-protein interaction that can be detected by several in vivo and in vitro assays. This is again in contrast to physical association of RBP-J with EBNA-2. Several models to explain functional interaction between RBP-L and EBNA-2 are discussed.

Amino Acid Sequence↗
PubMed1997

Pathophysiological significance of the obese gene product, leptin, in ventromedial hypothalamus (VMH)-lesioned rats: evidence for loss of its satiety effect in VMH-lesioned rats.

To explore the pathophysiological significance of the obese (ob) gene product, leptin, in ventromedial hypothalamus (VMH)-lesioned rats, we examined the synthesis and secretion of leptin and its satiety effect in VMH-lesioned rats compared with those in sham-operated rats. Northern blot analysis revealed that ob gene expression is markedly augmented in the mesenteric and sc white adipose tissue, but remained unchanged in the epididymal white adipose tissue during the development of obesity in VMH-lesioned rats. Plasma leptin levels were relatively constant in sham-operated rats, but were elevated during the development of obesity in VMH-lesioned rats. In sham-operated rats, a single i.v. (1.0 mg/rat) or intracerebroventricular (2.0 micrograms/rat) injection of recombinant human leptin reduced food intake and body weight gain in sham-operated rats. By contrast, no significant effect on food intake or body weight gain was observed in VMH-lesioned rats. The present study provides evidence that VMH-lesioned rats overproduce leptin and increase its release but cannot respond to it and suggests that the loss of its satiety effect contributes to the development of obesity and the obesity-related phenotypes in VMH-lesioned rats.

Animals↗
PubMed1997

Cardiovascular effects of YM430, a 1,4-dihydropyridine derivative with beta-adrenoceptor blocking activity, in dogs and rats.

We evaluated the cardiovascular effects of YM430, a novel 1,4-dihydropyridine derivative with beta-adrenoceptor blocking activity, in dogs and rats. In anesthetized dogs, YM430 (0.01-0.3 mg/kg, i.v.) dose-dependently decreased mean blood pressure, total peripheral resistance and double product without increasing the heart rate. YM430 (0.01-0.3 mg/kg, i.v.) increased coronary artery as well as vertebral artery blood flow, whereas its effects on carotid, mesenteric, femoral and renal blood flow were small. At the same dose range as that which induced vasodilation effects, YM430 had little effect on the max. dp/dt or PQ-interval. In conscious dogs, YM430 (0.1-1 mg/kg, i.v.) produced dose-dependent hypotension with tachycardia. In conscious rats, oral dosing of YM430 (100 mg/kg p.o.) produced a long-lasting hypotensive effect with slight tachycardia. YM430 also inhibited isoproterenol (0.1 micrograms/kg i.v.)-induced tachycardia. These two effects of YM430 may be attributable to its calcium entry blocking and beta(1)-adrenoceptor blocking activity, respectively. The time course of the hypotensive (calcium entry blocking) effect of YM430 after oral dosing was very similar to that of its inhibition of isoproterenol-induced tachycardia (beta(1)-adrenoceptor blocking effect). These results indicate that the ratio of the two activities (calcium entry blocking and beta(1)-adrenoceptor blocking) of YM430 is constant after oral administration. In conclusion, YM430 could be both an antianginal and antihypertensive agent, because of its dual activities.

Adrenergic beta-Antagonists↗
PubMed1997

Synthesis of novel succinamide derivatives having the 5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one skeleton as potent and selective M2 muscarinic receptor antagonists. I.

A series of 5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one derivatives containing the succinamide skeleton has been synthesized and evaluated for M1, M2 and M3 muscarinic receptor binding affinities (in vitro) and M2 and M3 muscarinic receptor antagonistic activities (in vivo). Some of them showed higher and more selective binding affinities for M(2) muscarinic receptors than that of AF-DX 116. Among them, 11-[3-[N-[2-(N-benzyl-N- methylamino)ethyl]-N-ethylcarbamoyl]propionyl]-5,11-dihydro-6H-pyr ido [2,3-b][1,4]benzodiazepin-6-one (68) was found to be the most potent and selective M2 muscarinic receptor antagonist in vitro. This compound also strongly inhibited the oxotremorine-induced bradycardia after intravenous administration and showed 130-fold selectivity for M2 muscarinic receptors over M3 muscarinic receptors in vivo.

Amides↗