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Biomedical subjects

S Ohta

Publications and source records attributed to S Ohta.

At least 109 records · Page 6Linked to original sources
PubMed2001

Bcr in vascular smooth muscle cells involvement of Ras and Raf-1 activation by Bcr.

Bcr gene was originally identified by its presence in the chimeric Bcr/Abl oncogene. In vascular smooth muscle cells, platelet-derived growth factor-BB (PDGF) stimulated Bcr kinase activity. The mutant PDGF receptor for PI3-K, but not for PLC-gamma binding sites, showed significantly reduced Bcr kinase activity. Bcr wild-type enhanced, whereas Bcr kinase negative form inhibited PDGF-stimulated ERK1/2 activity. A dominant negative Ras did not inhibit Bcr kinase activation, and overexpression of Bcr increased Ras/Raf-1 activity and DNA synthesis. These results demonstrated the importance of Bcr in PDGF-mediated events such as activation of Ras, Raf-1, and ERK1/2 and stimulation of DNA synthesis.

Animals↗
PubMed2000

The super anti-apoptotic factor Bcl-xFNK constructed by disturbing intramolecular polar interactions in rat Bcl-xL.

A powerful artificial anti-apoptotic factor will be useful for medical applications of the future therapies for many diseases by prolonging survival of sick cells. For constructing it, we designed the super anti-apoptotic factor by disturbing three intramolecular polar interactions among alpha-helix structures of Bcl-x(L). The resultant mutant Bcl-x(L), named Bcl-xFNK, was expected to make the pore-forming domain more mobile and flexible than the wild-type. When overexpressed in Jurkat cells, Bcl-xFNK was markedly more potent in prolonging survival following apoptosis-inducing treatment with a kind of cell death cytokines (anti-Fas), a protein kinase inhibitor (staurosporine), cell cycle inhibitors (TN-16, camptothecin, hydroxyurea, and trichostatin A), or oxidative stress (hydrogen peroxide and paraquat) than wild-type Bcl-x(L). Furthermore, the transfectants of bcl-xFNK became more resistant against a calcium ionophore and even a heat treatment than wild-type Bcl-x(L). In addition, Bcl-xFNK showed marked anti-apoptotic activity in Chinese hamster ovary and Jurkat cells deprived of serum. Thus, Bcl-xFNK may be the first mutant generated by site-directed mutagenesis of Bcl-x(L) with a gain-of-function phenotype. Interestingly, Bcl-xFNK was found to allow interleukin-3-dependent FDC-P1 to grow without interleukin-3, but not BaF/3. In Bcl-xFNK transfectants of FDC-P1 and Jurkat, the p42/p44 mitogen-activated protein kinase was activated by 2 to 5 times, but not in those of BaF/3 and Chinese hamster ovary. Bcl-xFNK might gain a new function to activate the mitogen-activated protein kinase in a cell-type specific manner. The findings of this study suggest that the central alpha5-alpha6 pore-forming region of anti-apoptotic factor Bcl-x(L) has a pivotal role in suppressing apoptosis.

Animals↗
PubMed2000

Clip migration causes choledocholithiasis after laparoscopic cholecystectomy.

The migration of surgical clips after laparoscopic procedures was first reported in 1992, but such instances are extremely rare. We herein demonstrate a case of a migrated metal clip, which had been applied originally to the cystic duct, but thereafter had moved to the common bile duct. This clip caused choledocholithiasis in a patient 1 year after a laparoscopic cholecystectomy. A 63-year-old man underwent a laparoscopic cholecystectomy. During the operation, the inflamed cystic duct was divided accidentally, and three clips were applied immediately. The patient complained of upper abdominal pain from postoperative day 8. Endoscopic retrograde cholangiography demonstrated bile leakage from the cystic duct, but showed no clips or choledochal stones. The patient complained of severe upper abdominal and back pain 1 year after the operation. Endoscopic retrograde cholangiography showed a metal clip in the common bile duct and choledochal stones above the clip. The clip and the cholesterol stones were removed using a basket catheter. Three clips applied to the cystic duct should have been removed because of the necrosis in the remaining cystic duct. Thereafter, the clip may have migrated through the stump of the cystic duct into the lower part of the common bile duct. This clip seems to have later caused choledocholithiasis resulting from stagnation of the bile flow. Bile leakage after an operation seems to increase the risk of clip migration. Regardless of the primary lesion, a careful follow-up evaluation is necessary for patients demonstrating complications.

Cholangiography↗
PubMed2000

Synthesis and HPLC analysis of enzymatically cleavable linker consisting of poly(ethylene glycol) and dipeptide for the development of immunoconjugate.

A model compound of anti-tumor agent, segment B of duocarmycin derivative DU-86, was conjugated to tumor-specific antibody via a cleavable linker consisting of poly(ethylene glycol) (PEG) and dipeptide, L-alanyl-L-valine (Ala-Val), to confirm the feasibility of the linker for application to immunoconjugate. The release of segment B from the linker was evaluated by HPLC analysis. When segment B was derivatized to have an amino residue and then linked to PEG through a dipeptide, segment B was cleaved at the peptide bond by a particular enzyme, thermolysin (EC 3.4.24.4), but not by plasmin (EC 3.4.2 1.7.), indicating that certain protease specifically expressed at the tumor site would be capable of peptide-specific digestion and release of anti-tumor agent since a thermolysin-like enzyme has been reported to be expressed at many tumor cells. Furthermore, the results showing that cell extract from G361 human melanoma had an ability to digest the linker peptide while the linker was stable in normal human serum suggested the tumor-specific activation of the conjugated agent. Segment B was conjugated via the linker to murine monoclonal antibody KM641 reactive to GD3 ganglioside to form immunoconjugate and the quantitative release of segment B under the treatment with the enzyme was also confirmed. These results indicate the possibility of double targeting based on both the recognition ability of tumor specific antibody and tumor specific activation of the anti-tumor agents to enhance tumor treatment efficacy and to decrease unwanted side effects.

Antibodies, Monoclonal↗
PubMed2000

A pathogenic point mutation reduces stability of mitochondrial mutant tRNA(Ile).

Point mutations in mitochondrial tRNA genes are responsible for individual subgroups of mitochondrial encephalomyopathies. We have recently reported that point mutations in the tRNA(Leu)(UUR) and tRNA(Lys) genes cause a defect in the normal modification at the first nucleotide of the anticodon. As part of a systematic analysis of pathogenic mutant mitochondrial tRNAs, we purified tRNA(Ile) with a point mutation at nucleotide 4269 to determine its nucleotide sequence, including modified nucleotides. We found that, instead of causing a defect in the post-transcriptional modification, a pathogenic point mutation in the mitochondrial tRNA(Ile) reduced the stability of the mutant tRNA molecule, resulting in a low steady-state level of aminoacyl-tRNA. The reduced stability was confirmed by examining the life-span of the mutant tRNA(Ile) both in vitro and in vivo, as well as by monitoring its melting profile. Our finding indicates that the mutant tRNA(Ile) itself is intrinsically unstable.

Acylation↗
PubMed2000

A novel transcriptional factor with Ser/Thr kinase activity involved in the transforming growth factor (TGF)-beta signalling pathway.

Transforming growth factor-beta (TGF-beta) shows a variety of biological activities in various organs or cells. Recently some factors such as Smads (Sma and Mad proteins) and TGF-beta activating kinase 1 ('TAK1') have been characterized as signalling molecules downstream of TGF-beta. Several TGF-beta response elements have been identified such as cAMP response element, Smad binding element, and recognition sites for activating protein-1 and stimulating protein-1 in various gene promoters. We also reported a TGF-beta response element in the human C-type natriuretic peptide (CNP) gene promoter. In this paper, we report on a novel factor which regulates the TGF-beta response promoter. This factor, named TSF1 (TGF-beta stimulated factor 1), possessed DNA-binding ability and activated the TGF-beta responsive CNP promoter or vascular endothelial growth factor gene promoter which possesses a sequence element analogous to the TGF-beta responsive GC-rich element of the CNP promoter. TSF1 did not directly activate a Smads-dependent promoter from plasminogen activator inhibitor 1 gene, but it showed enhancement in co-operation with Smad3 and Smad4. Interestingly, this factor had the structural features of a Ser/Thr kinase and actually exhibited protein kinase activity. TSF1 mRNA as well as its protein level were stimulated by TGF-beta treatment. Thus, TSF1 is an unique factor with two biological functions, transcriptional regulation and protein phosphorylation, that may be involved in TGF-beta signals.

Amino Acid Sequence↗
PubMed2000

Metabolic activation of the proestrogens trans-stilbene and trans-stilbene oxide by rat liver microsomes.

A liver microsome-mediated activation of the proestrogens trans-stilbene and trans-stilbene oxide was found in this study. trans-Stilbene and trans-stilbene oxide were negative in estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7 and growth assay in rat pituitary tumor cell line MtT/E-2. However, these compounds exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH. In contrast, cis-stilbene and cis-stilbene oxide did not show estrogenic activity after such incubation. When trans-stilbene was incubated with the liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH, two metabolites were detected by HPLC. They were identified unequivocally as trans-4-hydroxystilbene and trans-4, 4'-dihydroxystilbene by mass and UV spectral comparison with authentic samples. The oxidase activity of the liver microsomes toward trans-stilbene was inhibited by SKF 525-A and alpha -naphthoflavone. Minor activity was observed when liver microsomes of untreated or phenobarbital-treated rats were used instead of those from 3-methylcholanthrene-treated rats. trans-4-Hydroxystilbene and trans-4,4'-dihydroxystilbene exhibited significant estrogenic activities. These results suggest that the estrogenic activities of trans-stilbene and trans-stilbene oxide were due to formation of hydroxylated metabolites.

Animals↗
PubMed2000

Deficiency in mitochondrial aldehyde dehydrogenase increases the risk for late-onset Alzheimer's disease in the Japanese population.

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) deficiency is caused by a mutant allele in the Mongoloids. To examine whether genetic constitutions affecting aldehyde metabolism influence the risk for late-onset Alzheimer's disease (LOAD), we performed a case-control study in the Japanese population on the deficiency in ALDH2 caused by the dominant-negative mutant allele of the ALDH2 gene (ALDH2*2). In a comparison of 447 patients with sex, age, and region matched nondemented controls, the genotype frequency carrying the ALDH2*2 allele was significantly higher in the patients than in the controls (48.1% vs 37.4%, P = 0.001). Logistic regression analysis indicates that carriage of the ALDH2*2 allele is an independent risk for LOAD of the epsilon4 allele of the apolipoprotein E gene (APOE-epsilon4) (P = 0.002). Moreover, the odds ratio for LOAD in carriers of the ALDH2*2 allele was almost twice that in noncarriers, irrespective of status with regard to the APOE-epsilon4 allele. Among patients homozygous for the APOE-epsilon4 allele, age at onset of LOAD was significantly lower in those with than without the ALDH2*2 allele. In addition, dosage of the ALDH2*2 allele significantly affected age at onset of patients homozygous for the APOE-epsilon4 allele. These results indicate that the ALDH2 deficiency is a risk for LOAD, synergistically acting with the APOE-epsilon4 allele.

Age of Onset↗
PubMed2000

Increased CD40 expression on muscle cells of polymyositis and dermatomyositis: role of CD40-CD40 ligand interaction in IL-6, IL-8, IL-15, and monocyte chemoattractant protein-1 production.

In polymyositis (PM)/dermatomyositis (DM), T cells infiltrate the muscle tissues and interact with muscle cells via cell surface molecules. Recently, myoblasts have been reported to express CD40, but little is known about the role of CD40 in myoblasts. In the present study we examined the expression and involvement of CD40 and CD40 ligand (CD40L) in the interaction between muscle cells and T cells in PM/DM. Immunohistochemical staining revealed that CD40 was expressed on muscle cells in five of five PM and four of five DM patients, and that infiltrating mononuclear cells (MNCs) expressed CD40L in all cases of PM/DM. These CD40L-expressing MNCs were primarily CD4+ T cells. IFN-gamma, which is known to induce CD40 expression on various types of cells, was also expressed on the MNCs in four of the PM and four of the DM patients. Although cultured human myoblasts (SkMC 2859) did not express CD40 constitutively, IFN-gamma induced CD40 expression in a dose-dependent manner. To clarify the functional roles of CD40-mediated signals, the effects of a trimeric form of recombinant human CD40L on cytokine production were studied in SkMC 2859 that were prestimulated with IFN-gamma to express CD40. Recombinant human CD40L markedly increased the production of IL-6, IL-8, IL-15, and monocyte chemoattractant protein-1 of SkMC 2859. The expression of these humoral factors in muscle cells of PM and DM was demonstrated by immunohistochemistry. These results suggest that interaction between T cells and muscle cells via the CD40-CD40L system contributes to the immunopathogenesis of PM/DM by augmenting inflammation via cytokine production by the muscle cells.

Adjuvants, Immunologic↗
PubMed2000

Protein kinase cdelta and alpha are involved in the development of vasospasm after subarachnoid hemorrhage.

We have previously shown the enhanced activity of protein kinase C in the membrane fraction of the canine vasospastic artery after subarachnoid hemorrhage, which increased with progression of angiographic vasospasm. This study examined identification of protein kinase C isoforms in the canine basilar artery, and the changes in expression and/or translocation of each isoform during the development of vasospasm. Vasospasm was produced by using the "two-hemorrhage" canine model in the basilar artery, and angiographic progression of vasospasm was assessed consecutively. Two isoforms, protein kinase Calpha and delta were identified in basilar arteries by Western blotting. Densitometric analysis showed that the expression of protein kinase Cdelta in the membrane fraction was significantly increased in the earlier stage, and protein kinase Calpha was increased later as vasospasm progressed. These results indicate that protein kinase Cdelta and alpha isoforms may play a significant role in the development and maintenance of vasospasm.

Actins↗
PubMed2000

Localization of Bcl-xbeta in the developing and adult rat central nervous system.

bcl-xbeta is a novel apoptosis-regulating member of the bcl-x family that has recently been isolated from rats and mice. To explore the functional role of Bcl-xbeta, we raised a monoclonal antibody against rat Bcl-xbeta protein and investigated the cellular localization of the molecule in the rat CNS. Immunohistochemistry revealed that, in the fetal and neonatal stages, Bcl-xbeta was intensively and widely expressed in the CNS. Many neurons in the diencephalon and brain stem showed intense cytoplasmic labeling. The immunoreactivity decreased during the postnatal development and reached to the level of adulthood by P14. In the adult brain and spinal cord, labeling was restricted to specific types of neurons and distributed throughout their somata and dendrites. Weak immunoreactivity was present in many CNS regions such as the cerebral cortex, hippocampal dentate gyrus, caudate-putamen, globus pallidus, thalamus, locus ceruleus, pontine nuclei, inferior olive, reticular formation, cerebellar cortex and spinal anterior horn. Amygdaloid nuclei and hippocampal CA1 to CA3 sectors showed restricted expression of Bcl-xbeta in a subset of neurons. Neuronal labeling was almost undetectable in several regions, including the piriform cortex, hypothalamus, posterior column nuclei and spinal posterior horn. These results suggest that Bcl-xbeta plays an important role throughout the CNS in developing stage and may regulate the apoptosis of postnatal CNS neurons.

Animals↗
PubMed2000

Histochemical cytochrome c oxidase activity and caspase-3 in gerbil hippocampal CA1 neurons after transient forebrain ischemia.

We examined the cytochrome c oxidase (COX) activity in gerbil hippocampal CA1 neurons after 5-min ischemia by a histochemical method in the presence or absence of exogenous cytochrome c. In the CA1 neurons, COX activity without exogenous cytochrome c decreased from 1 h after ischemia, but was restored by the addition of exogenous cytochrome c in the following 6 h after ischemia. These results suggest that it is not COX activity but endogenous cytochrome c that is changed in the early phase after ischemia, and that COX activity begins to decrease 9 h after ischemia. We examined caspase-3 in the CA1 region by immunoblotting, as caspase-3 is known to take part in the cell-death cascade downstream from cytochrome c. Although pro-caspase-3 was strongly detected, active caspase-3 was not detected before and until 84 h after 5-min ischemia. Our data suggested that delayed neuronal death is likely to progress via cytochrome c-release but not via caspase-3 activation.

Animals↗
PubMed2000

Induction of urokinase-type plasminogen activator by lipopolysaccharide in PC-3 human prostatic cancer cells.

Urokinase-type plasminogen activator (uPA) plays a central role in many aspects of cellular regulation, such as fibrinolysis, cell migration, and metastasis. The uPA induction by inflammatory stimuli such as IL-1, TNFalpha, and lipopolysaccharide (LPS) has been reported in a number of human cells. We examined the effects of LPS on uPA expression in human PC-3 prostatic cancer cells that express uPA in a conditioned medium. LPS increased the uPA accumulation in PC-3 cells, whereas IL-1, IL-6, and TNFalpha did not. Northern blot analysis revealed that the peak induction of uPA mRNA occurred 5 hours after LPS stimulation. A protein synthesis inhibitor, cycloheximide, amplified the LPS-induced uPA mRNA, suggesting that LPS induces uPA by activating the gene expression in which de novo protein synthesis is not necessary.

Animals↗
PubMed2000

A role for nuclear PTEN in neuronal differentiation.

Mutations of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a protein and lipid phosphatase, have been associated with gliomas, macrocephaly, and mental deficiencies. We have assessed PTEN's role in the nervous system and find that PTEN is expressed in mouse brain late in development, starting at approximately postnatal day 0. In adult brain, PTEN is preferentially expressed in neurons and is especially evident in Purkinje neurons, olfactory mitral neurons, and large pyramidal neurons. To analyze the function of PTEN in neuronal differentiation, we used two well established model systems-pheochromocytoma cells and cultured CNS stem cells. PTEN is expressed during neurotrophin-induced differentiation and is detected in both the nucleus and cytoplasm. Suppression of PTEN levels with antisense oligonucleotides does not block initiation of neuronal differentiation. Instead, PTEN antisense leads to death of the resulting, immature neurons, probably during neurite extension. In contrast, PTEN is not required for astrocytic differentiation. These observations indicate that PTEN acts at multiple sites in the cell, regulating the transition of differentiating neuroblasts to postmitotic neurons.

Animals↗
PubMed2000

Defect in modification at the anticodon wobble nucleotide of mitochondrial tRNA(Lys) with the MERRF encephalomyopathy pathogenic mutation.

A mitochondrial tRNA(Lys) gene mutation at nucleotide position 8344 is responsible for the myoclonus epilepsy associated with ragged-red fibers (MERRF) subgroup of mitochondrial encephalomyopathies. Here, we show that normally modified uridine at the anticodon wobble position remains unmodified in the purified mutant tRNA(Lys). We have reported a similar modification defect at the same position in two mutant mitochondrial tRNAs(Leu)(UUR) in another subgroup, mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), indicating this defect is common in the two kinds of tRNA molecules with the respective mutations of the two major mitochondrial encephalomyopathies. We therefore suggest the defect in the anticodon is responsible, through the translational process, for the pathogenesis of mitochondrial diseases.

Anticodon↗
PubMed2000

Modification defect at anticodon wobble nucleotide of mitochondrial tRNAs(Leu)(UUR) with pathogenic mutations of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes.

The mitochondrial tRNA(Leu)(UUR) (R = A or G) gene possesses several hot spots for pathogenic mutations. A point mutation at nucleotide position 3243 or 3271 is associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes and maternally inherited diabetes with deafness. Detailed studies on two tRNAs(Leu)(UUR) with the 3243 or 3271 mutation revealed some common characteristics in cybrid cells: (i) a decreased life span, resulting in a 70% decrease in the amounts of the tRNAs in the steady state, (ii) a slight decrease in the ratios of aminoacyl-tRNAs(Leu)(UUR) versus uncharged tRNAs(Leu)(UUR), and (iii) accurate aminoacylation with leucine without any misacylation. As a marked result, both of the mutant tRNA molecules were deficient in a modification of uridine that occurs in the normal tRNA(Leu)(UUR) at the first position of the anticodon. The lack of this modification may lead to the mistranslation of leucine into non-cognate phenylalanine codons by mutant tRNAs(Leu)(UUR), according to the mitochondrial wobble rule, and/or a decrease in the rate of mitochondrial protein synthesis. This finding could explain why two different mutations (3243 and 3271) manifest indistinguishable clinical features.

Anticodon↗
PubMed2000

Cytochrome c release from mitochondria to the cytosol was suppressed in the ischemia-tolerance-induced hippocampal CA1 region after 5-min forebrain ischemia in gerbils.

Cytochrome c was detected by immunoblotting in the cytosolic fraction 3 h after 5-min ischemia in the non-ischemia-tolerant CA1 region in which about 96% of neurons had developed delayed neuronal death, while less cytosolic cytochrome c was detected in the ischemia-tolerance-induced CA1 region where many more neurons survived. In the immunohistochemical study using anti-non-native cytochrome c monoclonal antibody, immunoreactivity was observed throughout the cytoplasm in the non-ischemia-tolerant CA1 neurons, but not in the normal and ischemia-tolerant CA1 neurons. Then we determined whether Bcl-2, Bax, Bcl-xL and Bcl-xS, which regulate the release of cytochrome c from mitochondria, were altered in the ischemia-tolerant CA1 region. Bcl-2 and Bax were up-regulated in the ischemia-tolerant group, but Bcl-xL and Bcl-xS showed no apparent difference in their expression. These results suggest that cytochrome c release is prevented in CA1 neurons in gerbils in which ischemia-tolerance had been induced and that the altered ratio of Bcl-2 to Bax may play a part in this mechanism.

Animals↗