Biomedical subjects
Publications and source records attributed to M Yamaguchi.
[figure: see text] Beckmann rearrangement of oxime is catalyzed by [RhCl(cod)]2, trifluoromethanesulfonic acid, and tris(p-tolyl)phosphine in refluxing dichloroethane, giving the corresponding amide in good yield. Product/acid ratios of 10:20 can be attained in the reaction of benzophenone oximes.
The role of regucalcin in the regulation of protein kinase activity in rat brain neuronal cells obtained from primary culture was investigated. Protein kinase activity was assayed using the 5500 g supernatant fraction of the cell homogenate. Protein kinase activity was significantly raised by the addition of calmodulin (5 microg/ml) or dioctanoylglycerol (5 microg/ml) in the presence of CaCl2 (10(-4) M), indicating that Ca2+/calmodulin-dependent protein kinase and protein kinase C is present in the neuronal cells. The addition of regucalcin (10(-9)-10(-7) M) in the enzyme reaction mixture caused a significant decrease in protein kinase activity in the absence of calmodulin or dioctanoylglycerol without Ca(2+) addition. Moreover, regucalcin completely prevented the activation of protein kinase by the addition of calmodulin or dioctonoylglyceral in the presence of CaCl(2) (10(-4) M). The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) caused a significant elevation of protein kinase activity without CaCl2 addition. Such an effect was significantly inhibited by the addition of trifluoperazine (2x10(-5) M), an antagonist of calmodulin, or staurosporine (10(-6) M), an inhibitor of protein kinase C. The present study demonstrates that endogenous regucalcin in rat brain neuronal cells has an inhibitory effect on Ca2+ dependent protein kinase activity.
4-(6,7-Dihydro-5,8-dioxothiazolo[4,5-g]phthalazin-2-yl)benzoic acid N-hydroxysuccinimide ester was synthesized as a highly sensitive and selective chemiluminescence derivatization reagent for primary and secondary amines in liquid chromatography. Methyl-n-octylamine, n-nonylamine and n-decylamine were used as model compounds to optimize the derivatization, separation and chemiluminescence reaction conditions. This reagent reacts selectively with amines in the presence of triethylamine to give the highly chemiluminescent derivatives, which produce chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in an alkaline medium. The chemiluminescent derivatives of the three amines can be separated within 20 min by reversed-phase liquid chromatography with isocratic elution, followed by chemiluminescence detection. The detection limits (signal-to-noise ratio=3) for primary and secondary amines are at sub-fmol levels for a 20-microl injection. Furthermore, this method was applicable to the determination of amantadine in human plasma.
To investigate the acute changes in plasma oxidized low-density lipoprotein before and immediately after coronary angioplasty, we studied 132 consecutive patients who successfully underwent this procedure. Plasma oxidized low-density lipoprotein levels were significantly increased immediately after coronary angioplasty in patients with stable angina pectoris as well as in those with acute coronary syndromes.
BACKGROUND: The relationship between the extent of myocardial interstitial fibrosis, the percentage of each type of collagen, and cardiac function in patients with hypertrophic cardiomyopathy (HC) has not been established. HYPOTHESIS: The study aimed to establish that increases in some types of collagen may correlate with cardiac dysfunction. METHODS: Mallory-Azan staining and immunohistochemical staining by the avidin-biotin-complex (ABC) method using anticollagen antibodies were performed on the myocardial biopsy specimens in 35 patients with HC, and the percentage and type of collagen present was determined. Left ventricular (LV) function was evaluated by cardiac catheterization and ventriculography. RESULTS: The percentage of myocardial interstitial fibrosis correlated highly with indices of LV diastolic and systolic function. The amount of type III collagen correlated significantly with the peak negative dp/dt, the rapid filling volume/stroke volume, and the ejection fraction (EF). Significant correlations also were noted between the amount of type VI collagen and peak negative dp/dt, peak positive dp/dt, and EF. Type I collagen did not correlate with any of the LV function indices, and type IV collagen correlated only with peak ejection rate. Type V collagen did not accumulate substantially in the myocardial interstitium. CONCLUSIONS: The progression of myocardial interstitial fibrosis in the HC heart adversely impacts both the diastolic and systolic function of the LV. Increases in the percentage of type III and VI collagen correlate with cardiac dysfunction.
BACKGROUND: Mutations in the cardiac troponin T gene causing familial hypertrophic cardiomyopathy (HCM) are associated with a very poor prognosis but only mild hypertrophy. To date, the serial morphologic changes in patients with HCM linked to cardiac troponin T gene mutations have not been reported. HYPOTHESIS: The aim of this study was to determine the long-term course of patients with familial HCM caused by the cardiac troponin T gene mutation, Arg92Trp. METHODS: In all, 140 probands with familial HCM were screened for mutations in the cardiac troponin T gene. RESULTS: The Arg92Trp missense mutation was present in 10 individuals from two unrelated pedigrees. They exhibited different cardiac morphologies: three had dilated cardiomyopathy-like features, five had asymmetric septal hypertrophy with normal left ventricular systolic function, one had electrocardiographic abnormalities without hypertrophy, and one had the disease-causing mutation but did not fulfill the clinical criteria for the disease. The mean maximum wall thickness was 14.1 +/- 6.0 mm. The three patients with dilated cardiomyopathy-like features had progressive left ventricular dilation. Three individuals underwent right ventricular endomyocardial biopsy. There was a modest degree of myocardial hypertrophy (myocyte diameter: 18.9 +/- 5.2 microm), and minimal myocardial disarray and mild fibrosis were noted. CONCLUSION: The Arg92Trp substitution in the cardiac troponin T gene shows a high degree of penetrance, moderate hypertrophy, and early progression to dilated cardiomyopathy in Japanese patients. Early identification of individuals with this mutation may provide the opportunity to evaluate the efficacy of early therapeutic interventions.
The role of endogenous regucalcin, which is a regulatory protein in calcium signaling, in the regulation of protein kinase activity in the proliferation of the cloned rat hepatoma cells (H4-II-E) was investigated. Hepatoma cells were cultured for 6-72 h in the presence of fetal bovine serum (FBS; 1 or 10%). The number of cells and protein kinase activity in the 5500 g supernatant of cell homogenate was significantly increased 24 and 48 h after the culture with FBS (1 or 10%); the culture with 10% FBS was potent effect as compared with that of 1% FBS. FBS (10%)-increased protein kinase activity preceded a significant elevation of cell number of 6 h after culture. Serum stimulation-induced increase in protein kinase activity was significantly decreased in the presence of trifluoperazine (50 microM), staurosporine (10(-6) M) or genistein (10(-5) M) in the enzyme reaction mixture. The presence of anti-regucalcin monoclonal antibody (40 or 80 ng/ml) in the reaction mixture caused a significant increase in protein kinase activity in the cells cultured with FBS (1 or 10%). This increase was completely blocked by addition of regucalcin (10(-6) M), which can reveal an inhibitory effect on protein kinase activity. Moreover, the effect of antibody in increasing protein kinase activity was significantly inhibited in the presence of trifluoperazine, staurosporine, or genistein, indicating that endogenous regucalcin has an inhibitory effect on Ca(2+)/calmodulin-dependent protein kinase, protein kinase C, and protein tyrosine kinase. The present study suggests that endogenous regucalcin plays a suppressive role in the enhancement of various protein kinase activities associated with a proliferation of the cloned rat hepatoma cells (H4-II-E).
The role of endogenous regucalcin, which is a regulatory protein of calcium signaling, in the regulation of protein phosphatase activity in the cytosol of rat renal cortex was investigated. Protein phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine was found in the cytosol of kidney cortex. The addition of regucalcin (50-250 nM) in the enzyme reaction mixture caused a significant decrease in protein phosphatase activity toward three phosphoamino acids. The effect of calcium (25 microM) and calmodulin (2.5 microg/ml) in increasing protein phosphatase activity toward three phosphoamino acids was significantly decreased by the addition of regucalcin (100 nM). Protein phosphatase activity toward three phosphoamino acids was significantly increased in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the enzyme reaction mixture. The effect of antibody (25 ng/ml) in increasing the enzyme activity was significantly inhibited by cyclosporin A (10(-5) M) or vanadate (10(-5) M). Regucalcin in the kidney cortex cytosol was clearly decreased by the administration of saline (0.9% NaCl) for seven days in rats. Protein phosphatase activity toward three phosphoamino acids was significantly decreased by saline administration. The effect of anti-regucalcin antibody (25 ng/ml) in increasing protein phosphatase activity toward three phosphoamino acids was not seen in the renal cortex cytocol of saline-administered rats. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in the cytoplasm of rat kidney cortex.
The role of endogenous regucalcin in the regulation of deoxyribonuleic acid (DNA) synthesis in the nuclei of the cloned rat hepatoma cells (H4-II-E) with proliferative cells was investigated. Cells were cultured for 6-96 h in a alpha-minimum essential medium (alpha-MEM) containing fetal bovine serum (FBS; 1 or 10%). Cell number was significantly increased between 24 and 96 h after culture with 10% FBS; cell proliferation was markedly stimulated by culture with 10% FBS as compared with that of 1% FBS. In vitro DNA synthesis activity in the nuclei of cells was significantly elevated 6 h after culture with 10% FBS and its elevation was remarkable at 12 and 24 h after the culture. Nuclear DNA synthesis activity was significantly reduced in the presence of various protein kinase inhibitors (PD98059, staurosprine, or trifluoperazine) in the reaction mixture containing the nuclei of cells cultured for 12 and 24 h with FBS (1 and 10%). The addition of regucalcin (10(-7) and 10(-6)M) in the reaction mixture caused a significant inhibition of nuclear DNA synthesis activity. The presence of anti-regucalcin monoclonal antibody (25-100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS resulted in a significant increase in nuclear DNA synthesis activity. This increase was completely blocked by the addition of regucalcin (10(-6) M). The effect of anti-regucalcin antibody (100 ng/ml) in increasing nuclear DNA synthesis activity was significantly inhibited in the presence of various protein kinase inhibitors. DNA synthesis activity was significantly enhanced in the presence of anti-regucalcin antibody (100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS in the presence of Bay K 8644 (2.5 x 10(-6) M). Culture with Bay K 8644 did not cause a significant increase in DNA synthesis activity in the absence of anti-regucalcin antibody. The present study demonstrates that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis with proliferative cells.
The role of endogenous regucalcin (RC) in the regulation of cell proliferation was investigated in the cloned rat hepatoma H4-II-E cells overexpressing RC stably. H4-II-E cells were transfected with RC/pCXN2 vector and the multiple neomycin-resistant clones which overexpress stably RC were selected. The RC content of RC/pCXN2-transfected cells used in this study was 19.7-fold as compared with that of the parental wild type H4-II-E cells. Wild type H4-II-E cells, pCXN2 vector-transfected cells (mock type), and RC/pCXN2-transfected cells (transfectants) were cultured for 24, 48, and 72 h in the presence of fetal bovine serum (10% FBS). Cell numbers of wild and mock type were significantly increased with the time course of culture. Cell numbers of transfectants was significantly suppressed as compared with that of wild and mock type. Deoxyribonucleic acid (DNA) synthesis activity in the nuclear fraction of H4-II-E cells was significantly suppressed in transfectants with culture for 12-48 h. The presence of anti-RC monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant increase in DNA synthesis activity in the nuclei of wild type and transfectants; this increase was remarkable in transfectants. The effect of anti-RC monoclonal antibody (50 ng/ml) in increasing DNA synthesis activity in transfectants was completely prevented by the addition of regucalcin (1 microM). This study demonstrates that cell proliferation is suppressed in the cloned rat hepatoma H4-II-E overexpressing RC stably.
OBJECTIVE: The purpose of this study was to examine the relationship of hepatocyte growth factor/scatter factor (HGF/SF) to cell motility and invasion in uterine cervical cancer. METHODS: We examined the expression of HGF/SF and its receptor, c-met, in cervical cancer cell lines SKG-IIIa (squamous cell carcinoma) and Hela-S3 (adenocarcinoma) and in stromal cells of the cervical cancer tissue by reverse transcription-polymerase chain reaction. We studied the effect of HGF/SF on invasiveness of SKG-IIIa and Hela-S3 in an invasion model of the modified Boyden chamber method and by electron microscopy. SKG-IIIa cells were also seeded on the thick Matrigel-coated layer to evaluate the invasion patterns in three-dimensional directions. To investigate the mechanism of an inductive effect of HGF/SF on the invasiveness of SKG-IIIa, we examined the effect of HGF/SF on the expression of intercellular adhesion molecule E-cadherin, cell-substrate adhesion molecules CD44, alpha2beta1, and alpha6beta1, and intracellular skeleton fiber actin in SKG-IIIa in cell enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining. RESULTS: HGF/SF messenger RNA (mRNA) was detected in stromal cells, and c-met mRNA was detected in SKG-IIIa and Hela-S3. Hela-S3 that initially showed weak intercellular contact freely invaded the Matrigel-coated multiporous membrane without the addition of HGF/SF. In contrast, SKG-IIIa that initially showed strong intercellular adhesion could invade the membrane after the addition of HGF/SF. The same results were represented by an addition of HECD-1, an anti-human E-cadherin antibody. In an experiment with cell culture in a thick Matrigel layer, control SKG-IIIa showed a mirror-ball-like invasion pattern, whereas HGF/SF-stimulated SKG-IIIa spread horizontally over the membrane and migrated through the membrane holes, presenting a tentacular invasion pattern. Migration of SKG-IIIa under the membrane was confirmed by scanning and transmission electron microscopy. The addition of HGF/SF in cell ELISA assay decreased the expression of E-cadherin and actin in SKG-IIIa, but it did not change the expression of CD44, alpha2beta1, and alpha6beta1. Immunofluorescence staining revealed that the expression of E-cadherin in cell membrane was disturbed by HGF/SF. CONCLUSIONS: Our data indicate that HGF/SF produced by stromal cells influences the mode of stromal invasion of squamous cervical cancer by selectively decreasing the expression of both E-cadherin and actin.
We report a case of concomitant mantle cell lymphoma (MCL) and multiple myeloma (MM) in which we investigated the possibility of a clonal relationship. A 76-year-old man was diagnosed with MCL [immunoglobulin (Ig)M,D-kappa; stage IVB] and MM (IgG-kappa; stage I). Ig heavy chain (IgH) gene complementarity-determining region 3 in DNA from both the MCL tumor and from single MM cells from bone marrow smears was amplified to investigate whether there was a clonal relationship between MCL and MM. Sequence analysis revealed no clonal relationship between MCL and MM in our patient.
Human gingival fibroblast (hGF) cells reside in gingival tissues which are challenged frequently by oral bacteria. Lipopolysaccharide (LPS) from periodontal pathogens can penetrate gingival tissues and stimulate the production of interleukin-1beta (IL-1beta), which has been implicated in inflammation and bone resorption. The anti-inflammatory effects of low-energy laser irradiation have been reported, but the mechanisms of this biostimulatory effect have not been fully elucidated. Primary cultured hGF cells were challenged with LPS isolated from Campylobacter rectus, a known periodontal disease-associated pathogen, and irradiated by a Ga-Al-As diode low-energy laser (830 nm, 3.95-7.90 J/cm2). The hGF cells cultured medium showed a marked elevation of IL-1beta production by LPS, which was significantly inhibited by laser irradiation in a dose-dependent manner. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, this inhibitory effect was involved in the reduction of IL-1beta mRNA levels but not that of the IL-1beta converting enzyme.
The effect of zinc on in vitro deoxyribonucleic acid (DNA) synthesis activity in the femoral-diaphyseal and metaphyseal tissues of newborn rats was investigated to determine a role of zinc in bone growth. In vitro DNA synthesis was assayed in a reaction mixture containing the 100 g centrifugation supernatant, which includes the nucleus of bone cells, of bone issue homogenate with incorporation of [3H]-deoxythymidine 5'-triphosphate (dTTP). DNA synthesis activity in the femoral-diaphyseal and metaphyseal tissues of newborn rats was significantly raised with increasing age (1-21 days) after birth. The presence of dipicolinate (10(-3) M), a chelator of zinc, in the reaction mixture caused a significant decrease in DNA synthesis activity in the diaphyseal and metaphyseal tissues of newborn rats at 7 and 14 days after birth. The addition of zinc sulfate (10(-6) - 10(-4) M) resulted in a significant increase in DNA synthesis activity in the diaphyseal and metaphyseal tissues. When the diaphyseal and metaphyseal tissues of newborn rats at 7 days after birth were cultured for 24 hours in a serum-free medium containing either vehicle, zinc sulfate (10(-4) M), insulin-like growth factor-I (IGF-I; 10(-8) M) or transforming growth factor-beta (TGF-beta; 10(-10) M), bone DNA synthesis activity was significantly elevated. Culture with both zinc and IGF-I enhanced additively bone DNA synthesis activity. Such an effect was not seen in the case of zinc and TGF-beta. The effect of zinc, IGF-I, or zinc plus IGF-I in increasing bone DNA synthesis activity was completely prevented by culture with PD98059 (10(-5) M), an inhibitor of mitogen-activated protein (MAP) kinase. Also, the effect of zinc, TGF-beta. or zinc plus TGF-beta in elevating bone DNA synthesis activity was significantly inhibited by culture with staurosporine (10(-6) M), an inhibitor of protein kinase C. The present study demonstrates that zinc, like bone growth factors, has a stimulatory effect on bone DNA synthesis in newborn rats.
Tubular aggregates (TAs) originate from the sarcoplasmic reticulum (SR) and form polymorphic double (or single) -walled structures in cross section. TAs are involved in various human skeletal muscle disorders including periodic paralysis, congenital myasthenic syndromes, inflammatory myopathies, and malignant hyperthermias. Horse lumbrical muscle (LM) is a slender fusiform muscle that shows varying degrees of regression due to its limited activity in the limb. Double-walled TAs were found in degenerating spindle fibers and with a range of 80-116 nm (average 92 nm, n=135) for outer layer and 50-78 nm (average 59 nm, n=135) for the inner layer. TAs exhibit degradation of myofibrillar proteins, disruption of mitochondria with cristae lost, glycogen accumulation, electron-dense metabolic products, blebbing appearance of sarcolemma, and presence of various vacuoles. LM fibers also show a similarly degenerative state. The disassembly of the SR structure probably produces a large accumulation of SR proteins which remain as molecules without being further degraded and which could aggregate to form the orderly structure of TAs. We believe that TA formation may be an adaptation to store unbalanced extra proteins by forming ordered aggregates in degeneration caused by stress in cells.
Transgenic flies were established in which ectopic expression of boundary element-associated factor (BEAF) 32A was targeted to the Drosophila eye imaginal disc. The eyes of the adult fly displayed a severe rough eye phenotype. When these eyes were sectioned, most ommatidia were found to be fused and irregularly shaped rhabdomeres were observed. In the developing eye imaginal disc, expression of BEAF32A inhibited differentiation of photoreceptor cells. Expression of BEAF32A also induced extensive apoptosis of eye imaginal disc cells and, consistent with this, co-expression of baculovirus P35 in the eye imaginal disc suppressed the BEAF32A-induced rough eye phenotype. To investigate the effects of BEAF32A on regulation of chromatin structure, genetic crosses of the BEAF32A-overexpressing flies with loss-of-function mutants for genes encoding other boundary element-binding factors or regulators of chromatin structure were conducted. Interestingly, half-dose reduction of the su(Hw) gene strongly enhanced the rough eye phenotype induced by BEAF32A. Furthermore, genetic crosses of the transgenic flies with loss-of-function mutants for genes interacting with Polycomb revealed specific links between BEAF32A and genes such as Distalless and kohtalo, suggesting a relation to the chromatin insulator function of BEAF. In addition, genetic crosses of transgenic flies expressing BEAF32A with a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the BEAF32A-induced rough eye phenotype. The transgenic flies established in this study should be useful to identify targets of BEAF32A and its positive or negative regulators in Drosophila.
The role of endogenous zinc in protein synthesis in the bone tissues of newborn rats was investigated in the present study. Femoral-diaphyseal and metaphyseal tissues were obtained at 1, 7, 14, 21, and 28 days after birth. Many protein molecules were found to be present in the diaphyseal and metaphyseal tissues using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Bone protein synthesis activity was enhanced by increasing age, and reached a plateau 21 days after birth. Protein synthesis in the diaphyseal and metaphyseal tissues obtained from 7- or 14-day-old rats was significantly decreased by the addition of dipicolinate (10(-3) M), a chelator of zinc ion, into the reaction mixture. while it was significantly enhanced by zinc sulfate (10(-4) M). When the diaphyseal and metaphyseal tissues obtained from 7- or 14-day-old rats were cultured for 48h in a medium containing dipicolinate (10(-3) M), bone protein synthesis was significantly reduced. This decrease was blocked completely by culture with the addition of zinc (10(-4) M). Culture with zinc (10(-5) and 10(-4) M) alone had a stimulatory effect on the bone protein synthesis. Zinc (10(-4) M)-induced increases in bone protein synthesis were completely blocked by culture with cycloheximide (10(-6) M) or actinomycin D (10(-7) M). The present study suggests that bone protein synthesis is enhanced with increasing age of newborn rats, and that endogenous zinc in bone tissues has a stimulatory role in the enhancement of protein synthesis with bone growth.
Activating transcription factor 3 (ATF3) is a member of the CREB/ATF family of transcription factors. Previously, we demonstrated that the expression of the ATF3 gene is induced by many stress signals. In this report, we demonstrate that expression of ATF3 is induced by cardiac ischemia coupled with reperfusion (ischemia-reperfusion) in both cultured cells and an animal model. Transgenic mice expressing ATF3 under the control of the alpha-myosin heavy chain promoter have atrial enlargement, and atrial and ventricular hypertrophy. Microscopic examination showed myocyte degeneration and fibrosis. Functionally, the transgenic heart has reduced contractility and aberrant conduction. Interestingly, expression of sorcin, a gene whose product inhibits the release of calcium from sarcoplasmic reticulum, is increased in these transgenic hearts. Taken together, our results indicate that expression of ATF3, a stress-inducible gene, in the heart leads to altered gene expression and impaired cardiac function.