Biomedical subjects
Publications and source records attributed to H Fukazawa.
Cardioembolism is most likely to occur after waking in the morning. The identification of periods of high risk for cardioembolism may have important therapeutic implications, such as matching drug effects with vulnerability.
We have described a microplate colorimetric assay to quantitate anchorage-independent cell growth, using plates coated with an anti-adhesive polymer poly (2-hydroxyethyl methacrylate) (polyHEMA).We investigated whether this method could be applied to human cancer cells of epithelial origin. HAG-1, a non-tumorigenic human gall-bladder carcinoma cell line, and its pSVneo and c-H-ras transfectants, which are also non-tumorigenic, did not grow on a polyHEMA-surface. In contrast, the v-src transformant which produced tumors in nude mice and formed colonies in soft agar, was able to proliferate on the coated surface, suggesting that tumorigenicity of human cancer cells correlates with the ability to grow on a polyHEMA-coated surface. We report on the feasibility of this method as a screening system for inhibitors of oncogenic transformation. Herbimycin A and radicicol, which have been reported to block Src function, suppressed the growth of v-src-transformed NRK and HAG-1 cells on the non-adhesive polyHEMA-surface at concentrations significantly lower than on plastic. Differences in the inhibitory concentrations were not observed with KB cells, and cytotoxic agents such as adriamycin did not show any selectivity between the 2 surfaces. Growth of ras-transformed cells on the coated surface was selectively blocked by L-739,749, a farnesyltransferase inhibitor. The results demonstrate that compounds causing reversion of transformed cells to normal, hence, selectively inhibiting cell growth in anchorage-independent conditions, can be screened using this microtiter plate assay.
Aspirin 330/mg/day suppressed platelet function in patients with atrial fibrillation, although it did not affect the increased coagulation activity in these patients. Findings support the premise that anticoagulant therapy is more appropriate than aspirin for the prevention of systemic embolism in patients with atrial fibrillation.
Individuals with hyperthyroidism exhibit red blood cell concentrations of carbonic anhydrase I (CAI) that reflect the integrated serum thyroid hormone concentration over the preceding few months. Furthermore, T3, at a physiological free concentration, decreases the CAI concentration in human erythroleukemic YN-1 cells. The effect of T3 on CAI concentration in burst-forming unit-erythroid (BFU-E)- derived cells, obtained by culturing peripheral blood mononuclear cells with various cytokines, including erythropoietin, has now been investigated. BFU-E-derived cells contained a high concentration of CAI (mean +/- SE, 4.8 +/- 0.8 x 10(-12) mol/10(6) cells; n = 8). The CAI in BFU-E-derived cells was immunologically identical to that present in mature red blood cells. T3 decreased the CAI concentration in BFU-E-derived cells in a dose-dependent manner (28%, 47% and 75% decreases at 3 x 10(-10), 1 x 10(-9), and 3 x 10(-9) mol/liter T3, respectively). These results suggest that BFU-E-derived cells may be used to study the effect of T3 on human red blood cell CAI. This system may prove useful in the tissue diagnosis of resistance to thyroid hormone.
Triterpene carboxylic acids were isolated from the methanol extract of Hoelen, Poria cocos, and found to inhibit 12-O-tetradecanoylphorbol 13-acetate (TAP)-induced mouse ear edema. Their chemical structures were identified as 3 beta,-16 alpha-dihydroxylanosta-7,9(11),24-trien-21-oic acid, 16 alpha-hydroxydehydropachymic acid, 16 alpha-hydroxytrametenolic acid and dehydrotumulosic acid.
We developed a 96-well microplate assay to quantitate anchorage-independent growth of transformed cells. Wells of tissue culture microtiter plates are coated with poly(2-hydroxyethyl methacrylate) (poly-(HEMA)) to prevent cell attachment, and cells suspended in liquid media are seeded into the treated plates. Cell growth is assessed by tetrazolium dye reduction or by counting [3H]thymidine incorporation into DNA. There appeared to be a close correlation between growth in poly(HEMA)-coated plates and colony formation in soft agar, i.e., fibroblasts transformed by various oncogene proliferated in the coated plates, whereas their normal counterparts did not. Transformed cells on the nonadhesive surface were round and formed multicellular spheroids which resembled colonies in soft agar. Cells carrying either temperature-sensitive v-src or inducible v-Ki-ras oncogene proliferated on poly(HEMA)-coated plates only when they displayed transformed phenotype. This method is simple, quick, quantitative, and economical and in many cases might be more practical than conventional soft agar colony formation assay for measurement of anchorage-independent growth.
Intercellular adhesion molecule (ICAM-1), a ligand for lymphocyte function-associated antigen-1 (LFA-1), plays an important role in a variety of immune-mediated mechanisms such as lymphocyte attachment to cultured Graves' thyroid cells. We report the detection of a soluble form of the ICAM-1 molecule (sICAM-1) in sera from patients with Graves' disease (GD) and other thyroid disorders. The mean (+/- SD) sICAM-1 concentration in 28 euthyroid control subjects was 1931 +/- 681 pmol/L. The mean sICAM-1 concentration in 25 untreated hyperthyroid patients with GD was significantly elevated (3065 +/- 890 pmol/L), and decreased significantly (2489 +/- 845 pmol/L) after treatment with antithyroid drugs and/or 131I. Of 14 GD patients who had been in remission following administration of antithyroid drugs, 12 had recurrent disease. In 10 of the 12 patients in whom GD recurred, the sICAM-1 concentration (3807 +/- 796 pmol/L) increased significantly. The mean sICAM-1 concentration in patients with hypothyroidism due to chronic thyroiditis (n = 15:2895 +/- 569 pmol/L) was significantly elevated over that of control subjects, and not different from untreated hyperthyroid patients. The mean sICAM-1 concentration in patients with subacute thyroiditis (n = 13: 3036 +/- 441 pmol/L) was significantly elevated, while the mean sICAM-1 concentration in patients with nodular goiter (n = 10: 2318 +/- 490 pmol/L) was within the normal range. These results indicate that mean serum sICAM-1 concentration was significantly elevated in patients with untreated GD, and it decreased after treatment and increased at the time of recurrence. Therefore, the elevated serum concentration of sICAM-1 in patient with GD probably reflects ongoing immune processes.
OBJECTIVE: Graves' disease is recognized as an organ-specific autoimmune disorder caused by the presence of TSH receptor antibodies. The long-term effects of 131I treatment for Graves' disease on TSH receptor antibodies have not previously been studied. We have measured the TSH-binding inhibitory immunoglobulin (TBII) index and thyroid stimulating antibody (TSAb) activity in patients with Graves' disease following treatment with 131I. DESIGN: A retrospective study. PATIENTS: Two hundred and twenty-five patients with Graves' disease who were treated with 131I 1-13 years earlier were studied (1 year: 27 patients; 2-5 years: 42 patients; 6-9 years: 79 patients; 10-13 years: 77 patients). MEASUREMENTS: The TBII index was measured as the percentage 125I-TSH bound to pig thyroid membranes and TSAb activity as the amount of cAMP produced by cultured FRTL-5 cells. RESULTS: TBII was detected in 78% of patients prior to 131I administration. Following 131I administration, the incidence of positive TBII was 85% at the end of the first year decreasing to 40, 19, and 17% at 2-5, 6-9 and 10-13 years, respectively. The frequency of a positive TSAb was 74% at the end of the first year, and also decreased to 49, 27 and 29% at 2-5, 6-9 and 10-13 years, respectively. At more than 2 years after 131I therapy, the frequencies of hyperthyroidism in TBII and TSAb positive patients were 42% (19/45) and 30% (19/63), respectively, which were significantly higher than those in TBII and TSAb negative patients (8%: 12/153 and 8%:11/131, respectively). The frequency of hyperthyroidism after 131I treatment in patients with negative TBII before treatment (7%:2/29) was significantly lower than that (29%:30/102) in patients with positive TBII before treatment. CONCLUSIONS: These results indicate that (1) the TBII index and TSAb activity decreased over a period of more than 2 years after 131I therapy for Graves' disease, and (2) the TBII index before treatment may influence the long-term outcome of 131I therapy.
The effect of Madopar (benserazide and L-dopa, 1:4) on the disposition of the new selective inhibitor of catechol-O-methyltransferase, tolcapone, in rats was investigated. There was no statistically significant difference in the pharmacokinetic parameters of tolcapone in the presence or absence of Madopar except for a change in the mean residence time after oral administration. Thus, we rejected the hypothesis that the consumption of S-adenyl-L-methionine by Madopar would change the disposition of tolcapone. There were no statistically significant differences in the cumulative amount absorbed of drug and the absorption rate in the presence or absence of Madopar. We concluded that there was no interaction between tolcapone and Madopar.
The mechanism of aberrant GH responses to TRH was indirectly evaluated in 7 patients with primary hypothyroidism. All patients showed GH response to TRH. When TRH was administered together with GHRH, the plasma GH response was much greater than after a single administration of TRH or GHRH (TRH+GHRH vs. TRH or GHRH: max. delta GH, 16.4 +/- 3.2 vs. 5.4 +/- 1.3 or 6.0 +/- 0.8 microgram/L; AUC, 1282.7 +/- 234.7 vs. 384.0 +/- 95.0 or 441.8 +/- 66.2, both P < 0.01). The combined administration of TRH and GHRH caused an additive, but not a synergistic, GH response. In contrast, 8 normal subjects showed neither any plasma GH response to TRH nor enhancement by TRH of GHRH-induced GH response following combined administration. It is concluded that the sites of action of TRH seemed to be different from GHRH in patients with primary hypothyroidism.
The kinetics of type III iodothyronine deiodinase (5-D) in rat placenta and brain and the role of phospholipids in enzyme activity were determined. Pregnant Sprague-Dawley rats were given either vehicle (control group) or T4 (15 micrograms/100 g bw/day; hyperthyroid group) from the 14th to the 21st day of gestation. Mitochondrial-microsomal fractions of the placenta and brain were used as the source of T4 5-D. Placental T4 5-D activity in the hyperthyroid group was increased when determined at 13 nM T4, but it was not significantly different from the control value when assayed at 1.3 microM T4. In contrast, T4 5-D in the brain was significantly increased in the hyperthyroid group regardless of the substrate concentration. Hyperthyroid rats showed decreased Km for placental 5-D and increased Vmax for brain 5-D. CM-Sephadex chromatography of solubilized placental microsomes was performed to determine whether phospholipids cause a reduction in the Km of placental 5-D in hyperthyroid rats. T3 5-D activity was undetectable unless protein-containing fractions were combined with phospholipid-containing fractions in the reaction mixture. Kinetic studies revealed that phospholipids had no effects on either Km or Vmax of placental T3 5-D. These data indicate that 5-D activity in the rat placenta is increased in hyperthyroidism with different kinetic changes from those in the brain, and that phospholipids have no effects on the kinetic parameters of placental 5-D whereas they are essential for the enzyme activity.
We examined effect of the tyrosine kinase inhibitor herbimycin A on A431 human epidermoid carcinoma cells which over-express epidermal growth factor (EGF) receptor. Herbimycin A inhibited the autophosphorylation of EGF-stimulated receptors in intact cells both time- and dose-dependently. The inhibition was found to be due to a decrease in the level of expression of the receptor amount, because herbimycin A equally decreased the receptor quantity and EGF-stimulated receptor kinase activity in intact cells, but did not exhibit a direct inhibitory effect on EGF receptor kinase activity in vitro. The decrease of the level of EGF receptor was also confirmed by 125I-EGF binding to herbimycin A-treated cells, and Scatchard analysis showed that the decrease in the receptor number occurred in the major population of the low-affinity binding ones, whereas the number with high-affinity binding was unaffected. Interestingly, although the proliferation of A431 cells was inhibited by EGF, herbimycin A converted EGF into a stimulatory ligand for cell growth, as determined by both cell number and DNA synthesis. These findings indicated that herbimycin A decreased the level of expression of EGF receptor by a mechanism other than inactivation of the receptor kinase and reversed the transformed phenotype of A431 cells to a normal one in the proliferative response to EGF.
The ansamycin antibiotic, herbimycin A, selectively inactivates cytoplasmic tyrosine kinases, most likely by binding irreversibly to the reactive SH group(s) of kinases. To further investigate the mechanism of herbimycin A action, we attempted to label tyrosine kinases with [14C]herbimycin A. p60v-src and p210BCR-ABL in immune complexes were labeled with [14C]herbimycin A, demonstrating that the antibiotic binds directly to tyrosine kinases. Digestion of [14C]herbimycin A-labeled p60v-src with Staphylococcus aureus V8 protease revealed that the herbimycin A binding site is within the C-terminal 26-kDa fragment of p60v-src, which contains the tyrosine kinase domain. Herbimycin A treatment inhibited labeling of p60v-src by [14C]fluorosulfonylbenzoyl adenosine, an affinity labeling reagent of nucleotide binding sites, indicating that herbimycin A-modified p60v-src cannot interact with ATP. The results suggest that herbimycin A inactivates tyrosine kinases by binding directly to the kinase domain, thereby inhibiting access to ATP.
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Catechol-O-methyltransferase (COMT) catalyses the transfer of the methyl group from S-adenyl-L-methionine (SAM) to one of the hydroxy groups of a catechol, usually the hydroxy group in position 3. COMT is present mainly in a soluble form (S-COMT) in the cytosol, but a small fraction is bound to cell membranes (MB-COMT). MB-COMT has higher affinity for the catechol substrate than does S-COMT by a factor of > 10, and high MB-COMT activity is observed in the intestinal muscle layer. The present study investigates the effect of the administration route on the disposition of the tolcapone 3-O-methylated metabolite following intravenous and oral tolcapone administration in rats. Tolcapone is a substrate for COMT although the 3-O-methylated metabolite produced has no pharmacological actions. The 3-O-methylated metabolite was eliminated very slowly following oral administration of tolcapone, and its concentration approached a plateau level, which was in contrast to the situation following intravenous administration of tolcapone. It is thought that the oral dose of tolcapone receives a high exposure to MB-COMT in the intestinal muscle layer during its absorption, and tolcapone seems to form a complex with MB-COMT having a high affinity constant (i.e. a very low Ki). The fraction of the intravenous dose of tolcapone metabolized to the 3-O-methylated metabolite at 10 mg kg-1 was 2.6%, whereas those of the oral doses, which were corrected by the bioavailability, were 5.4% for 20 mg kg-1 and 2.7% for 40 mg kg-1.
We recently have reported that patients with hyperthyroidism have red blood cell carbonic anhydrase I (CAI) levels that reflect the patient's integrated thyroid hormone level over the preceding few months. In the present study, the effect of T3 on CAI concentrations in the human erythroleukemic cell line, YN-1, was studied. The presence of high affinity (1.9 x 10(9) L/mol), low capacity (20.4 fmol/10(6) cells) T3 binding sites was demonstrated in YN-1 cell nuclei. T3 at a physiological free T3 concentration (0.54 nmol/L) significantly (32 +/- 7%, P < 0.01) decreased CAI concentrations in YN-1 cells before induction of differentiation. The effect of T3 on CAI levels was more obvious (42 +/- 7%, P < 0.01) in the presence of transforming growth factor-beta 1 (TGF-beta 1), which induces hemoglobin synthesis and differentiation in YN-1 cells. These results suggest that the YN-1 cell line may be used to study T3 action on human red blood cell CAI.
The effects of methanol extracts of 80 herbs on hair growth were investigated, using normal C3H/He mice from which telogen hair on the back had been removed. Eighteen of the extracts apparently promoted hair regrowth on the mice. As one of active principles in Polyporus umbellatus F., 3,4-dihydroxybenzaldehyde was isolated by column chromatography on Amberlite XAD-2, Sephadex LH-20 and silica gel.
The present study was performed to examine living conditions of elderly diabetic patients in rural areas in Iwate and Miyagi in northern Honsyu and compared them with similar cases in Tokyo. One hundred and nine cases over 60 years old were interviewed, and results were compared to 109 age- and sex-matched random samples in the Tokyo area. The original Tokyo subjects consisted of 383 outpatients of the Diabetic Clinic of Tokyo Metropolitan Geriatric Hospital. Types of family structure, educational background and total income were quite different in the 3 areas, and patterns of social support depended on area-specific characteristics. Attitudes toward diabetic treatment (acceptance of importance and compliance) in Tokyo were excellent, but the sensation of social burden and dissatisfaction were also at a high level. In contrast, the level of compliance in Iwate was low. Total morale scale scores were similar in the 3 areas. Multivariate analysis (dependent variable is the total morale score) showed significant contribution of levels of ADL and adherent behavioral style only in Tokyo. In contrast, the feeling of social burden was a negative factor in all areas. The quality of life of elderly people with chronic diseases such as diabetes, is affected by ability to adaptation to living conditions.