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Biomedical subjects

H Fukui

Publications and source records attributed to H Fukui.

At least 415 records · Page 23Linked to original sources
PubMed1988

Blood clotting factor IX Niigata: substitution of alanine-390 by valine in the catalytic domain.

Factor IX Niigata is a mutant factor IX responsible for the moderately severe hemophilia B in a patient who has a normal level of factor IX antigen with reduced clotting activity (1-4% of normal). We reported previously that the purified mutant protein could be converted to the factor IXa beta form by factor XIa/Ca2+ at a rate similar to that in the case of normal factor IX, but the resulting mutant factor IXa beta could not activate factor X in the presence of factor VIII, Ca2+, and phospholipids (Yoshioka, A. et al. (1986) Thromb. Res. 42, 595-604). In the present study, we analyzed factor IX Niigata at the structural level to elucidate the molecular abnormality responsible for the loss of clotting activity. Amino acid sequence analysis of a peptide obtained on lysyl endopeptidase digestion, coupled with subsequent SP-V8 digestion, demonstrated that the alanine at position 390 was substituted by valine in the catalytic domain of the factor IX Niigata molecule.

Alanine↗
PubMed1988

Marked increase in ascorbate oxidase protein in pumpkin callus by adding copper.

Ascorbate oxidase from pumpkin (Cucurbita sp.) was purified from a commercially available preparation. A single polypeptide band with M(r) 64,000 was detected after sodium dodecylsulfate-polyacrylamide gel electrophoresis of the purified enzyme. In double immunodiffusion tests, antiserum against the purified preparation formed a single precipitin line with the crude extract from pumpkin fruit tissue or the callus as well as with the purified preparation. Immunological blotting method showed that mol wt of ascorbate oxidase subunit in pumpkin callus was the same as that of the purified preparation. Analysis with the single radial immunodiffusion method showed that the increase in ascorbate oxidase activity during the growth of pumpkin callus correlated with an increase in the enzyme protein. Furthermore, enzyme protein in the callus grown in the presence of 10 micromolar CuSO(4) for 2 weeks was about eight times that grown in the presence of 0.1 micromolar CuSO(4). The synthesis of ascorbate oxidase in pumpkin callus may be induced by copper, a prosthetic metal of the enzyme, or copper may help stabilize the enzyme against proteolytic breakdown.

Journal Article↗
PubMed1988

Factor VIII polypeptide specificity of monoclonal anti-factor VIII antibodies.

Four monoclonal antibodies against factor VIII, NMC-VIII/1, NMC-VIII/2, NMC-VIII/3 and NMC-VIII/4, were produced. The first three antibodies were of the IgG1 immunoglobulin subclass, while the fourth was IgM. The affinity of each antibody for factor VIII was high and anti-factor VIII clotting activity was detected in NMC-VIII/2, NMC-VIII/3 and NMC-VIII/4. NMC-VIII/1 had no inhibitory effect on factor VIII clotting activity. Immunoblotting using purified intact and thrombin-treated factor VIII identified the antibodies' factor VIII polypeptide specificities. With thrombin-treated factor VIII the factor VIII fragments for NMC-VIII/1 and NMC-VIII/2 were 80 kDa and 54 kDa, respectively, while both NMC-VIII/3 and NMC-VIII/4 recognised a 44 kDa fragment. With intact factor VIII, antibodies NMC-VIII/2-4 bound to polypeptides larger than 90 kDa, especially NMC-VIII/2, which reacted with a chain of 330 kDa thought to be a mature form of factor VIII protein. We also detected a mature form of factor VIII in hepatic sinusoidal endothelial cells by immunoperoxidase staining. Identification of the factor VIII polypeptides bearing these fragments enabled us to clarify the localization of the factor VIII thrombin cleavage site, and suggested the existence of factor VIII in hepatic sinusoidal endothelial cells.

Animals↗
PubMed1988

Disappearance of inhibitor to factor IX in a patient with severe haemophilia B and immunological characterization of the inhibitor.

Disappearance of an inhibitor to factor IX in an 11-year-old boy with haemophilia B is described. He had been given a total of 14,200 units of a prothrombin complex concentrate (PCC) before an inhibitor to factor IX developed. He subsequently received four separate infusions of PCC and his inhibitor titre rose in response to the treatment for the following 4 years. No inhibitor is presently detected despite repeated administration of PCC. Immunological characterization of the inhibitor by inhibitor neutralization assays, modified crossed-immunoelectrophoresis and enzyme-linked immunosorbent assay demonstrated that it contained IgG2 and IgG4 heavy chains and kappa and lambda light chains. No large deletion of the factor IX gene in the patient was observed using cDNA (cVII).

Child↗
PubMed1988

Solubilization, characterization and partial purification of [3H]mepyramine-binding protein, a possible histamine H1 receptor, from rat liver membrane.

[3H]Mepyramine binding protein, a possible subtype of histamine H1 receptors, was solubilized from rat liver membrane with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and Tween 60 as detergents and glycerol as an enhancer of solubilization. The optimal concentration of CHAPS was 10 mM and that of glycerol was 20% or more (v/v). The molecular weight of the [3H]mepyramine binding protein-detergent complex was determined to be 670K by Sepharose CL-4B gel filtration and 800K by sucrose density gradient sedimentation. By target size analysis, the molecular weights of both the membrane-bound and solubilized [3H]mepyramine binding protein were determined to be 162K. These values are similar to those of other well-characterized H1-receptor proteins, though slightly different. Simultaneous computerized analysis of the data obtained by [3H]mepyramine binding to the solubilized [3H]mepyramine binding protein indicated the presence of a single binding site with a KD value of 19.0 +/- 5.6 nM and a binding capacity (Bmax) of 6.6 +/- 2.1 pmole/mg protein. The Ki value of cold mepyramine for [3H]mepyramine binding to the solubilized receptor was 20 +/- 4 nM, whereas those of diphenhydramine, d-chlorpheniramine and triprolidine were all 2.9 +/- 0.8 microM, or about 150 times that of mepyramine. These data on the molecular and binding characteristics of the solubilized protein reported here suggest that there is a subtype of histamine H1 receptor in rat liver membrane. The solubilized preparation retained 90% and 75% of its [3H]mepyramine binding activity after storage at -80 degrees C and 4 degrees C, respectively, for 20 days. The solubilized [3H]mepyramine binding protein was purified 30-fold by Sepharose CL-4B gel filtration, Bio Gel HTP hydroxylapatite, Octyl Sepharose 4B and hydroxylapatite HPLC column chromatographies.

Aminopyridines↗
PubMed1988

17-beta-estradiol treatment maintains differentiative potential of virgin mouse mammary gland in culture and its responsiveness to insulin.

Synthesis of the milk proteins, casein and alpha-lactalbumin was not induced in cultured mammary explants from C3H/HeN castrated virgin mice in the presence of lactogenic hormones such as insulin, cortisol and PRL. Replacement therapy with 17-beta-estradiol to the castrated mice completely restored the differentiative potentials of mammary explants, inducing synthesis of the two milk proteins. [3H]thymidine incorporation into DNA synthesized in cultured mammary explants was also decreased by castration to less than 50% (P less than 0.001) of that obtained in intact mice, and 17-beta-estradiol treatment to castrated animals restored DNA synthesis to 90% of the intact level. The addition of insulin to culture medium significantly (P less than 0.001) enhanced [3H]thymidine incorporation into DNA in cultured mammary explants from both intact and 17-beta-estradiol-treated castrated mice but not from castrated animals. Insulin binding sites (1710 +/- 260 sites/cell) to mammary epithelial cells from castrated mice were significantly (P less than 0.05) lower than those from both intact (2870 +/- 300 sites/cell) and 17-beta-estradiol-treated castrated animals (2860 +/- 190 sites/cell). The present findings suggest that 17-beta-estradiol maintains growth and differentiative responses of mammary epithelial cells to insulin, which may be through preserving the number of insulin binding sites in the cells.

Animals↗
PubMed1988

[Multidisciplinary treatment of esophageal carcinoma].

From 1965 until Aug. 1987, a total of 1,249 cases of primary thoracic esophageal carcinoma were resected. Some 70%-80% of these cases treated before 1980 underwent preoperative irradiation. From 1976-1987, in addition to preoperative irradiation, postoperative irradiation of the neck and upper mediastinum was performed in 20% of resected cases. (1) Evaluation of combined radiotherapy: In C greater than 0 resected cases, the postoperative T-shape irradiated group was significantly better than the nonirradiated and preoperative irradiation--only groups, with a risk factor of p less than 0.05, However in comparison to historical controls, no significant improvement was obtained and in the preoperative irradiated group. (2) Multidisciplinary treatment based on preoperative staging: The effectiveness of CDDP treatment in cases of recurrence or non-resectable cancer of the esophagus was 44.4%. Based on these findings, we have administered adjuvant CDDP + VDS in resected cases from 1985 and have obtained encouraging 1-year survival. Since 1985, extended dissection in 3 regions (cervical, thoracic and abdominal) has been carried out. Compared to C greater than 0 resected since 1981, in which dissection of lymph-nodes in two major regions was performed, dissection of lymph-nodes in the three major regions has resulted in an improved 1-2 year survival, despite the increased numbers of stage IV cases. The findings thus indicate that the presence of lymph-node metastasis significantly affects prognosis. Therefore, in cases that are preoperatively evaluated as sm or more extensive invasion, it is necessary to combine extensive lymph-node dissection, postoperative irradiation or chemotherapy.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1988

[Multidisciplinary treatment of thoracic esophagus carcinoma based on preoperative staging].

Recently, with developments in CT scanning and endoscopic ultrasound (EUS), in addition to conventional ultrasound (US), significant advances have been made in the evaluation of preoperative staging. Evaluation of the presence of A3 invasion is made on the basis of X-ray, CT and EUS findings. In superficial cases of ep, mm or sm, EUS with particular reference to the X-ray and endoscopic appearance is very effective, and in our department the diagnostic accuracy in cases of mucosal lesions is 88%, while it is 84% in sm cases. Diagnosis of metastasis to cervical and abdominal lymph nodes is performed by US, while diagnosis of mediastinal lymph node metastasis is performed by EUS. The accuracy of US for detecting cervical and abdominal lymph node metastases, including those approximately 0.5 cm in size, is 95%, while that of EUS in the diagnosis of metastasis to mediastinal nodes is 89%. The 5-year survival of patients with lymph node metastasis significantly affects prognosis. Based on this, since 1985, extended dissection in the cervical, thoracic and abdominal regions has been carried out. Since 1985, some resected cases have been treated with pre- or postoperative adjuvant chemotherapy regimens, centered primarily on CDDP (majority of cases with CDDP + VDS). A significant improvement has been obtained in the 1-2-year survival of C greater than O resected cases treated since 1985, compared with cases in 1980-1984. This improvement is thought to be due to dissection of lymph nodes in the three major regions, based on more accurate preoperative evaluation and the introduction of postoperative adjuvant chemotherapy.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1987

Different localization of receptors for omega-conotoxin and nitrendipine in rat brain.

The bindings of radioiodinated omega-conotoxin GVIA and [3H]-nitrendipine to subcellular fractions of rat brain were examined. The results indicated that omega-conotoxin binding site was mainly present in the mitochondrial fraction, whereas nitrendipine binding site was rich in the mitochondrial but also present in the post-mitochondrial fraction. Fractionation of the mitochondrial fraction on a sucrose density gradient centrifugation showed that the both binding sites were localized in the heavy synaptosomal fraction. These results strongly suggest that the N- and L-type voltage-sensitive calcium channels have different localizations.

Animals↗
PubMed1987

In vitro characterization of various heat-treated prothrombin complex concentrates (PCC).

All of 6 heat-treated prothrombin complex concentrates (PCC) tested contained adequate levels of factor IX but factor VII content was low. Levels of factors II, X, protein C and protein S were variable and antigen levels were always greater than those of functional activities. On crossed-immunoelectrophoresis factor IX showed variable anodal shift in all concentrates tested and in some activated factor IX was demonstrated by immunoblotting technique. These findings suggested some activation and/or denaturation during production and/or heating. Modest amount of factor VIII clotting activity by solid-phase amidolytic method and of factor VIII antigen was demonstrated in some concentrates but none contained more than a trace factor VIII inhibitor bypassing activity. The results suggested that heat-treated PCC should provide safe therapeutic products for hemophilia B.

Blood Coagulation Factors↗
PubMed1987

A simple method for analyzing factor IX activation in the patients with hemophilia B variants.

A simple method for analyzing the activation mechanism of FIX in patients with hemophilia B variants is described. The procedure consists of rapid partial purification of FIX by BaCl2 adsorption-elution from only 3 ml of plasma, incubation with FXIa/Ca2+, SDS-PAGE, western blotting and subsequent autoradiography using monoclonal anti-FIX antibody. Abnormal FIX from the plasma of 7 unrelated patients with hemophilia BR, B+ or BM was investigated. A time course study showed that FIX in the patient with hemophilia BM (Nagoya I), BM (Nagoya II) and B Kawachinagano seemed not to be cleaved by FXIa, FIX in the patient with hemophilia B Kashihara was partially cleaved, FIX in the patient with hemophilia BM (Takatsuki) showed delayed cleavage, and that FIX in the patient with hemophilia BM (Niigata) and BM (Kiryu) was cleaved completely at a rate similar to normal FIX. These findings were identical to those previously observed for the respective factors in a purified system. The procedure used here is useful for screening for a defective activation mechanism of abnormal FIX.

Factor IXa↗
PubMed1987

Demonstration of histaminergic neurons in horizontal cells of guinea pig retina.

The existence of L-histidine decarboxylase (HDC, EC 4.1.1.22)-like immunoreactive (HDC-I) cells in guinea pig retina was demonstrated using antiserum raised against HDC purified from fetal rat liver. The anti-HDC antiserum partially cross-reacted guinea pig L-DOPA decarboxylase (DDC, EC 4.1.1.28), so the histaminergic neurons were carefully identified. Comparison of HDC-I and DDC-like immunoreactive (DDC-I) cell types in adjacent sections revealed that HDC-I structures were found in some horizontal cells and amacrine cells, and double-staining procedures with anti-HDC antiserum and monoclonal anti-DDC antibody showed that HDC-I horizontal cells had no DDC-I structures, but all the HDC-I amacrine cells had DDC-I structures. From the results, some horizontal cells (with HDC-like immunoreactivities but without DDC-like immunoreactivities) were concluded to be histaminergic.

Animals↗