Biomedical subjects
Publications and source records attributed to M Fujimaki.
A novel MspI DNA polymorphic site has been found in intron 22 of the human factor VIII gene. This site is informative almost exclusively in the Japanese population (heterozygosity 0.45) and will be of considerable importance in carrier detection and prenatal diagnosis of hemophilia A in this population.
As basic studies of hyperthermia and hypothermia on malignant tumor, the kinetics of proliferative activity, the morphologic changes in the two cell lines, SGF-3 and SGF-5, established in our department after the change of culture temperature were examined. The results obtained were: a) A significant difference was found in the sensitivity to temperatures between the two cell lines originated from human esophageal squamous cell carcinoma. The temperature range allowing cultured cell to proliferate were from 31 degrees to 39 degrees C in SGF-3 and from 29 degrees to 41 degrees C in SGF-5. b) Minor difference occurred in the results between the two cell lines examined during the recovery of proliferative activity, but no proliferative activity was discovered after the cells were exposed to 42 degrees C for 72 h. Two cell lines resumed their proliferation after having been exposed to 27 degrees or 28 degrees C for 72 h. c) Morphologic changes of the cell lines cultured at high temperature were cytoplasmic vacuolation and cell aggregation by phase contrast microscope and the increase of heterochromatin, the decrease of granular formation in nucleoli, and nucleolar vacuolation by transmission electron microscopy (TEM). At low temperatures the changes observed included cytoplasmic ballooning and circumnuclear halo formation by phase contrast microscope, and the increase of heterochromatin, nucleolar segregation, swelling of mitochondria, and dilatation of rough endoplasmic reticulum (rER) by TEM.
1. After separate administration of R(+)-carvedilol, S(-)-carvedilol and (+/-)-14C-carvedilol to rats at an oral dose of 10 mg/kg, the metabolic pattern in the bile was studied using h.p.l.c. with radioactivity and u.v. monitoring. 2. Two major metabolites, M-1 and M-2, present in the bile, accounted for 39% and 22% dose, respectively. 3. M-1 and M-2 were characterized as 1-hydroxycarvedilol O-glucuronide and 8-hydroxycarvedilol O-glucuronide, respectively, from FAB-mass spectrometry, 1H-n.m.r. and enzymic hydrolysis. 4. Oral administration of R(+)-carvedilol led to highly selective excretion of M-1 in bile whereas S(-)-carvedilol resulted predominantly in excretion of M-2 rather than M-1.
Reported is the case of a 46 year old woman with no significant family medical history, who presented a neck tumor that she had had for about ten years. This large tumor was noted on her admittance to hospital due to a bone fracture. She subsequently underwent a resection of this giant neck tumor. A histologic examination of the specimen revealed a medullary carcinoma of the follicular variety. Silver impregnation by the Grimelius method caused a positive reaction, and an electron-microscopic examination revealed round, electron-dense, endocrine granules about 195 nm in diameter, Immunohistochemically, the carcinoembryonic antigen (CEA) and the presence of calcitonin proved the cells to be cancerous.
We have developed Tc-99m labeled activated carbon microspheres (99mTc-CH44) for use in mammary lymphoscintigraphy and detailed analysis of mammary lymph flow. 99mTc-CH44 was prepared according to the EDTA complex method. The labeling rate immediately after preparation was 97.7 +/- 2.6% at 37 degrees C and was 94.9 +/- 2.6% after 24-hour continuous shaking. The release of 99mTcO4- from 99mTc-CH44 was determined in vitro, using a simulation model. In this assay, the 99mTcO4- release rate in one hour was 6.6% and that in 6 hours was 38%. When 2ml (2mCi, 74MBq) of 99mTc-CH44 was injected into the mammary glands of 12 patients with breast tumors, the axillary, subclavian and parasternal lymph nodes of 10 patients were visualized 60 minutes later. In analysis of lymph flow in 25 rats using 99mTc-CH44 the lymph flow from the foot pad through the popliteal to the intraperitoneal lymph nodes was known to have only one route when examined by gross finding and histology. However, the examination using RI-technique indicated two routes. The examination in 9 patients with breast cancer indicated that the use of 99mTc-CH44 allowed detailed analysis of lymph flow. These results suggest that 99mTc-CH44 is useful for the preoperative mammary lymphoscintigraphy and detailed analysis of mammary lymph flow.
Polymerase chain reaction amplification and nucleotide sequencing were used to identify the molecular defect in a Japanese patient with mild hemophilia A and an alteration of a TaqI site in exon 26 of the factor VIII gene. The mutation was a G-to-T transversion in codon 2326 of the factor VIII gene resulting in an Arg-to-Leu substitution at amino acid 2307 of the protein. The mutation, which is not of the common CG-to-TG type, is at the same codon in which both nonsense and a different missense (Arg to Gln) have previously been observed.
An immunoadsorbent method has been developed for the direct analysis of normal and variant plasma factor VIII. Using this method, the molecular defect responsible for mild hemophilia A has been identified for a patient whose plasma factor VIII activity is 0.05 unit/ml, even though the factor VIII antigen content is 3.25 units/ml. Although the variant factor VIII has an apparently normal molecular mass and chain composition, the 92-kDa heavy chain accumulates when the variant protein is incubated with thrombin and the 44-kDa heavy chain fragment cannot be detected. In contrast, thrombin cleavage of the 80-kDa light chain to the 72-kDa fragment is normal. As these data indicate a loss of factor VIII cleavage by thrombin at arginine-372, the genetic defect was determined by polymerase-chain-reaction amplification of exon 8 of the factor VIII gene and direct sequencing of the amplified product. A single-base substitution (guanine----adenine) was identified that produces an arginine to histidine substitution at amino acid residue 372. These data identify the molecular basis of an abnormal factor VIII, "factor VIII-Kumamoto," that lacks procoagulant function because of impaired thrombin activation.
1. The metabolic fate of midaglizole, 2-[2-(4,5-dihydro-1H-imidazole-2-yl)-1-phenylethyl]pyridine dihydrochloride sesquihydrate, was studied in rats after a single oral dose of 10 mg/kg. 2. After oral administration of 14C-midaglizole to rats, 63% of the dose was excreted in the urine and 41% in the faeces within 72 h. The major radioactive compound in the urine was unchanged midaglizole and accounted for 38.1% of the dose. In the faeces, two major radioactive compounds, M-VII and unchanged midaglizole, were present. These accounted for 17.2 and 14.1% of the dose, respectively. M-VII is a new metabolite, identified as 2-[2-(4,5-dihydro-1H-imidazole-2-yl)-1-(4-hydroxyphenyl)ethyl]pyridine by n.m.r. and mass spectrometry. 3. The biliary excretion of the radioactivity after oral administration of 14C-midaglizole to bile-duct cannulated rats amounted to 53% of the dose. Of the total amount of radioactivity excreted in the bile, 48% was calculated to be subject to enterohepatic recycling. 4. Four biliary metabolites were new metabolites and were identified by n.m.r., mass spectrometry and enzymic hydrolysis. These compounds are 2-[2-(4,5-dihydro-1H-imidazole-2-yl)-1-(4-hydroxyphenyl)-ethyl]pyridine O-glucuronide (M-XI), 2-[2-(4-hydroxyphenyl)-2-(2-pyridyl)]ethyl-2-imidazole O-glucuronide (M-XII),3-(4-hydroxyphenyl)-3-(2-pyridyl)propioimidamide O-glucuronide (M-XIII) and 2-[2-(4,5-dihydro-1H-imidazole-2-yl)-1-(4-hydroxy- 3-methoxyphenyl)ethyl]pyridine O-glucuronide (M-XIV). These glucuronides accounted for 35.4% of the dose. 5. Midaglizole was metabolized in rats mainly via phenyl ring para-hydroxylation followed by glucuronidation, with or without the biotransformation of the imidazoline ring moiety.
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Multidisciplinary treatment for advanced esophageal carcinoma, which is not resectable in many cases, has become an important subject to study. We have reported the effectiveness of thermotherapy in treatment of esophageal carcinoma by basic experiments using three cell strains of human esophageal carcinoma designated as SGF-3,4,5. Based on the results of the basic experiments, we have performed thermotherapy combined with radiotherapy, chemotherapy and immunotherapy on cases of advanced esophageal carcinoma since October 1985. As for the basic experiment, proliferation of SGF-3 was inhibited at more than 40 degrees C, SGF-4 at more than 41 degrees C and SGF-5 at more than 42 degrees C. We did not observe the recovery of the growth of SGF-3 after heating at 41 degrees C for 72 hours and 42 degrees C for more than 24 hours. And the growth of SGF-4 after heating at 42 degrees C for more than 48 hours did not recover either. No recovery of the growth of SGF-5 after heating at 42 degrees C for 72 hours was noted. Based on the data, combined therapy was performed in 12 patients with advanced esophageal carcinoma. The evaluation of the local response to the combined thermotherapy revealed CR in five cases, PR in five and NC in two.
AOM was administered subcutaneously once a week for 11 consecutive weeks to the rat. On the 15th and 30th week after starting of AOM injection, PGE2 content of the colonic mucosa, tumor and blood of portal vein and NK activity of the spleen and mesenteric lymph node (MLN) were evaluated. On the 15th week, a significant high value of the PGE2 content of colonic mucosa was shown when compared with that of the AOM non-administered group (control group). However, no significant difference was observed in the PGE2 content of blood of portal vein and NK activity of the spleen and MLN. On the 30th week, significant high values of the PGE2 content of AOM-induced tumor and blood of portal vein and low values of NK activity of the spleen and MLN were shown when compared with that of the control group. When indomethacin (IND) was administered intrarectally twice a week for 19 consecutive weeks after completion of AOM injection, induced-colon tumors was significantly suppressed. For this reason, it is important to administer IND at the point when the PGE2 content of colonic mucosa begins to augment.
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Cathepsin B was purified from rabbit skeletal muscle by ammonium sulfate fractionation and successive chromatographies on Sephadex G-75, phosphocellulose, peptide-conjugated Sepharose, DEAE-Toyopearl and Sephadex G-100. The purified enzyme gave a single protein band on SDS/polyacrylamide gel electrophoresis. The enzyme did not abolish the Ca sensitivity of the ATPase activity of myofibrils. The molecular mass of the enzyme was found to be 27 kDa on gel filtration and SDS/polyacrylamide gel electrophoresis. The optimum pH for the hydrolysis of N alpha-benzoyl-DL-arginine-beta-naphthylamide was 6.5. The enzyme was stable in the range of pH 4.5-5.5. Tetrathionate reacted with thiol groups of the enzyme reversibly so that it stabilized the enzyme. The enzyme was strongly inhibited by iodoacetate, HgCl2, antipain, leupeptin, N alpha-p-tosyl-L-lysine chloromethane and L-tosylphenylalanylchloromethane, but not by pepstatin or trypsin inhibitor.
Murine RCT(+) sarcoma cells were sorted using a fluorescence-activated cell sorter with regard to the expression of H-2 antigens and then an increased H-2-expressing subclone was established, and named RCT(+)H-2+. The experimental metastasis of RCT(+) cells was compared with that of RCT(+)H-2+ cells by counting pulmonary colonies on the 21st day after i.v. inoculation of tumor cells (5-10 x 10(4)/mouse). When mice were inoculated with RCT(+) cells, mean numbers of pulmonary colonies were 2.1(range 0-6), 2.8(range 0-7) using 5 x 10(4) and 1 x 10(5) cells, respectively. On the other hand, in the mice inoculated with RCT(+)H-2+ cells, figures obtained were 7.0(range 4-16), 31.9(range 13-79), using 5 x 10(4) and 1 x 10(5) cells, respectively. The survival rate of RCT(+)H-2+ cells was higher than that of RCT(+) cells, when this was assayed in the early stage after i.v. injection of 51Cr-labeled cells (1 x 10(5) cells/mouse). In addition, RCT(+)H-2+ cells were more resistant than RCT(+) cells to lysis mediated by natural killer cells. These data suggest that an increase in metastatic ability was paralleled by an increase in the H-2 antigen expression and a decrease in sensitivity to the natural killer cells.
Myo-inositol uptake by erythrocytes from humans, rabbits and rats was studied with an isotope technique. In human erythrocytes, the inhibitory effect on myo-inositol uptake was stronger with glucose than with ouabain. However, an aldose reductase inhibitor (ONO-2235, 100 microM) or insulin (200 microU/ml) failed to correct the decrease in myo-inositol uptake in packed RBC, produced by either 10 mM glucose or 2mM ouabain. Ten mM ouabain had an inhibitory effect on myo-inositol uptake in all species, but an inhibitory effect was not observed with 20 mM glucose in rabbit erythrocytes. The results suggest that myo-inositol uptake by erythrocytes may be dependent on the active transport system via sodium-ATPase and that erythrocytes may not be a suitable model to monitor the possible effect of an aldose reductase inhibitor on myo-inositol concentrations in other tissues concerned with diabetic complications.
Two kinds of dosage forms of commercially available suppositories containing ketoprofen (KP), fatty suppositories (FS) and gelatin capsulated suppositories (GCS), were administered to patients immediately after anal surgery, and results obtained were compared. No difference was found in each corresponding pharmacokinetic parameter of the two dosage forms. However, when these parameters were compared with those from healthy subjects, significant differences were found in the values of peak level (Cmax), peak time (Tmax) and terminal phase half-life (t1/2). Cmax decreased by one half, and Tmax and t1/2 increased two and four times longer, respectively, those from healthy subjects. The absorption rate constant (ka) in patients was significantly (p less than 0.01) smaller than that in healthy subjects. However, the distribution volume/bioavailability (Vd/F), elimination rate constant (kel), and area under the curve (AUC) differed only slightly. Consequently, the flip-flop phenomena could be seen in the time profiles of plasma KP concentration of patients. These results suggested that the rectal suppository of KP should be administered with care, especially in the patients operated on under spinal anesthesia.