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Biomedical subjects

N Miwa

Publications and source records attributed to N Miwa.

At least 91 records · Page 5Linked to original sources
PubMed1983

The effect of interaction between human urokinase and its competitive inhibitor, N alpha-benzoyl-L-arginine amide, on reduction of a specific SS bond related to enzymatic activity.

The 55- (H-UK) and 36-kDa forms (L-UK) of human urinary urokinase lost most of esterase activity toward acetyl-glycyl-L-lysine methyl ester upon reductive cleavage of 3 SS bonds with dithiothreitol in the presence of the competitive inhibitor, N alpha-benzoyl-L-arginine amide (BAA), bound to polyacrylyl azide with C16N3-arm (PAA) at 0.3 M guanidine, a threshold point of the native state where a protein-denaturating transition began. One of the 3 SS bonds was protected from reduction, with an unaltered activity, under the similar conditions except for replacement of BAA-PAA conjugate by glycine-PAA conjugate. This "specific" SS bond was reduced and, after the other SH groups produced were blocked with iodoacetamide (IAM), selectively reoxidized, which resulted in complete reactivation. The intact B-chain isolated from H-UK was completely inactivated when its specific SS bond was reduced and selectively alkylated with IAM after the other SH groups were reversibly blocked with 5, 5'-dithiobis (2-nitrobenzoic acid), which was finally removed. The results indicate that a single specific SS bond is essential for retaining a conformation necessary to activity exhibition.

Arginine↗
PubMed1983

Receptor-mediated introduction of pepstatin-asialofetuin conjugate into lysosomes of rat hepatocytes.

Pepstatin was linked through a carboxyl group to asialofetuin (PS-ASF). An analysis by separation of hepatocytes from nonparenchymal cells showed that PS-ASF was taken up by hepatocytes, following intravenous injection into rats. After the injection of PS-ASF, pepstatin concentration in the liver reached a maximum at 2 h and then decreased. In an analysis by differential centrifugation of the liver homogenate from rats injected with PS-ASF, pepstatin showed a lysosomal type subcellular distribution pattern. Isolation studies of tritosomes clearly demonstrated the exclusive accumulation of pepstatin within the lysosomes of livers from rats given PS-ASF (at 2 h after administration). Pepstatin contained in tritosomes was in a free form, as determined by column chromatography of Sephadex G-15. The activity of cathepsin D in the livers was markedly inhibited in rats given PS-ASF. However, the treatment of rats with PS-ASF had no effect on the hepatic lysosomal degradation of endocytosed FITC-labeled asialofetuin (FITC-ASF). Introduction of PS-ASF into the hepatocytes was followed by the immediate and time-dependent excretion of free pepstatin into the bile. Quantification of pepstatin excreted into the bile revealed that the biliary excretion route can account for the disappearance of pepstatin from the liver.

Animals↗
PubMed1983

Differential cytotoxicity to human lung normal diploid, virus-transformed and carcinoma cells by the antitumor antibiotics, auromomycin and macromomycin, and their non-protein chromophores.

With the use of three human lung cultured cell lines: normal diploid fibroblasts (WI38), their SV40-transformants (VA13) and carcinoma cells (A549), whose doubling times were similar, the cytotoxicity of the protein antitumor antibiotics, auromomycin (AUR) and macromomycin (MCR), was studied by colony formation method. The susceptibilities of the three cell lines to these antibiotics were in the order: WI38 less than VA13 less than A549. This differentiality in the cytotoxic effect was similar between AUR and MCR. The differential cytotoxic effect did not depend on cell densities of each cell line at the time of the antibiotic-treatment. Alcohol-extracted chromophores of the antibiotics also showed a similar differential cytotoxic effect, while the apo-proteins of the antibiotics showed no cytotoxicity. It is concluded that the differential cytotoxic effect of AUR and MCR to normal, transformed and carcinoma cells is attributed to the chromophore moiety and the protein moiety is not involved in this effect.

Anti-Bacterial Agents↗
PubMed1983

Role of pronase-resistant peptide segments of the antitumor protein antibiotics, auromomycin and macromomycin, in stabilizing cytocidal activity of the chromophore moieties to carcinoma cells.

The free chromophores isolated from the antitumor protein antibiotics, auromomycin (AUR) and macromomycin (MCR), were rapidly inactivated by incubation in serum-containing medium at 37 degrees C in the dark with respect to cytocidal activity to human lung carcinoma A549 cells. Under the same conditions, the intact antibiotics, their pronase-hydrolysates and reconstituents from the chromophores and apo-proteins were stable. Intact and reconstituted AUR and MCR were more resistant to pronase digestion than the apo-proteins. The analyses of the pronase-hydrolysates of AUR and MCR by SDS-polyacrylamide gel electrophoresis and ultrafiltration showed that the antibiotics (13 kilodaltons (kDa] were degraded to produce peptide fragments (1-3 kDa) in which most cytotoxicity of the pronase-hydrolysates resided. The pronase-hydrolysates exhibited a differential cytocidal activity to normal diploid fibroblasts (WI38), their SV40-transformants (VA13) and carcinoma cells (A549) of human lung origin as was observed for the intact antibiotics. These results indicate that specific interaction between the chromophores and the pronase-resistant peptide segments (1-3 kDa) of the protein moiety stabilizes the cytocidal activity of the chromophores and also protects the peptide segments from pronase digestion.

Anti-Bacterial Agents↗
PubMed1983

Differential enhancement of cytotoxicity by combination of the carcinostatic agent benzaldehyde and hyperthermia in simian virus 40-transformed and normal cell lines.

The effect of the carcinostatic drug benzaldehyde (BA) on hyperthermia-induced cytotoxicity was studied in various types of cultured mammalian cell lines by colony formation assay. The treatment of HeLa cells with nontoxic doses of BA (1 to 4 mM) at 42 and 43 degrees for up to 3 hr induced enhancement of cell killing. BA-induced enhancement of hyperthermic cell killing was much greater in SV40-transformed rat fibroblast cells than in the untransformed cell line. This BA-induced differential enhancement of hyperthermic cell killing increased as the time and dose of BA treatment were increased at elevated temperatures. For example, BA (2 mM) treatment for 4 hr at 42 degrees decreased the survival of transformed cells to 2 X 10(-4), but decreased the survival of untransformed cells to only 0.1. Furthermore, BA (4 mM) treatment for 2 hr at 42 degrees reduced the survival of transformed cells to less than 10(-5), but reduced the survival of untransformed cells to 5 X 10(-2). Differential enhancement of hyperthermic cytotoxicity by BA was also evident between normal human diploid fibroblast W138 cells and its SV40-transformed cell line, although the magnitude of the enhancement was less than that observed with rat cell lines. The determination of BA remaining in the medium after incubation with transformed and untransformed rat cells at 37, 42, and 43 degrees suggested that greater amounts of BA reacted with or were consumed by transformed cells irrespective of temperature.

Animals↗
PubMed1982

Conformations of protein moieties and chromophore-protein interactions in the antitumor antibiotics, macromomycin and auromomycin, characterized by IR and CD spectral analysis.

The free chromophores (chr) extracted from macromomycin (MCR) and auromomycin (AUR) showed different IR spectra in KBr tablets and different CD spectra in ethanol or methanol solution. Chr-MCR contained a greater amount of alkyl moieties than chr-AUR as shown by much stronger IR absorbance at 2960 cm-1. The IR spectra of MCR, AUR and their apo-proteins (apo) in D2O showed that major parts of the proteins formed antiparallel beta-pleated sheets. Apo-AUR exhibited a faster H-D exchange than AUR, indicating existence of more extensive "nonmotile parts" of the beta-sheet formed through the chromophore-protein interaction in AUR. However, apo-MCR exhibited a slightly faster H-D exchange than MCR. This difference presumably was associated with the lower content of the chromophore (1.4 wt%) in MCR than that in AUR (8.2 wt%). Chr-AUR was extracted from the intact antibiotic with methanol more efficiently than chr-MCR. Further, a marked bathochromic effect by the chromophore-protein interaction was observed in the CD and UV absorption for MCR, but not for AUR. These results indicated that chr-MCR was tightly bound to the protein moiety while most of chr-AUR was loosely bound.

Anti-Bacterial Agents↗
PubMed1976

Studies on an alpha-amylase from a thermophilic bacterium. I. Purification and characterization.

An amylase-producing thermophilic bacterium was isolated from a Japanese hot spring. The thermophilic cells were gram-negative, nonsporulating, nonpigmented rods and were motile with flagella. Alpha-Amylase [EC 3.2.1.1) purified from the thermophile was studied. The enzyme was more heat-stable than the corresponding enzymes from mesophilic sources, and 50% loss of activity was observed after incubation for 1 hr at 90 degrees. The thermophilic enzyme resembled the corresponding mesophilic enzymes in many respects, such as kinetic parameters, physicochemical properties, and amino acid composition. The molecular weight of the enzyme was 50,000 and the enzyme contained 2 moles of half-cystine residues per mole of protein, but there were no disulfide crosslinkages. The alpha-helix content of the enzyme was estimated to be about 20% from CD spectra, a value similar to those of other alpha-amylases. The isoelectric point of the enzyme was at pH 9.2.

Amino Acids↗
PubMedDate not supplied

Paramagnetic water-soluble metallofullerenes having the highest relaxivity for MRI contrast agents.

Water-soluble gadolinium (Gd) endohedral metallofullerenes have been synthesized as polyhydroxyl forms (Gd@C(82)(OH)(n)(), Gd-fullerenols) and their paramagnetic properties were evaluated by in vivo as well as in vitro for the novel magnetic resonance imaging (MRI) contrast agents for next generation. The in vitro water proton relaxivity, R(1) (the effect on 1/T(1)), of Gd-fullerenols is significantly higher (20-folds) than that of the commercial MRI contrast agent, Magnevist (gadolinium-diethylenetriaminepentaacetic acid, Gd-DTPA) at 1.0 T close to the common field of clinical MRI. This unusually high proton relaxivity of Gd-fullerenols leads to the highest signal enhancement at extremely lower Gd concentration in MRI studies. The strong signal was confirmed in vivo MRI at lung, liver, spleen, and kidney of CDF1 mice after i.v. administration of Gd-fullerenols at a dose of 5 micromol Gd/kg, which was 1/20 of the typical clinical dose (100 micromol Gd/kg) of Gd-DTPA.

Animals↗