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K Fukuyama

Publications and source records attributed to K Fukuyama.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of two dipeptidases purified from hepatic schistosome egg granulomas in mice. Leukotriene D4 hydrolases of granulomatous tissue.

Extracts prepared from tissue with granulomatous inflammation experimentally produced in liver of CBA-strain mice showed increased hydrolysis of leukotriene D4 (LTD4), Leu-Leu and Ala-Gly as compared with normal hepatic cells. Two dipeptidases, Leu-Leu dipeptidase and Ala-Gly dipeptidase, were purified from hepatic granulomas, and quantitative conversion of LTD4 into leukotriene E4 (LTE4) by both enzymes was demonstrated. M(r) values of the purified enzymes were 178,000 for Leu-Leu dipeptidase and 183,000 for Ala-Gly dipeptidase. The enzymes showed homogeneity, appearing as a single band on SDS/PAGE, and the M(r) values of the subunits were 56,000 and 57,000 for Leu-Leu and Ala-Gly dipeptidase respectively. The amino acid compositions of the two enzymes differed considerably from each other. The activity of Leu-Leu dipeptidase was inhibited by bestatin and captopril and stabilized with MnCl2. The Km for LTD4 was 25 microM with a V(max.) of 49.0 mumols/min per mg. In contrast, the activity of Ala-Gly dipeptidase was inhibited by cilastatin, cytinylglycine, EDTA and dithiothreitol, and also by captopril. The Km for LTD4 was 5.3 microM with a V(max.) of 50.4 mumols/min per mg. The findings indicate that the conversion of LTD4 into LTE4 by microsomal dipeptidases is elevated during granulomatous tissue reaction. This enzyme activity may become useful for biochemical quantification of the pathological tissue reaction that occurs in organized granulomas.

Amino Acid Sequence↗
PubMed1992

Crystal structure of oxidized flavodoxin from a red alga Chondrus crispus refined at 1.8 A resolution. Description of the flavin mononucleotide binding site.

In order to describe the detailed conformation of the oxidized flavodoxin from a eukaryotic red alga, Chondrus crispus, the crystal structure has been refined by a restrained least-squares method. The crystallographic R factor is 0.168 for 13,899 reflections with F greater than 2 sigma F between 6.0 and 1.8 A resolution. The refined model includes 173 amino acid residues, flavin mononucleotide (FMN) and 110 water molecules. The root-mean-square deviation in bond lengths from ideal values is 0.015 A, and the mean co-ordinate error is estimated to be 0.2 A. The FMN is located at the periphery of the molecule. The orientation of the isoalloxazine ring is such that the C-7 and C-8 methyl groups are exposed to solvent and the pyrimidine moiety is buried in the protein. Three peptide segments, T8-T13, T55-T58 and D94-C103, are involved in FMN binding. The first segment of T8-T13 enfolds the phosphate group of the FMN. The three oxygen atoms in the phosphate group form extensive hydrogen bonds with amide groups of the main chain and the O gamma atoms of the side-chains in this segment. T55 O and W56 N epsilon 1 in the second segment form hydrogen bonds with O-2 in the ribityl moiety and one of the oxygen atoms in the phosphate group, respectively. The O gamma H of T58 forms a hydrogen bond with the N-5 atom in the isoalloxazine ring, which is expected to be protonated in the semiquinone form. The third segment is in contact with the isoalloxazine ring. It appears that the hydrogen bond acceptor of the NH of Asp94 in the third segment is O-2 rather than N-1 in the isoalloxazine ring. The isoalloxazine ring is flanked by the side-chains of Trp56 and Tyr98; it forms an angle of 38 degrees with the indole ring of Trp56 and is almost parallel to the benzene ring of Tyr98. The environment of the phosphate group is conserved as in other flavodoxins whereas that of the isoalloxazine ring differs. The relationship between the hydrogen bond to the N-5 in the ring and the redox potential for the oxidized/semiquinone couple is discussed.

Amino Acid Sequence↗
PubMed1992

Prolyl endopeptidase purified from granulomatous inflammation in mice.

Activity of prolyl endopeptidase (EC 3.4.21.26) which hydrolyses the Pro7-Phe8 bond in angiotensin II has been found to elevate in experimentally produced granulomatous inflammation in liver and skin. We purified the enzyme 1,536-fold by 6 steps from murine hepatic granulomas. The purified enzyme has a molecular weight of 79 kDa and physicochemical properties equivalent to those previously reported for prolyl endopeptidase purified from other sources. By HPLC analysis, the cleavage of Phe8-Leu10 and Phe8 from angiotensin I and II, respectively, was detected and quantified. Monospecific IgG was prepared from serum of rabbits injected with purified enzyme. Concentration of the enzyme was immunohistochemically detected in cells which form granulomatous organization, but not in inflammatory cells surrounding the foci. The antibody, however, cross reacted with the enzyme in adjacent liver cells and weakly stained their cytoplasm. The findings indicate that this enzyme, in addition to angiotensin converting enzyme, may serve as a useful biochemical marker for granulomatous tissue reactions.

Angiotensin I↗
PubMed1992

Peripheral acting muscle relaxants alter the effects of baclofen on the electrocorticograms in the rat.

The effects of intraperitoneally administered baclofen on behavior and electrocorticograms (ECoG) were studied in freely moving and paralyzed rats. In the freely moving rats, 10 mg/kg of baclofen suppressed ECoGs with behavioral sedation, and 20 mg/kg caused whole body twitching accompanied with spike discharges in low voltage slow waves background. In the freely moving rats, baclofen acted as depressant in low dose and convulsant in high dose. In the paralyzed rat, 10 mg/kg of baclofen first suppressed ECoGs. Though fast waves components recovered with slow waves background in the ECoGs of rats paralyzed with gallamine (50 mg/kg) 3 hours after the baclofen injection, fast waves activities were not observed in the ECoG of rats paralyzed with succinylcholine (50 mg/kg), d-tubocurarine (3 mg/kg) or pancuronium (1 mg/kg). Under paralysis with d-tubocurarine, baclofen suppressed ECoG first, and then spike discharges started 90 min after the injection. These results suggest that the effects of baclofen on ECoG were different depending on the muscle relaxant used.

Animals↗
PubMed1992

Detection of human papillomavirus genome and analysis of expression of c-myc and Ha-ras oncogenes in invasive cervical carcinomas.

Invasive carcinomas of the uterine cervix of 38 patients were examined for the presence of human papillomavirus (HPV) genomes and for the state of the c-myc and Ha-ras oncogenes. A combination of Southern blot hybridization and polymerase chain reaction revealed the presence of the genome of HPV type 16 in 17 tumors (45%), that of HPV type 18 in 3 tumors (8%), and that of unknown types in 16 others (42%), while no viral DNA sequences were detected in 2 tumors. Of the 38 tumors, c-myc amplification was found in only 1 tumor, while there was no Ha-ras amplification. Overexpression of the c-myc gene was observed in 15 (44%) of the 34 tumors analyzed, while there was no overexpression of Ha-ras. Of the 23 squamous cell carcinomas analyzed, relapse-free rates at 24 months were 55% in tumors with c-myc overexpression and 100% in case of tumors with no c-myc overexpression, respectively. The results suggest the possibility that activation of the c-myc oncogene is involved in tumor progression.

Base Sequence↗
PubMed1992

Direct inactivation of herpes simplex virus type-2 by rat epidermal protein.

Proteins were extracted from corneocytes of skin of 2-day-old rats and fractionated by gel filtration and cation exchange column chromatography. The different protein fractions were tested for direct inactivation of herpes simplex virus infectivity as determined by reduction of plaque formation. The most effective protein fractions against herpes simplex virus were a neutral pH buffer soluble and mol. wts. ranging from 20 K to 30 K. Amino acid composition of the proteins were virtually identical to epidermal histidine-rich proteins. The activity was significantly (P less than 0.001) stronger against type-2 than type-1. The activity was most stable at pH 7.2 and the rate of inhibition increased in a time-dependent manner up to 4 h. The 50% effective dose was estimated as 1.1 micrograms protein/ml.

Amino Acids↗
PubMed1992

Altered structure and expression of the p53 gene in human neuroepithelial tumors.

The p53 gene, located on chromosome 17p13.1, may be important in the pathogenesis of human neuroepithelial tumors, because it is a tumor suppressor gene and genetic alteration is essential for certain human cells to acquire the neoplastic phenotype. The structure and expression of the p53 gene were investigated in cultured human glioma cells and biopsied specimens of neuroepithelial tumors. Immunocytochemical examination of p53 gene expression revealed positive nuclear staining in six of seven glioma cell lines tested. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis demonstrated unequivocal heterogeneity of migration rate in p53 bands. Pulse-chase analysis clearly showed an increased half-life of p53 in cultured human glioma cells. These abnormalities are presumably due to genetic alterations in the p53 gene. Nucleotide substitutions in exon 5, 7, or 8 of the p53 gene could be detected by polymerase chain reaction-single strand conformational polymorphic analysis in four of seven (57%) human glioma cell lines, and nine of 29 (31%) biopsied specimens of neuroepithelial tumors examined. The present results indicate that genetic alterations in the p53 gene are responsible for the tumorigenesis of at least some human neuroepithelial tumors.

Astrocytoma↗
PubMed1992

Isolation and characterization of granuloma initiation factor.

A soluble component that transfers granulomatous tissue reaction was fractionated from Schistosoma mansoni egg-induced hepatic granulomas (SMHG) by Sephacryl S-300 column chromatography. The fractions separately bound to inert, Affi-Gel agarose beads were inoculated subcutaneously in naive mice. The low molecular weight fraction, consisting of proteins 23 kd, 20 kd, and 16 kd, produced organized granulomas 6 to 7 weeks after inoculation. This fraction was further purified by high-pressure liquid chromatography (HPLC) gel filtration and gave three fractions eluting at retention times of 44, 46, and 48 minutes. Each fraction contained all low-molecular-weight proteins in varying amounts and induced skin granulomas when inoculated subcutaneously. Amino acid sequence of the major 20-kd protein showed 11 N-terminal residues identical to those of cyclophilin. Antisera raised to the protein with retention time of 46 minutes, reacted with cells in the granulomas but not surrounding liver tissue as detected by immunofluorescence microscopy. The findings indicate a low molecular weight soluble fraction of SMHG can induce new granuloma formation when injected in an immobilized form into skin of naive mice. The results suggest granuloma initiation factor is a homolog of the cyclophilin gene family.

Animals↗
PubMed1991

Crystallization and preliminary X-ray crystallographic studies of bovine heart mitochondrial cytochrome bc1 complex.

Cytochrome bc1 complex (ubiquinol:ferricytochrome c oxidoreductase, EC. 1.10.2.2) from bovine heart mitochondria was crystallized by a batchwise method from protein solution containing sucrose monolaurate using polyethylene glycol-4000 as a precipitant. The red parallelepiped crystals grew to a size of approximately 1 mm x 1 mm x 1 mm. The crystalline protein showed enzymic activity catalyzing electron transfer from ubiquinol-2 to cytochrome c. The subunit composition and absorption spectrum of the crystalline enzyme were identical to those reported previously for the enzyme in solution. The crystal diffracted X-rays to 7.5 A resolution. The diffraction pattern indicated a monoclinic form, space group P2(1), and unit-cell constants of a = 196 A, b = 179 A, c = 253 A and beta = 97 degrees. Most probably four functional units are present in an asymmetric unit.

Animals↗
PubMed1991

Macrophage chemotactic factor partially purified from granulomatous inflammation.

Pathophysiological roles of macrophage chemotactic factor (MCF) in granulomatous inflammation were investigated. MCF was extracted in 10 mM phosphate-buffered saline, pH 7.4, from experimentally produced epithelioid cell granulomas in the liver and skin of mice. MCF activity reached a peak in the lesions prior to the time when granulomatous inflammation became maximal. MCF was then purified from 10-week-old hepatic granulomas and 2-week-old skin lesions by gel filtration, ion exchange column chromatography, and HPLC gel filtration. MCF from either liver or skin had a molecular weight about 650 kDa. MCF from hepatic granulomas was coupled to Affi-Gel beads and transplanted subcutaneously into naive mice. In vivo macrophage chemotaxis was observed around the beads and the cells formed a sheet, but organization of macrophages into granulomas did not occur with the MCF-active fractions. Macrophage chemotaxis alone is insufficient to elicit granulomatous inflammation.

Animals↗
PubMed1991

An improved noninfectious murine skin model of organized granulomatous inflammation.

An improved model of granulomatous inflammation in skin was developed by second passage skin grafting of isolated, lyophilized skin granulomas, originally elicited in naive mice by inoculations of lyophilized hepatic schistosome egg granulomas. The tissue reaction is caused by a single exposure to a noninfectious, acellular granulomagenic stimulus and occurs in healthy mice free of systemic disease. The model should prove useful for isolation of granuloma initiation factor(s). Furthermore, because there is a time lag before new granuloma formation begins, a window exists for analytical dissection of the initiation process. In this study we described the responses of host cells by autoradiography, and light and electron microscopy. The activity of angiotensin-converting enzyme and proline-specific endopeptidase showed a modulation during granuloma formation. In addition we found that severe immunosuppression with high dose cyclosporine therapy did not alter granuloma formation, supporting the idea that initiation of organized granulomas is T-cell independent.

Animals↗
PubMed1991

Immunogenetic influences on skin granuloma formation in mice.

Genetic influence on the development of granulomatous tissue reaction was investigated in C57BL/6 mice. Granulomas developed in the skin of euthymic C57BL/6 mice by transplantation of lyophilized hepatic granulomas were excised and lyophilized. The tissue mass free of parasite egg antigen and living cells was grafted into the skin of euthymic, athymic (nu/nu), and beige (bg/bg) C57BL/6 mice. Histological changes at the skin sites were studied weekly by light microscopy, and cells in newly developed granulomas at 6 weeks after grafting were examined by electron microscopy. Granulomatous inflammation occurred in all the variants but morphometric analysis showed that granulomatous inflammation was the most extensive in beige mice and least in athymic mice. The differences in the degree of tissue reaction were also quantified by measuring angiotensin converting enzyme and prolyl endopeptidase. Statistically significant differences among the animals with varying genetic background were confirmed by the marker enzyme activity. The findings confirm that initiation of a granulomatous response does not require T cells but T cell function is important for full expression of the reaction, while NK cell activity seems to suppress granuloma formation.

Animals↗
PubMed1991

Crystallization and preliminary X-ray diffraction study of cytochrome c552 from Hydrogenobacter thermophilus.

Cytochrome c552 from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus, exhibits remarkable thermostability. The oxidized cytochrome c552 has been crystallized in an ethanol/water mixture by means of the vapor diffusion method. The crystals belong to the orthorhombic system, space group P2(1)2(1)2, with unit cell dimensions of a = 93.4 A, b = 52.9 A, and c = 32.4 A. Most probably the asymmetric unit contains two molecules of cytochrome c552. The crystals diffract X-rays to better than 2.5 A resolution and are stable to X-ray irradiation.

Bacteria, Aerobic↗
PubMed1991

Dural arteriovenous malformation associated with occlusion of the superior sagittal sinus--case report.

The authors report a rare case of dural arteriovenous malformation (AVM) associated with occlusion of the superior sagittal sinus. A 78-year-old female developed transient aphasia, followed by generalized convulsion. Common carotid angiography showed a dural AVM fed by the bilateral middle meningeal arteries, draining to the superior sagittal sinus, and sinus occlusion. 123I-single photon emission computed tomography demonstrated decreased blood flow in the bifrontal-parasagittal regions. Transcatheter embolization via the feeding arteries improved the cerebral blood flow around the lesion, and the symptoms disappeared.

Aged↗
PubMed1990

Structure of the [2Fe-2S] ferredoxin I from the blue-green alga Aphanothece sacrum at 2.2 A resolution.

Crystals of a [2Fe-2S] ferredoxin (Fd) I with a relative molecular mass of 10,480 were obtained from the blue-green alga Aphanothece sacrum. Each asymmetric unit of the crystal contains four molecules. An electron density map calculated by the single isomorphous replacement method with the anomalous dispersion at 2.5 A resolution was refined by averaging the four molecules in the asymmetric unit. Positional and isotropic thermal parameters for the non-hydrogen atoms of the four molecules and 158 water molecules were refined to an R-factor (R = sigma[Fo-Fc[/sigma Fo) of 0.23 by the restrained least-squares method. The estimated root-mean-square (r.m.s.) error for the atomic positions is 0.3 A. The r.m.s. deviations of equivalent C alpha atoms of the asymmetric-unit molecules superposed by the least-squares method average 0.35 A. The Fd molecule has a structure like the beta-barrel in the molecule of the [2Fe-2S] Fd from Spirulina platensis. A [2Fe-2S] cluster is bonded covalently to the protein molecule by four Fe-S, in which three of the Fe-S bonds are in a loop segment from position 38 to 47. The hydrophobic core inside the beta-barrel is formed by seven conservative residues: Val15, Val18, Ile24, Leu51, Ile74, Ala79 and Ile87. The molecular surface around Tyr23, Tyr80 and the active center may interact with ferredoxin-NADP+ reductase. One of the two iron atoms of the [2Fe-2S] cluster should be more easily reduced than the other because of differences in the hydrogen-bonding scheme and the hydrophobicity around the atoms.

Amino Acid Sequence↗
PubMed1990

Tertiary structure of oxidized flavodoxin from an eukaryotic red alga Chondrus crispus at 2.35-A resolution. Localization of charged residues and implication for interaction with electron transfer partners.

The crystal structure of the oxidized form of a flavodoxin from an eukaryotic red alga, Chondrus crispus, has been determined by multiple isomorphous replacement and anomalous scattering methods. A model of the 173 residues and flavin mononucleotide (FMN) has been refined by a restrained least squares method to a crystallographic R-factor of 22.6% using 6236 reflections between 6.0 and 2.35 A with F greater than 3 sigma F. This molecule has a sheet consisting of five parallel beta-strands with two alpha-helices on one side of the sheet and three on the other side, and has a (beta alpha)5 structure. The molecule incorporates a substantial insertion in beta 5, as in Anacystis nidulans flavodoxin, which distinguishes these flavodoxins from the short-chain type. The isoalloxazine ring of FMN is sandwiched between the side chains of Trp-56 and Tyr-98, with its C-7 and C-8 methyl groups being exposed to solvent. The phosphate group of FMN is located at the N-terminal end of alpha 1, and forms extensive hydrogen bonds with the loop (T8-T13) between beta 1 and alpha 1 of the protein. Six of the total 11 lysine residues are clustered at the opposing face to the FMN-binding site, while about two-thirds of the total 35 acidic residues are located in the half of the molecule which includes the FMN-binding site. Such localization of charged residues produces a dipole within the molecule, which may be important in its recognition of the other proteins participating in electron transfer reactions.

Amino Acid Sequence↗