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Biomedical subjects

K Fukuyama

Publications and source records attributed to K Fukuyama.

At least 109 records · Page 6Linked to original sources
PubMed1989

The amino acid sequence of a flavodoxin from the eukaryotic red alga Chondrus crispus.

The amino acid sequence of the constitutive flavodoxin from the red alga Chondrus crispus was determined from the analyses of peptide fragments derived by enzymic digestions of the carboxymethylated protein. This is the first sequence reported for a flavodoxin from a eukaryote. The protein is composed of 173 amino acid residues and is a member of the longer-chain group of flavodoxins. The extent of sequence homology to the three other flavodoxins in the group for which sequences are available is in the range 36-39%, with the most strongly conserved regions being those implicated in binding of the FMN, the redox-active prosthetic group. Nevertheless, Chondrus crispus flavodoxin stands apart in a number of respects, in particular the possession of an unusually high content of proline, with these residues distributed more or less regularly along the peptide chain.

Amino Acid Sequence↗
PubMed1989

Crystallization and preliminary X-ray diffraction study of the heat-labile enterotoxin B subunit produced by enterotoxigenic Escherichia coli.

Heat-labile enterotoxin LT produced by enterotoxigenic Escherichia coli is composed of A and B subunits. The A subunit is enzymatically active; whereas, through the action of the B subunit, the toxin binds to the receptor, a GM1 ganglioside present on the cell surface. Crystals of the LT-B subunit were formed at room temperature by vapor diffusion with polyethylene glycol in the presence of the non-ionic detergent beta-octylglucoside. The crystals were characterized by X-radiation as orthorhombic, space group P2(1)2(1)2(1), with unit cell dimensions of a = 224.1 A, b = 65.3 A, c = 118.4 A. They diffract X-rays to a resolution of at least 2.5 A and are stable to X-rays.

Bacterial Toxins↗
PubMed1989

Purification and characterization of carboxypeptidase from terminally differentiated rat epidermal cells.

A tissue carboxypeptidase-A-like enzyme was purified to apparent homogeneity from terminally differentiated epidermal cells of 2-day-old rats by potato inhibitor affinity chromatography followed by FPLC Mono Q column chromatography. The enzyme has an Mr of 35,000 as determined by SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It has a pH optimum of 8.5 for hydrolysis of benzyloxycarbonyl-Phe-Leu (Km = 0.22 mM, kcat = 57.9 s-1). The enzyme does not hydrolyze substrates with Arg, Lys and Pro at the C-terminal and Pro at the penultimate position. Angiotensin I was effectively hydrolyzed (Km = 0.06 mM, kcat = 6.48 s-1) and produced both des-Leu10-angiotensin I and angiotensin II. The enzyme activity, relatively stable at 4 degrees C and pH 8.0-10.5, was inactivated at pH values higher than 12.0 and lower than 5.0 or at 65 degrees C for 10 min. Inhibitor profiles of the epidermal enzyme also differed slightly from those of tissue carboxypeptidase A of pancreatic or mast cell origin.

Angiotensin I↗
PubMed1989

Dynamic changes of cell-surface glycoconjugates in human palmar epidermis following friction-blisters.

Damage and repair of cell-surface glycoconjugates were examined in human palmar skin following friction-blister injury, using biotinylated lectins and the avidin-biotin complex method. In normal skin, concanavalin A, Ricinus communis, and Triticum vulgaris bound to the surface of cells from the basal layer to the granular layer. After injury, binding of concanavalin A was absent in the plasma membrane, but appeared in the cytoplasm at perinuclear sites. The surface reaction was recovered in basal and spinous cells, but not in granular cells, when cell maturation began at 5 days after injury. In contrast, binding of Ricinus communis and Triticum vulgaris was, in general, much more resistant to tissue damage. Even in some cells, where the surface staining became obscure at an early period, a normal staining pattern reappeared by 6 h after injury. Staining of Ulex europeus I and Glycine max, detected on the surface of upper spinous and granular cells in normal skin, disappeared immediately after the injury, but recovered quickly on the surfaces of the differentiated cells. These findings suggest that at least 2 oligosaccharide sequences, one binding with concanavalin A, and the other with Ricinus communis and Triticum vulgaris, may exist on epidermal cells. Addition of terminal carbohydrates, detectable with binding of Ulex europeus I and Glycine max, appears to occur on the Ricinus communis I and Triticum vulgaris-bound oligosaccharide chain.

Biopsy↗
PubMed1989

Crystallization and preliminary X-ray diffraction studies of oxidized flavodoxin from Chondrus crispus, a red alga.

Crystals of the oxidized form of flavodoxin from a red alga, Chondrus crispus, have been grown in ammonium sulfate solution by the dialysis method. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions of a = 63.6, b = 48.8, and c = 56.8 A. The asymmetric unit contains one molecule of flavodoxin. The crystals diffract X-rays to about 2.0 A resolution and are stable to X-ray beams. The diffraction patterns changed significantly upon soaking the crystal in a solution of a platinum complex. The major heavy-atom sites in the platinum derivative crystal have been identified from the difference Patterson function calculated at 4 A resolution.

Crystallization↗
PubMed1989

Isolation and sequence studies of cysteinyl peptides from Spirulina glutathione reductase: comparison of active site cysteine peptides with those of other flavoprotein disulfide oxidoreductases.

The amino acid sequences of the cysteinyl peptides of Spirulina sp. glutathione reductase were determined. Spirulina glutathione reductase was covalently bound to Thiopropyl-Sepharose 6B in the presence of 8M urea through thiol-disulfide exchange. After tryptic digestion, 4 distinct cysteinyl peptides were finally isolated from NADPH-reduced glutathione reductase and 2 from oxidized glutathione reductase. The amino acid sequences of the two cysteinyl peptides which could not be isolated from the oxidized glutathione reductase were very similar to those around the active site disulfide of the other flavoprotein disulfide oxidoreductases and a unique replacement of asparagine and valine by isoleucine and arginine between the two cysteine residues was found. The other two peptides isolated from both oxidized and reduced glutathione reductase also show considerable homology to the corresponding parts of human and Escherichia coli glutathione reductases.

Amino Acid Sequence↗
PubMed1989

Candidacidal activities of proteins partially purified from rat epidermis.

Proteins with approximate molecular weights of greater than 300,000 (EP greater than 300K) and 49,000 (EP 49K) were partially purified from terminally differentiated cells of 2-day-old rat epidermis. They were extracted in 0.34 M sucrose containing 0.01 M citric acid and purified by Sephacryl S-300 chromatography followed by reverse-phase column chromatography. The major constituents of EP greater than 300K and EP 49K were focused around pH 10 to 11 by sucrose gradient isoelectric focusing. Both proteins were effective at inhibiting colony formation of Candida albicans and C. tropicalis, but neither inhibited the growth of C. parapsilosis. The effect was maximum below pH 5.0 and reduced considerably above pH 5.0. The activity of EP greater than 300K on C. albicans TIMM 1623 (group A) was much stronger than that of EP 49K, whereas both proteins similarly inhibited C. albicans TIMM 1604 (group B). Their effects against C. albicans TIMM 1623 were dose dependent and were activated after a longer preincubation time, and NaCl concentration influenced their potency. At a low salt concentration and a 60-min preincubation at pH 4.5, the 50% effective dose for EP greater than 300K was calculated to be 1.7 x 10(-9) M, whereas that for EP 49K was 1.8 x 10(-7) M.

Amino Acids↗
PubMed1989

Immunosuppression and in vivo T cell function in granulomatous inflammation.

Subcutaneous grafts of fresh or freeze-dried hepatic schistosome egg granulomas in inbred mice caused increased numbers of total cells, T cell subsets, and elevated DNA synthesis in regional lymph nodes. This in vivo T cell activation was not apparent by in vitro tests. Spleen cells were unaffected both in vivo and in vitro. Immunosuppressive therapy with ciclosporin or monoclonal antibodies to T cell subsets profoundly depressed the T cell numbers and function, even when primed by a prior graft. Subsequent grafting partly reversed these effects, and T cells became activated. The findings indicate that in vitro assessment of activation and suppression of T cell function does not necessarily correspond to the events occurring in vivo.

Animals↗
PubMed1989

Immuno- and enzyme-histochemical detection of phosphoprotein phosphatase in rat epidermis.

A phosphoprotein phosphatase (PPase: EC 3.1.3.2) was recently purified from rat epidermis. The enzyme dephosphorylates phosphoprotein, and its properties, such as pH optimum, inhibitor spectrum, and Fe2+ activation, differ from those of other soluble phosphatases. We investigated in 2-day-old rat skin the distribution of immunologically detectable PPase and intracellular localization of PPase activity. The reaction of rabbit monospecific anti-PPase IgG was identified in granular and cornified cells by the avidin-biotin complex method. For activity staining, basic principles of the Gomori lead-salt method and azo dye technique with the substrates p-nitrophenylphosphate (p-NPP) and alpha-naphthyl phosphate (NP), respectively, were modified according to the biochemical properties of PPase activity which is resistant to formalin, Na tartrate, and NaF. Activity was detectable in granular cells including keratohyalin granules and the lower strata of cornified cells. The activity was inhibited by 1 mM CuSO4 and enhanced by a mixture of 0.5 mM FeSO4 and 1 mM ascorbic acid. We consider that PPase may be involved in dephosphorylation of histidine-rich proteins in granular and cornified cells and may play a key role in intracellular catabolism associated with epidermal cell differentiation.

Animals↗
PubMed1989

Partial purification and characterization of epidermal plasminogen activator and their inhibitor.

Plasminogen activator (PA) and PA inhibitor were partially purified from 2-d-old rat epidermis and characterized in order to elucidate the enzyme-inhibitor interaction in epidermis. PA extracted with buffer containing KSCN was first purified by Blue-Sepharose affinity chromatography. Separation of two PAs, with relative molecular mass (Mr) of 66000 and 44000, was accomplished by Con A-Sepharose column chromatography. The Mr 66000 enzyme had the properties of tissue-type PA (t-PA), while the Mr 44000 enzyme showed those of urokinase-type PA (u-PA) as determined by immunological and fibrin-binding studies. PA inhibitor was extracted in 1,4-piperazinediethanesulphonic acid buffer and purified by (NH4)2SO4 precipitation, gel filtration followed by a Mono Q column in an FPLC system. This inhibitor showed Mr 60000 and inhibited human u-PA activity in a dose- and time-dependent manner, but did not inhibit the activity of t-PA from human and murine melanoma cells or plasmin. It inhibited epidermal PA, Mr 44000, more effectively than it did the Mr 66000 epidermal PA. It was stable at 60 degrees C for 60 min or between pH 5 and 11. This study indicates that both u-PA and t-PA function in normal rat epidermis. On the other hand, an inhibitor which preferentially acts against u-PA exists, but inhibitor to t-PA does not appear to operate under normal epidermal functions.

Animals↗
PubMed1989

Mechanisms of granulomatous inflammation.

The mononuclear phagocyte system is the central body defense called upon to deal with persistent foreign objects or irritants. Depending on circumstances, the macrophages function as phagocytes or secretory cells or both. Granulomas may form in the absence of immunologic modulation (foreign body granuloma) or they may develop under immunologic control (hypersensitivity granulomas). In either case, the secretory products of macrophages are qualitatively similar and differ mainly in the total amounts produced. If cell-mediated immunity dictates hypersensitivity granulomas as commonly taught, one has to explain what aberrent signals are sent from T cells or what abnormal message is perceived in the macrophages which causes them to differentiate to purely secretory epithelioid cells or produce such abnormal pathologic patterns as necrobiosis. Alternatively, some granulomatous hypersensitivity reactions such as organized epithelioid cell granulomas may be initiated primarily by a macrophage secretion product with cellular immunity acting to amplify or promote granuloma formation and organization. The known heterogeneity of macrophages is insufficient to account for the varied observations. Future experimentation will uncover the different differentiation programs of monocytes required for the varied and various granulomas seen in skin.

Antigen-Presenting Cells↗
PubMed1989

Possible role of natural killer cells in granulomatous inflammation.

Natural killer (NK) cell activity is down-regulated in patients with active chronic inflammatory processes, and appears abnormal in sarcoidosis. We investigated NK cell activity in lymph nodes (LN) and spleen cells of C57BL/6 mice by using a 51Cr-release assay against Yac-1 tumor target cells. Hepatic granulomas were caused by infection with 75 cercariae of Schistsoma mansoni ans skin granulomas formed by transplanting isolated liver granulomas into skin of another group of naive mice. Also, anti-NK-1.1 MoAb was injected intraperitoneally into some mice (1 mg IgG/mouse) 1 wk before and weekly for 4 wks after grafting; control mice received saline. Well-organized granulomas appeared in both liver and skin. NK activity (%) was markedly reduce in LN and spleen: (table; see text) The results showed a relationship between granuloma formation and reduction of NK cell activity; both systemically and locally. MoAb did not alter total LN and spleen cell numbers, but reduced NK cell activity by 70-90%. Mean diameter of skin granulomas developed in treated mice was greater (p less than 0.05). NK cells may act as suppressor cells in granulomatous inflammation.

Animals↗
PubMed1988

Soluble dipeptidyl peptidase IV from terminal differentiated rat epidermal cells: purification and its activity on synthetic and natural peptides.

In terminally differentiated epidermal cells dipeptidyl peptidase IV (EC 3.4.14.5) (DPP IV) is present mainly in a soluble form. We purified the enzyme from 2-day-old rat cornified cells to homogeneity by Sephadex G-200 and Mono-Q column chromatography and finally HPLC gel filtration on G3000SW. The enzyme was estimated to be Mr 190,000 by HPLC gel filtration and Mr 90,000 by sodium dodecyl sulfate-electrophoresis. The enzyme showed general properties reported for detergent-solubilized DPP IV from other tissues. It was Con A binding and almost completely inhibited by 1 mM diisopropyl fluorophosphate and Diprotin A. The pI was 5.6 and the pH optimum was 7.5. The specific activity for Gly-Pro-p-nitroanilide was 31.9 units/mg. HPLC analysis demonstrated the release of dipeptides of the N-terminal of substance P, beta-casomorphin, and their related peptides. A stoichiometric reaction of the enzyme on substance P was observed. The epidermal DPP IV had a Km of 0.3 mM and a kcat of 50.3 s-1 for substance P and the Km value decreased by shortening the peptide from the carboxyl-terminal amino acids. The enzyme hydrolyzed human and bovine beta-casomorphin with Km values of 0.025 and 0.05 mM, respectively. Shortening the bovine beta-casomorphin peptide chain did not affect enzyme affinity.

Animals↗
PubMed1988

Purification and characterization of bradykinin-hydrolyzing enzyme from 2-day-old rat epidermis.

Bradykinin-hydrolyzing enzyme was purified 200-fold from a soluble fraction of cornified cells from 2-day-old rat epidermis. The enzyme has an Mr of 80,000 as identified by SDS polyacrylamide gel electrophoresis and HPLC gel filtration. The isoelectric point of the enzyme is 5.05. The enzyme hydrolyzed Phe5-Ser6 of bradykinin and seven bradykinin-related peptides, and Tyr5-Ser6 of Tyr5-bradykinin. Production of bradykinin fragments, Arg-Pro-Pro-Gly-Phe and Ser-Pro-Phe-Arg, proceeded in a stoichiometric fashion. Km and Vmax values for bradykinin were 33 microM and 22.2 mumol/min per mg, respectively. The enzyme did not hydrolyze azocasein, denatured hemoglobin or synthetic substrates for other epidermal proteinases. The enzyme activity was enhanced by reducing agents and inhibited by sulfhydryl-blocking agents and divalent cations. Diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride had no effects. The enzyme has a pH optimum of 7.0-7.5 and is stable at 4 degrees C for 1 month, but loses activity completely at 60 degrees C for 10 min. The epidermal endopeptidase differs in several properties from endooligopeptidase A purified from brain which hydrolyzes Phe5-Ser6 of bradykinin.

Amino Acid Sequence↗
PubMed1988

Hydrolysis of histones by proteinases.

Hydrolysis of histones by proteinases from rat liver, skin and other sources was studied by using a rat thymus histone preparation as the substrate and polyacrylamide-gel electrophoresis and densitometric analysis as the methods to detect histone subtypes and their hydrolysis. The rat mast-cell proteinase I effectively hydrolysed histones except type H4. Thrombin hydrolysed effectively histones H1 and H2A, whereas plasmin hydrolysed all types of histones. Cathepsin D hydrolysed especially histone H2A. Cathepsins B and L hydrolysed all histones more slowly, and cathepsin H hydrolysed them extremely slowly. Epidermal aminoendopeptidase did not hydrolyse histones. Trypsin and chymotrypsin were used as reference enzymes, which hydrolysed all types of histones in very low concentrations. This study suggests that a variety of proteinases could play a role in histone hydrolysis. Hydrolysis of a specific subtype of histones, such as histone H2A at pH 6 by cathepsin D, may be directly involved in regulation of epidermal-cell differentiation.

Aminopeptidases↗
PubMed1988

Tertiary structure of Bacillus thermoproteolyticus [4Fe-4S] ferredoxin. Evolutionary implications for bacterial ferredoxins.

The structure of a low-potential [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus has been solved using anomalous scattering data from iron atoms in the diffraction data of native crystals and refined partially to a crystallographic R-factor of 0.33, with 2.3 A (1 A = 0.1 nm) resolution data. The least-squares refinement based on the Bijvoet differences has determined that the four iron atoms in the cluster are an equal distance, approximately 2.8 A, apart. The NH ... S hydrogen bonds between polypeptide nitrogen atoms, and both cysteine and inorganic sulfur atoms, are present, as in ferrodoxin from Peptococcus aerogenes. The polypeptide chain of the B. thermoproteolyticus ferredoxin has a fold closely similar to that of 2[4Fe-4S] ferredoxin from P. aerogenes. The structural correspondence indicates strongly that both types of ferredoxin evolved from a common ancestor. The second cluster-binding region in P. aerogenes ferredoxin corresponds to the alpha-helix in B. thermoproteolyticus ferredoxin. The secondary-structure predictions strongly suggest that the alpha-helix is generally present in the monocluster-type ferredoxins. The conformational change to alpha-helix, insertions of a loop and a protrusion, as well as the absence of the second cluster in B. thermoproteolyticus ferredoxin, result in the lack of 2-fold symmetry present in P. aerogenes ferredoxin. So, the track of gene duplication is no longer detectable in the tertiary structure alone. The evolutionary events that may have occurred in the ferredoxins with the [4Fe-4S] cluster are discussed.

Amino Acid Sequence↗
PubMed1988

Cysteine protease and its inhibitor in experimentally produced squamous cell carcinomas in hairless mouse skin.

Squamous cell carcinomas (SCC) were experimentally produced in hairless mouse skin, and cysteine protease and its inhibitor were simultaneously purified from extracts of 1 g of tissue of SCC and normal skin. Activity of cysteine proteinases, Mr greater than 50,000 and Mr 28,000, increased in SCC compared to those in normal skin. SCC also showed elevation of cysteine proteinase inhibitor activity and Mr 13,000 and Mr 82,000 inhibitors were purified. Mr 13,000 inhibitor was found to have biochemical properties which were the same as those of the inhibitor present in normal skin. Mr 82,000 inhibitor was not detectable in normal skin and it differed from a serum inhibitor with a similar Mr in terms of activity and stability at acidic pH. The findings suggest that the increased activity of both cysteine proteases and endogenous inhibitors may be involved in the regulatory mechanisms of malignant cell metabolism and tissue remodeling associated with SCC development.

Animals↗