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Biomedical subjects

K Fukuyama

Publications and source records attributed to K Fukuyama.

At least 163 records · Page 9Linked to original sources
PubMed1984

Appearance of T-cell subpopulations during the time course of beryllium-induced granulomas.

Employing monoclonal antibodies that identify T-helper/inducer or T-suppressor/cytotoxic cells, we investigated the T-lymphocyte subsets in hypersensitivity granulomas developed after beryllium salt injection. T-helper/inducer cells were detected at all stages of the lesions, including early nonspecific inflammatory lesions seen at 3 weeks after injection. They formed clusters intermingled with the mononuclear phagocytes in granulomatous lesions, but were not found among the epithelioid cells. In contrast, T-suppressor/cytotoxic cells were slow to appear in the lesions and constituted very small numbers in mature epithelioid cell granulomas of 9-11 weeks. These findings suggest that T-helper/inducer cells may be responsible for the evolution of the perivascular infiltrate of mononuclear cells. Furthermore, appearance of T-helper/inducer cells without T-suppressor/cytotoxic cells may contribute to the development and organization of epithelioid cells and their maturity in the granulomatous lesions.

Antibodies, Monoclonal↗
PubMed1984

Purification and properties of aminoendopeptidase from rat epidermis.

An aminoendopeptidase isolated from 2-day-old rat epidermis was purified to apparent homogeneity by the procedures of ammonium sulfate fractionation, DE-52 column chromatography, Sephadex G-200 gel filtration, and CM-52 and DEAE-Sepharose 6B column chromatography. Enzymatic activity was exhibited only in the presence of sulfhydryl compounds and further enhanced by addition of 5 mM EDTA. It was inhibited by p-chloromercuribenzoate, other sulfhydryl blocking reagents, and o-phenanthroline. The monomer form of the enzyme is Mr = 52,000 +/- 2,300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, but a native form was considered to be Mr = 400,000 +/- 26,000 having an isoelectric point of pH 5.25. Among synthetic substrates the enzyme hydrolyzed amino acid 2-naphthylamide derivatives and L-leucine amine (L-LeuNH2) most effectively. N-alpha-benzoyl-DL-arginine-2-naphthylamide (BANA) was the only endopeptidase substrate for the enzyme and a competitive inhibitor for its aminopeptidase activity. Protein substrates have not yet been found. The pH optimum is 7.5 and in a range of pH 6.5-7.5 it is stable at 37 degrees C for 30 min but loses about 50% of its activity at 50 degrees C.

Animals↗
PubMed1983

Structure of a T = 1 aggregate of alfalfa mosaic virus coat protein seen at 4.5 A resolution.

A T = 1 empty aggregate of alfalfa mosaic virus coat protein had been crystallized in a hexagonal unit cell and its orientation was determined with the rotation function. A single heavy-atom derivative has now been prepared and the position of the two Hg atoms per protein subunit were determined using a systematic Patterson search procedure, given the particle orientation. Phases, initially determined by single isomorphous replacement, were refined by six cycles of electron density averaging and solvent leveling to produce a 4.5 A resolution electron density map. The protein coat is confined between 95 and 58 A radius. The subunit boundary could be delineated easily. It has a central cavity reminiscent of the beta-barrel in other spherical plant viruses, but its topology could not be determined unambiguously. The spherical particle has large holes at the 5-fold axes, consistent with previous observations. The subunits have substantial interactions at the 2 and 3-fold axes. The structure of the elongated particles is discussed in relation to these results.

Capsid↗
PubMed1983

Reduction in eumelanin by the activation of glutathione reductase and gamma-glutamyl transpeptidase after exposure to a depigmenting chemical.

Topical application of 4-tertiary butyl catechol (TBC) causes vitiligo in the skin of man and animals, and previous electron microscopic studies showed pheomelanin formation in the affected areas. In the present study, we investigated changes of enzyme activities, eumelanin content and amount of sulfur in tissue cultured human melanoma cells exposed to the depigmenting chemical. TBC enhanced glutathione reductase activity without changing the eumelanin content by 24 hr after exposure and subsequently (by 42 hr) increased gamma-glutamyl transpeptidase activity and sulfur content in the cells with a decrease in eumelanin content. It is suggested that this chemical alters the types of melanin formed by modulation of these enzyme activities.

Catechols↗
PubMed1983

Glutathione reductase activity in skin exposed to 4-tertiary butyl catechol.

The effect of 4-tertiary butyl catechol (TBC), a potent depigmenting chemical, on glutathione reductase (GR) in pigmented ear skin of hairless mice was investigated. Three topical applications of TBC, which cause neither skin color changes nor melanocyte degeneration, induced an increase in enzyme activity. Since the same treatment resulted in pheomelanin formation as evidenced by electron microscopy, it is suggested that the GR increase correlates at least in part with changes in melanocyte metabolism. This enzyme generates a reduced form of glutathione which may be involved in the formation of glutathionedopas, substrates for hydrolytic enzymes to produce cysteinyldopas, and pheomelanin. Elevation of GR may be an indication of melanogenesis before clinical skin color changes due to TBC appear. It may be used for the early detection of occupational leukoderma.

Animals↗
PubMed1983

Autoimmune response directed against conserved determinants of nuclear envelope proteins in a patient with linear scleroderma.

We have studied the autoantibodies in the serum of a patient with linear scleroderma that specifically recognize the nuclear envelope of cultured cells. These antibodies bind to conserved determinants of nuclear lamins, the predominant mammalian nuclear envelope proteins. Of the three mammalian nuclear lamin proteins (970, P68, and P60), only P70 and P60 bind the autoantibodies. In addition, two proteins of the Drosophila embryonic nuclear matrix, P70 and P68, bind these autoantibodies. We have used nuclear matrices to isolate the autoantibodies from the patient's serum that react to the nuclear lamins. At least three different IgG heavy chains were found to be involved in this autoimmune response to nuclear lamins, indicating that this response is not due to the expansion of a single B-cell clone.

Adult↗
PubMed1983

Main chain fold of a [2Fe-2S]ferredoxin I from Aphanothece sacrum at 2.5 A resolution.

Crystal structure analysis of a [2Fe-2S]ferredoxin I from Aphanothece sacrum, a blue green alga, was carried out at 2.5 A resolution. The phase angle of each reflection was determined by the single isomorphous replacement method coupled with the anomalous dispersion effect for the uranium derivative. The four molecules in an asymmetric unit were clearly seen in a 3.9 A electron density map. The main chains of three molecules were traced at 2.5 A resolution. The structure of the remaining one molecule, however, remains unknown because of the poor electron density of the corresponding region. The three main chain folds exhibit the same topology as that in Spirulina platensis. The structural similarity between A. sacrum and S. platensis ferredoxins, whose amino acid sequences are different from each other by about 30%, strongly suggests that all plant-type ferredoxins have the same main chain fold.

Amino Acid Sequence↗
PubMed1983

The epidermal barrier to Schistosoma mansoni infection.

The stratified epithelium serves as protection for underlying tissues and organs, and the structural fitness of the epidermal cells has been extensively reported. We studied the possible roles played by the epidermis as a barrier against the migration of Schistosoma mansoni larvae. Freshly shed cercariae were collected and placed on the back skin of 2-day-old rats. Electron microscopy of biopsies taken at various intervals showed the larvae and their secretory granules in keratinocytes in which the cytoplasm had become homogeneous in appearance. SDS gel electrophoresis showed the digestion of purified epidermal keratin (60K protein) by a proteinase secreted from cercariae, but this activity was inhibited by an inhibitor purified from epidermal cells. These findings suggest that epidermal cells function both structurally and chemically as a barrier against cercariae invasion.

Animals↗
PubMed1983

Zinc-binding proteins of newborn rat epidermis.

Hematoxylin stain was used to demonstrate localization of Zn2+-binding proteins in the skin of the newborn rat. The binding protein showed different solubilities and the zinc binding was dissociated at pH 4.5. Epidermal proteins were extracted stepwise in Tris-HCl buffer containing NaCl at 4 degrees C and 37 degrees C, 1 M potassium phosphate, and 4 M urea. Zn2+-binding protein was obtained from each extract by means of Zn2+ chelate affinity chromatography. We detected elution of histidine-rich protein from the affinity column with a pH 4.5 buffer by 3H-histidine labeling and immunoreactivity to rabbit IgG directed to histidine-rich protein. Amino acid analysis demonstrated that the compositions of the Zn2+-binding proteins soluble in the different extracts not only resemble each other, but are essentially the same as those of histidine-rich protein of the newborn rat skin as previously reported. However, sodium dodecylsulfate gel electrophoresis showed multiple protein bands ranging from Mr less than 25,000 to Mr greater than 200,000. The findings indicate that histidine-rich protein of different forms and solubility exists, in the epidermis and may be involved in the biological function of the cells through metal-binding.

Amino Acids↗
PubMed1983

Generation of biologically active, complement-(C5) derived peptides by cathepsin H.

The thiol proteinase cathepsin H, isolated and purified from rat liver lysosomes, provokes acute inflammation characterized by the accumulation of polymorphonuclear leukocytes (PMN) when injected intracutaneously into newborn rats. We have examined the possibility that the accumulation of PMN at skin sites injected with cathepsin H is due, in part, to generation locally of C-derived chemotactic factors. We have found that cathepsin H acts in a concentration- and time-dependent fashion in whole human (and rat) EDTA-plasma to generate C5-derived peptides with chemotactic activity for PMN. Chemotactic activity was not generated in EDTA-plasma by either heat-inactivated cathepsin H or by a combination of active enzyme and a thiol proteinase inhibitor isolated from rat epidermis. Cathepsin H also acted in a concentration- and time-dependent fashion on isolated (functionally pure) human C5 to yield chemotactic activity for PMN as well as PMN lysosomal enzyme-releasing activity. Whereas 10 ng/ml cathepsin H generated significant chemotactic activity from isolated C5 (1000 CH50 U/ml), 7 to 10 micrograms/ml were required to generate chemotactic activity in whole EDTA-plasma. Cathepsin H not only was capable of generating biologically active, C5-derived peptides, but also was capable of degrading these peptides. Incubation of either whole EDTA-plasma or isolated C5 with high concentrations of cathepsin H (e.g., 25 micrograms/ml and 100 ng/ml, respectively) caused the rapid appearance of chemotactic activity followed by an equally rapid disappearance. PMN accumulated more rapidly in the skin of newborn rats injected with cathepsin H-treated C5 than in the skin of animals injected with cathepsin H alone. These data suggest that generation by cathepsin H of C-derived chemotactic activity contributes to the ability of this enzyme to induce dermal inflammation.

Animals↗
PubMed1983

Autoradiographic study of colchicine inhibition of DNA synthesis and cell migration in hairless mouse epidermis in vivo.

In vivo effects of colchicine on mitotic counts, DNA synthesis, and cell migration time were investigated in hairless mouse epidermis. [H3]TdR was used to examine DNA synthesis and measure the transit time. We found that colchicine (at a dose of 50 micrograms/25 g body weight) caused mitotic arrest, but had no effect on the number of cells in S phase during the first 4 hr after injection. Colchicine reduced the number of cells in S phase in normal epidermis during the 16-30 hr after injection, but the effect was no longer seen at 42 hr post injection. A similar depression in S-phase cell counts appeared in UVR-stimulated hyperplastic epidermis by 48 hr after injection of colchicine. In addition, migration of [H3]TdR-labelled cells from the basal layer to the superficial layers in UVR-stimulated hyperplastic epidermis was disturbed by colchicine injection. The delay in movement was not seen during the first 24 hr, but appeared by 48 hr after injection.

Animals↗
PubMed1982

Partial purification and characterization of an inhibitor from newborn-rat epidermis with activity against the proteinase of Schistosoma mansoni cercariae.

The penetration of cercariae through the skin initiates infection of the host with the human trematode parasite Schistosoma mansoni. Many larvae fail to migrate into the living epidermal cell layer. In order to determine if chemical as well as mechanical barriers to cercarial skin penetration exist, inhibitory activity of epidermal cell extracts against the proteinase obtained from cercarial secretions was assayed. An inhibitor was purified 50-fold by gel filtration on Sephadex G 75 and cation exchange chromatography at pH 5.8 and 4.9. The inhibitor has a relative molecular mass (Mr) of approx. 40 000-53 000. Oxidation of the inhibitor with N-chlorosuccinimide eliminated its inhibitory activity and thus indicated a critical methionine residue. The inhibitor was active against a wide spectrum of serine proteinases: porcine pancreatic elastase, human granulocyte elastase, bovine trypsin, and bovine alpha-chymotrypsin. However, no inhibition was detected against papain or clostridial collagenase. The inhibitor did not cross react with antiserum to human or rat serum alpha 1-proteinase inhibitor.

Animals↗
PubMed1982

Further characterization of cysteine proteinase inhibitors purified from rat and human epidermis.

Cysteine proteinase inhibitors isolated from rat and human epidermis were purified to homogeneity and had isoelectric points of pH 4.31 and pH 5.10, respectively, Both inhibitors caused noncompetitive inhibition to the same degree against papain (EC 3.4.22.2), but the activity of human inhibitor against rat liver cathepsins B (EC 3.4.22.1), H (EC 3.4.22.16), and L (EC 3.422.-) was more effective than that of rat inhibitor. Dependency on pH was observed with rat inhibitor for cathepsins B and H, and with human inhibitor for cathepsin L. The reaction of the inhibitors with papain and cathepsins H and L occurred immediately, while the inhibition reaction of cathepsin B increased progressively during a preincubation time up to 40 min. Incubation at pH 7.0 maximized the progressive inhibitory activity. These findings demonstrate that cysteine proteinase inhibitors from rat and human epidermis inhibited a variety of cysteine proteinases. However, the inhibitor and enzyme interaction depends upon the enzyme, inhibitor source, and experimental conditions such as pH and preincubation time.

Animals↗
PubMed1982

Ultrastructural demonstration of chemical modification of melanogenesis in hairless mouse skin.

We investigated chemical and physical modifications of the genetically determined ultrastructure of melanosomes. The flank skin of hairless mice was treated with ultraviolet energy (UV) shorter than 320 nm or with a combination of a photosensitizer and UV (PUVA treatment). All melanosomes in the induced melanocytes and those in resident melanocytes in the ear skin showed eumelanogenesis, although the degree of melanin deposition differed considerably according to the induction process. Eumelanogenesis was most advanced in the resident melanocytes while PUVA-induced melanocytes showed more immature premelanosomes. We then topically applied 4-tertiary butyl catechol on the skin. The depigmenting agent caused an appearance of pheomelanosomes. The alteration in melanogenesis was seen most distinctly in premelanosomes of the PUVA-induced cells. Altered ultrastructure was also observed in matured melanosomes; this change was most apparent in the resident melanocytes. These findings indicate that cells with eumelanogenesis may undergo pheomelanogenesis. The present study demonstrated effects of chemicals on genetically determined function of melanocytes by quantitative analysis of melanosome ultrastructure.

Animals↗
PubMed1982

Cellular localization of thiol-proteinase inhibitor in the epidermis of the newborn rat.

Subcellular distribution of a thiol-proteinase inhibitor protein was determined in the epidermis of the newborn rat by light and electron microscopy. This protein was highly soluble in basal cells and concentrated on ribosomes in the perinuclear region. Solubility in Tris buffer decreased in granular and cornified cells in which the protein appeared on polysomes which were attached on other cellular structures such as dense homogenous deposits and tonofilaments. The protein also appeared to be deposited on the plasma membrane and became insoluble in Tris buffer at 37 degrees C, but solubilized in 1 M phosphate buffer. Location of the protein around keratohyalin granules or by the plasma membrane suggested that the inhibitor protein bound to cysteine-rich protein of the epidermis with or without forming a thiol-proteinase inhibitor complex. The thiol-proteinase inhibitor protein seems to contribute to epidermal cell differentiation at multiple points through changes in its solubility and subcellular localization from basal cells to cornified cells.

Animals↗
PubMed1982

Structure-function relationship of [2Fe-2S] ferredoxins and design of a model molecule.

[2Fe-2S] ferredoxins isolated from various plants and algae comprise 93-99 amino acid residues and resemble each other not only in sequences, but also in physiological functions. One of them isolated from Spirulina platensis was subjected to X-ray analysis and its three dimensional structure is now known. [2Fe-2S] ferredoxins of a different type are found in halobacteria and comprise 128 amino acid residues. Both types of the [2Fe-2S] ferredoxins exhibit low redox potentials. By comparing the amino acid sequences of 28 [2Fe-2S] ferredoxins and the tertiary structure of S. platensis ferredoxin we predicted a common three-dimensional structure to the [2Fe-2S] ferredoxins and proposed a molecular surface area to be interacting with FNR. An artificial small molecule composed of 20 amino acid residues is designed on the basis of the tertiary structure of S. platensis ferredoxin. The amino acid sequence was predicted to be Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys-Ala-Gyl-Pro-Leu-Leu-Thr Cys-Val which should have a [2Fe-2S] cluster with a low redox potential.

Amino Acid Sequence↗