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Biomedical subjects

H Shinkai

Publications and source records attributed to H Shinkai.

At least 145 records · Page 8Linked to original sources
PubMed1980

Procollagen type III N-terminal endopeptidase in fibroblast culture.

A peptidase activity capable of excising in a single fragment the N-terminal extension of the precursor of collagen type III (p-N-collagen type III) was observed in calf tendon fibroblast culture medium. A new procedure was developed for detecting this peptidase (p-N-collagen type III peptidase). It is based on the use of 14C-labelled p-N-collagen type III obtained by carboxymethylation of the half-cystine residues with iodo-[14C]acetamide. The released labelled N-terminal extension is soluble in 27% (v/v) ethanol, whereas the uncleaved substrate and the collagen are precipitated under these conditions. The endopeptidase nature of p-N-collagen type III peptidase is supported by the similarity in molecular weight of the product of cleavage of p-N-collagen III by the enzyme to those obtained by cleavage with bacterial collagenase. An apparent Km of 0.3 X 10(-6)M was established. The pH optimum of p-N-collagen type III peptidase is similar to that of p-N-collagen type I peptidase, i.e. about 7.5. Both peptidases are inhibited by dithiothreitol and by Cu2+ and Zn2+, but not by other bivalent ions. p-N-collagen type III peptidase does not cleave p-N-collagen I or p-N-gelatin I. Partial purification of p-N-collagen type III peptidase from fibroblast culture medium was performed by sieve chromatography on Ultrogel AcA-34 to yield two peaks of activity, of mol.wts. 170000 and 100000. Part of the activity was retained on affinity chromatography on concanavalin A--Sepharose. Studied as a function of the age of the culture, p-N-collagen type III peptidase activity produced by tendon fibroblasts parallels that of p-N-collagen type I peptidase and collagen synthesis.

Animals↗
PubMed1980

Purification and characterization of subcomponent Clq of the first component of bovine complement.

Bovine complement subcomponent C1q was purified, in a highly hemolytically active form, by a combination of precipitation with EGTA, ion-exchange chromatography, and gel filtration. Yield ranged from 22 to 28% as protein amounts, and the activity of final preparations was in the range of 2 X 10(13)-4 X 10(13) effective molecules/mg. The molecular weight of undissociated C1q was 407,000, as determined by polyacrylamide gel electrophoresis containing sodium dodecyl sulfate (SDS). C1q was shown to be composed of two non-covalently linked subunits of approximately 46,000 and 45,000 molecular weights in a molar ratio of 2 : 1. On reduction, the higher molecular weight subunit gave two chains having approximate molecular weights of 23,600 and 22,200 in equimolar ratio, and the lower molecular weight subunit gave one chain with a molecular weight of approximately 22,000. C1q contained hydroxyproline, hydroxylysine, a high percentage of glycine and approximately 9% carbohydrate and 14.8% nitrogen. The absorption coefficient (A 1% 1cm) in 300 mM NaCl was found to be 7.3 +/- 0.12 at 280 nm. From these results, overall molecular structure of bovine C1q looks similar to that of human complement subcomponent C1q.

Amino Acids↗
PubMed1979

Hydroxylysine-linked glycosides of human complement subcomponent C1q and various collagens.

1. Human C1q, a subcomponent of the first component of complement, contains 67 disaccharides (glucosylgalactose) and 2.4 monosaccharides (galactose) linked to hydroxylysine in one molecule. It was found that 82.6% of the hydroxylsine residues were glycosylated. The suggestion of the possible existence of glucosylgalactosylhydroxylysine reported previously [Yonemasu, Stroud, Niedermeir & Butler (1971) Biochem. Biophys. Res. Commun. 43, 1388--1394] was confirmed. 2. The hydroxylysine-glycosides are not detected in the C-terminal, non-collagen-like, globular regions, but only in the collagen-like regions in the subcomponent C1q molecule. 3. Alpha 1(I) and alpha 2 in pig skin, alpha 1(II) in bovine cartilage and alpha 1(III) in bovine skin collagens contain 2.0, 2.2, 13.2 and 2.0 residues of hydroxylysine-glycosides per molecule, respectively. The percentage of hydroxylysine residues glycosylated in each of these chains is relatively low (on average 38%). 4. Neither the high percentage of hydroxylysine residues glycosylated nor the high values for the ratios of disaccharides to monosaccharides in the subcomponent C1q resembles that in alpha 1(I), alpha 2, alpha 1(II) and alpha 1(III). 5. Similarities between the extent of glycosylation of hydroxylysine residues in collagen-like regions in the subcomponent C1q molecule and that of the collagenous constituents of human glomerular basement membranes, aortic intima, skin A- and B-chains and of bovine anterior lens capsule are discussed.

Animals↗
PubMed1979

Immunobiological aspects of Clq in sera of patients with cutaneous vasculitis and collagen diseases.

Clq was comparatively quantified with CH50 or C3 in sera of patients with various types of cutaneous vasculitis and collagen diseases. The following results were found: 1) Elevated levels of Clq were seen much more frequently in cutaneous vasculitis and PSS. 2) No significant correlations were found between Clq and CH50 or C3, except for a moderate rank correlation between Clq and C3 in SLE. 3) The amount of hydroxyproline in serum (collagen-like protein) is nearly identical with the calculated value of that present in Clq.

Adolescent↗
PubMed1978

Acidic glycosaminoglycans in skin and urine of a patient with wide-spreaded lichen sclerosus et atrophicus.

Changes of dermal and urinary acidic glycosaminoglycans in Lichen Sclerosus et Atrophicus (LSA) were investigated with the following results: 1. Hyaluronic acid (HA) was excreted in urine of a patient with LSA. 2. HA and low sulfate chondroitin were eluted with 0.5 M NaCl by Bio Rad AG 1- X 2 (Cl- form) column chromatography. 3. Involved dermal tissue contained only about 50% of HA in the skin of a healthy adult.

Chondroitin↗
PubMed1978

Smooth muscle cells in the development of plasmatic arterionecrosis, arteriosclerosis, and arterial contraction.

Plasmatic arterionecrosis, the causative lesion of hypertensive cerebral hemorrhage, follows upon medial muscle cell necrosis. The development of medial muscle cell necrosis, the earliest cerebral arterial change seen in hypertensive rats, was inhibited when these animals were fed a cholesterol and lard-supplemented diet. Insudation of fibrin was noted in the arterial intima of hypertensive rats with bilaterally constricted renal arteries. Removal of the constriction induced a fall in the elevated blood pressure and an increase of intimal muscle cells. These were responsible for the dissolution of the deposited fibrin, leading to arteriosclerosis. These myointimal cells may originate from the endothelium. Arterial contraction caused by methoxamine hydrochloride often induced the intrusion of one medial muscle cell into another and increased endothelial permeability. 12-24 h after contraction, the arterial segments showed medial muscle cell necrosis, endothelial desquamation with platelet adhesion, and blood plasma infiltration.

Animals↗
PubMed1978

Effect of age on C1q and C3 levels in human serum and their presence in colostrum.

The initiating complement component (C1q) in the classical pathway of 730 subjects and the first essential component (C3) in the alternative pathway of 461 subjects in Japan were examined. The study population consisted of normal healthy newborns, infants, children, adults and the old (from birth up to 75 years of age). In cord sera, both C1q and C3 were about 60% to the total mean level. At 3 days of age, C1q markedly increased to the mean level which remained relatively invariable up to 40 years of age. And above 40 years, the C1q level increased steadily with age up to about 75 years of age. C3 reached the mean level at about 1 month of age, and was highest during infancy. This level declined at about puberty and then continued to increase steadily with age up to around 55 years. In normal healthy subjects, a moderate positive rank correlation was found between C1q and C3 amounts. No significant differences of C1q and C3 levels were evident between male and female. No C1q was demonstrable in the colostrum from which lipids were previously removed, but after concentration by precipitation in a chelating agent with a low ionic strength, C1q, measured immunochemically, was detectable at concentrations of 300 ng/ml of colostrum. C3 was also detected at concentrations of about 200 microgram/ml of colostrum.

Adolescent↗
PubMed1978

Acidic glycosaminoglycans in skin and urine of a patient with wide-spreaded lichen sclerosus et atrophicus.

Changes of dermal and urinary acidic glycosaminoglycans in Lichen Sclerosus et Atrophicus (LSA) were investigated with the following results: 1. Hyaluronic acid (HA) was excreted in urine of a patient with LSA. 2. HA and low sulfate chondroitin were eluted with 0.5 M NaCl by Bio Rad AG 1- x 2 (Cl- form) column chromatography. 3. Involved dermal tissue contained only about 50% of HA in the skin of a healthy adult.

Atrophy↗
PubMed1977

Biochemical characterization of connective tissue macromolecules derived from cutaneous fibrosarcoma.

Tumor tissue of cutaneous fibrosarcoma was solubilized with salt, acetic acid and salt-extracted after pepsin treatment. Type III collagen was observed in neutral soluble collagen, 1.5 M and 2.4 M NaCl precipitated fractions after pepsin treatment. Type III collagen fraction precipitated with 1.5 M NaCl eluted in the alpha2 region when chromatographed on CM-cellulose without reduction, while the type III collagen partially purified with 1.5 M NaCl eluted between the position of alpha1(I) and beta12 after reduction and alkylation. Analysis of amino acid showed the presence of cysteine and the high content of hydroxyproline following CM-chromatography of 1.5 M NaCl precipitated type III collagen fraction. Acidic glycosaminoglycans were composed of hyaluronic acid, chondroitin sulfate, dermatan sulfate and heparan sulfate, and the first two were major components of fibrosarcoma.

Aged↗
PubMed1977

Characterization of the synthesized collagen by mouse L-929 (L-5) cells.

In 48 h incubation L-929 (L-5) cells secreted collagenous protein accounting for 16% of the total protein. CM-chromatography, SDS-acrylamide gel electrophoresis and DEAE-cellulose chromatography revealed that the collagenous protein was mainly composed of type I collagen, having no collagen of type III. In addition, precursor-form collagen and tropocollagen molecules were found to be present in the incubate.

Alkylation↗
PubMed1977

A complex of collagenase with low molecular weight inhibitors in the culture medium of embryonic chick skin explants.

Collagenase inhibitors with molecular weights of about 6,000 and 12,000 were isolated from latent chick skin collagenase treated with 3 M NaI and from the culture medium of embryonic skin explants. It is suggested that these inhibitors, which are possibly derived from connective tissue macromolecule metabolites, are candidates for regulating factors of collagenase activity in vivo.

Animals↗
PubMed1977

A latent collagenase from embryonic human skin explants.

Collagenase released from embryonic and adult human skin explants has been studied with special reference to the latency of the enzyme. 1) Embryonic human skin explants showed a much higher capacity for collagenase production than did adult skin, on the basis of unit weight of tissue. 2) Culture medium from embryonic skin explants contained latent collagenase at almost twice the concentration of the active form. No appreciable amount of latent enzyme was observed in the adult skin system. 3) The molecular weights of active and latent collagenases were about 40,000 and 50,000, respectively. 4) The latent collagenase was found to be activated by simple passage through a Sephadex G-50 column after adding NaI to a final concentration of 3 M. The degree of activation produced by this treatment was as high as that by limited proteolysis with trypsin. It was concluded that no activating enzyme system was involved in the activation of latent collagenase during NaI treatment, and that the latent enzyme was composed of an enzyme-inhibitor complex. 5) The physiological significance of latent enzyme in the regulation of collagenase activity in vivo is discussed.

Adult↗
PubMed1976

Connective tissue metabolism in culture fibroblasts of a patient with Ehlers-Danlos syndrome type I.

Study on connective tissue metabolism was conducted with a patient with Ehlers-Danlos syndrome (E-D) of Type I who visited our institute. The conversion of procollagen into tropocollagen in the medium of cultured fibroblasts was assayed by the chase technique using 3H-proline. The conversion was inhibited in the cultured fibroblasts of the patient. The components of glycosaminoglycans in cultured medium and fibroblasts from E-D were within normal ranges, however, the ratio of glycoprotein to glycosaminoglycans of intra- and extra-cellular fractions of E-D fibroblasts was higher than the normal one. These findings suggest that the insufficient maturation of collagen fiber may be considered fundamental disorders of E-D.

Adolescent↗
PubMed1976

An electron microscopic study of plasmatic arterionecrosis in the human cerebral arteries.

An electron microscopic study of the intracerebral arteries from 9 hypertensive cases was performed in order to elucidate the morphogenesis of the plasmatic arterionecrosis which was considered to be the direct cause of hypertensive intracerebral hemorrhage. In the preceding stage of the arterial lesions, marked necrosis of medial smooth muscle cells and increase of basement membrane-like substance in the intima and media were observed. The lumina of these arteries were slightly dilated. The dilatation and hemodynamic factors were supposed to cause endothelial injury resulting in blood plasma insudation into the intima through the opened spaces between endothelial cells. The insudated blood plasma dispersed and dissolved the basement membrane-like substance, collagen and elastic fibers in the arterial wall, leading to the development of the plasmatic arterionecrosis.

Aged↗