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Biomedical subjects

E Kulonen

Publications and source records attributed to E Kulonen.

At least 37 records · Page 2Linked to original sources
PubMed1980

Effects of granulation-tissue extracts on collagen synthesis.

The effect of granulation-tissue extract on the synthesis of collagen in embryonic-chick tendon cells was found to depend on (i) the developmental phase of the granulation tissue, (ii) the concentration of the extract and (iii) the presence of diffusible components in it. The suppressing effect was maximal with concentrated extracts from 2--3-week granulation tissue (where the collagen synthesis is also maximal). The suppressing factor could be enriched by gel filtration chromatography. Cultured human synovial cells were affected like the embryonic-chick cells. When the sponge implant was imbibed with granulation-tissue extract in advance, the developing granuloma contained less collagen than the control. The stimulating effects with dilute extracts from granulation tissue were due to diffusible components. Blood serum and rheumatoid synovial fluid samples also stimulated collagen synthesis in granulation-tissue slices, less after dialysis.

Animals↗
PubMed1980

A rapid assay to measure collagen synthesis in cell cultures.

Limited pepsin digestion and precipitation of resistant parts of proteins with perchloric acid on glass-fiber filters has been used as a rapid way to determine the radioactive collagen secreted into fibroblast culture media. The specificity of the pepsin cleavage was tested by digesting [14C]- or [3H]proline- and [3H]tyrosine-labeled procollagens. Radioactivities obtained with this method were comparable with those obtained with collagenase digestions or hydroxyproline determinations. Dialysis of the samples is avoided and the radioactive collagen can thus be determined from the small medium samples obtained from microtest plates. The method was used to localize a collagen synthesis-increasing factor in preparative isoelectric focusing of microphage culture media.

Animals↗
PubMed1980

Macrophages and the synthesis of connective tissue components.

1. Macrophage homogenate, added to the incubation medium of granulation tissue slices, decreases the incorporation of labelled proline to collagen and other proteins, to a greater extent in the presence of certain prostaglandins and in oxygen atmosphere, but to a lesser degree if serotonin and cyclic AMP are added. 2. Rheumatoid synovial fluid, connective tissue active peptide (CTAP) of Castor and an extract from CCl4-damaged liver stimulate collagen synthesis in the presence of macrophages. 3. Preparations from non-treated macrophages stimulate the incorporation of glucosamine to acid mucopolysaccharides, mainly hyaluronate. 4. In a developing rat granuloma, the formation of collagen was stimulated by homogenized macrophages and by macrophage culture medium, but especially by whole macrophages in the presence of SiO2.

Animals↗
PubMed1980

Effects of purified macrophage RNase on granuloma fibroblasts with reference to silicosis.

Two alkaline RNases, designated RNase 1 and RNase 2, were isolated from the culture media of silica-treated and non-treated macrophages. The yield of RNase from the medium of silica-treated macrophages was 30% of that from the non-treated control. The effects of these RNases on cultured granuloma fibroblasts and on granulation-tissue nuclei were studied. RNase 1 inhibited thymidine incorporation into fibroblasts except at low concentrations, where it was observed to be stimulatory. RNase 1 also inhibited the protein synthesis of fibroblasts. The incorporation of cytidine into RNA in cultured fibroblasts was not affected by RNase 1, but the incorporation into isolated nuclei was decreased. In pulse chase experiments RNase 1 increased the release of cytidine, but not that of thymidine, from the cells. RNase 2 had no effect on the protein or nucleic acid metabolism of the fibroblasts or on the RNA metabolism of isolated nuclei, perhaps because of impermeability. These experiments confirm that macrophage RNase activity is able to regulate the metabolism of granulation-tissue fibroblasts by increasing RNA degradation. Through this action it also regulates DNA and protein synthesis and other metabolic functions of those cells.

Animals↗
PubMed1980

Liberation of a fibrogenic factor from human blood monocytes, ascites cells, cultured histiocytes and transformed mouse macrophages by treatment with SiO2.

Human monocytes and ascites macrophages from cirrhotic patients were isolated in Percoll-gradient and cultured with and without silica. Similar experiments were carried out also with cultured malignant human histiocytes and transformed mouse macrophages. The fibrogenic activity of the culture media was tested by measuring the incorporation of [3H]proline and [3H]thymidine into cultured rat granuloma and human synovial cells. Media from silica-treated monocytes, ascites macrophages and certain histiocyte and mouse macrophage lines caused an increase in the incorporation of both [3H]proline and [3H]thymidine into collagen and DNA, respectively, in both cell systems. Alkaline RNase activities were decreased markedly in the media from silica-treated ascites macrophages but not in the media of the monocytes or histiocytes.

Adult↗
PubMed1980

Influence of extracellular proline on collagen synthesis in rat liver slices.

The synthesis of collagen was measured in incubated rat liver slices. The concentration of proline in the medium was varied, but the specific radioactivity of proline was kept constant. The synthesis of total protein was found to be about 500-fold that of collagen. The incorporation into hydroxyproline--that is, the synthesis of collagen--and the total incorporation of proline into protein were dependent on the concentration of proline in the medium, by Michaelis-Menten kinetics, with an apparent Km of 0.40 mM for the synthesis of collagen and 0.43 mM for the synthesis of total protein.

Animals↗
PubMed1979

Glycoproteins from experimental granulation tissue and their effects on collagen synthesis in embryonic chick tendon cells.

Buffer-soluble and pronase-liberated glycoproteins from experimental granulation tissue were fractionated by gel filtration and DEAE-cellulose chromatography. The age of the granuloma was reflected in the gel filtration pattern. Two glycoproteins were isolated, purified to homogeneity and analyzed for their carbohydrate and amino acid compositions. The collagen synthesis in embryonic chick tendon cells was measured in the presence of these fractions, which were found to be inhibiting even at 10(-6) M. These glycoproteins may be significant in the feedback regulation of the development of granulation tissue and fibrosis.

Amino Acids↗
PubMed1979

Subcellular targets of the soluble SiO2-liberated macrophage factors in experimental granulation tissue.

The supernatant from SiO2-treated macrophages increased the incorporated radioactivity of collagen in the incubated experimental granulation tissue slices, especially in the rough endoplasmic reticulum fraction, markedly over that in the respective control preparation. The effect was observed after a 20 min incubation and increases linearly at least up to 3 h. The amount of RNA and phospholipids in the rough endoplasmic reticulum, calculated per protein, increased simultaneously in the incubated slices. The incorporation of the radioactive precursors into DNA in the slices and in the nuclei from proliferating granulation tissue was enhanced significantly by the soluble fraction from SiO2-treated macrophages. This effect could be seen after a 40 min incubation in the slices and after 5 min in the nuclei, when the incorporated radioactivity in DNA began to decrease in the control experiments.

Animals↗
PubMed1979

Effect of SiO2-liberated macrophage factor on protein synthesis in connective tissue in vitro.

1. To elucidate the role of macrophages in fibrosis the effect of the SiO2-liberated macrophage factor was assessed on protein synthesis in granulation-tissue slices. SiO2 could be replaced by chrysotile asbestos. The active factor is found in the 45/55% sucrose interface of macrophage homogenate. 2. There is no evidence of the involvement of collagenase. 3. The SiO2 effect is not influenced by the addition of yeast RNA to the incubation medium. 4. At a small concentration of polyvinylpyridine-N-oxide (PVNO) protein synthesis in the granulation tissue slices is stimulated, but at higher concentrations PVNO prevented the liberation of the fibrogenic factor from macrophages by SiO2. 5. Phospholipase C and trypsin inhibited the effect of SiO2, which was partially abolished also by heating, and by repeated freezing and thawing. 6. The macrophage RNAse, its inhibitors and fibroblast mRNA are suggested as key factors in the development of fibrosis.

Animals↗
PubMed1979

Ribonuclease activities in developing experimental granulation tissue with reference to polysomes.

The Mg2+-precipitable polysomes in experimental granulation tissue increase up to three weeks and decrease slowly thereafter. Polysomes from young granulation tissue are, on a weight basis, more active in cell-free protein synthesis than polysomes from old granulation tissue. RNase activity is highest in polysomes from 1-week tissue. The polysomes contain both acid and alkaline RNase, but the alkaline RNase is the latent form, manifested by the addition of p-chloromercuribenzoate (pCMB). The high RNase activity of granulation tissue renders the analysis of polysomal patterns and functions of the polysomes difficult. In the post-polysomal supernatant of granulation tissue the RNase activity is highest in 2-4 week old tissue; it is maximal at pH 7-8 and only slightly influenced by pCMB.

Animals↗
PubMed1979

Preparation of subcellular fractions from granulation tissue by density gradient centrifugation.

The 7000 g pellet of homogenized mature sponge-induced granulation tissue was fractionated by centrifugation in a stepwise sucrose gradient in order to study the synthesis and secretion of collagen and other components of this tissue on the subcellular level. As indicated by chemical and enzymatic assays, by electron microscopy and by incorporation experiments, the collagen-synthethesizing rough endoplasmic reticulum fraction was isolated free from the secretory vesicles (smooth endoplasmic membranes and Golgi elements) and fibrous extracellular matrix. Collagen differed from other proteins in the distribution among the subcellular fractions. In pulse-chase experiment the translocation of 14C-labelled collagen was demonstrated from the rough endoplasmic reticulum through the secretory vesicles to extracellular fibrillar collagen. This fractionation method will be used to study the modulation of collagen synthesis and secretion in the reparative tissue.

Animals↗
PubMed1979

Fractionation of connective-tissue-activating factors from the culture medium of silica-treated macrophages.

The medium of cultured, SiO2-treated peritoneal macrophages contained a factor which enhances the incorporation of labelled proline to collagen and other proteins in granulation tissue slices, cells and polysomes. Simultaneously, the activity of alkaline RNase in the whole medium was decreased in comparison with the corresponding control. Polyvinylpyridine-N-oxide, PVNO, protected the macrophages against SiO2. Latex-particles and E. coli lipopolysaccharide decreased the RNase activity in the macrophage medium, but unlike SiO2 did not cause liberation of the collagen synthesis-stimulating factor. Fractionation of the medium by gel filtration chromatography showed the SiO2-pretreatment to have caused a very significant decrease in the aggregation state of RNase. The fraction from gel filtration chromatography that contained the SiO2-liberated factor stimulating collagen synthesis also contained the disaggregated RNase. There was no RNase-activity in the control sample. A homogenous protein (mol. wt. 14,300) was isolated with repeated gel filtrations from the medium of silica-treated macrophages. It increased the incorporation of 3H proline and 3H thymidine into cultured granuloma cells.

Animals↗
PubMed1979

Isoelectric focusing of macrophage culture media and the effect of the fractions on the synthesis of DNA and collagen by fibroblasts.

Macromolecules from rat peritoneal macrophage culture media were separated into 30 fractions by flat bed isoelectric focusing (IEF). The fractions were tested for their influence on thymidine and proline incorporation into cultured rat granuloma fibroblasts. Three fractions, stable after freezing and lyophilization, were of interest: one inhibiting thymidine incorporation (focusing at pH 7.3-7.6), another stimulating thymidine incorporation (focusing at pH 4.4-5.3), and the third stimulating proline incorporation into cells and medium collagen (focusing at pH 10.2-10.7). The last also exhibited a ribonuclease (RNase) activity with a pH-optimum of 7.0-7.5.

Animals↗
PubMed1978

Dependence of collagen synthesis by embryonic chick tendon cells on the extracellular concentrations of glutamine.

An agent in rheumatoid synovial fluid which stimulates collagen synthesis by embryonic chick tendon cells was identified as glutamine. The stimulating effect was observed only at a subnormal concentration of proline (0.02 mM). The concentration of glutamine in rheumatoid synovial fluid is about 2.5 mM but it falls rapidly during storage even at -20 degrees C. Collagen synthesis is shown to depend on the extracellular concentrations of glutamine and proline in addition to any connective tissue activating factors.

Animals↗
PubMed1978

Alkaline ribonuclease associated with polyribosomes in fibroblasts of experimental granulation tissue.

Alkaline ribonuclease (RNase) from polyribosomes derived from experimental granulation tissue has been purified 1900-fold through affinity chromatography. The preparation was homogeneous in sodium dodecyl sulfate (SDS) polyacrylamide-gel electrophoresis with an estimated molecular weight of 15 000. Purified RNase was completely inhibited in the presence of divalent ions Mg2+(100 mM) and Ca2+(100 mM) but activated slightly with Na+(50 mM). The enzyme is an endonuclease and the best substrates were poly(U), mixed RNA from yeast, rRNA from granulation tissue and poly(C). The estimated apparent Km-values were 0.037, 0.064, 0.13 and 0.27 g1-1, respectively. In polyribosomes RNase occurred in both free and p-chloromercuribenzoate (pCMB)-liberated forms. The total activity was at the highest but the proportion of the free activity minimal in the granulation tissue during the maximal synthesis of collagen.

Animals↗