Biomedical subjects

A Malmström

Publications and source records attributed to A Malmström.

At least 73 records · Page 4Linked to original sources

PubMed1985

The core proteins of large and small interstitial proteoglycans from various connective tissues form distinct subgroups.

Large and small proteoglycans were separately isolated from a number of connective tissues and compared to determine the extent of structural similarity. This was studied by enzyme-linked immunosorbent assays and by the peptide patterns obtained when 125I-labelled proteoglycans were digested with trypsin. All the large proteoglycans, i.e. from tendon, sclera, cartilage and aorta, appear to contain the structure typical for the hyaluronic acid-binding region, both shown by enzyme-linked immunosorbent assay and by content of peptides unique for this region. These proteoglycans also share other structural features of the protein core, as indicated by immunological cross-reactivity and similar peptide patterns. The large proteoglycans from aorta in addition show the presence of unique structures both upon immunoassay and with regard to peptide pattern. Among the small proteoglycans two groups can be identified, although amino acid composition and protein core sizes are grossly similar. One group consists of the small proteoglycans from aorta and cartilage having similar peptide maps and showing immunological cross-reactivity in enzyme-linked immunosorbent assay. The other distinctly different group consists of the small proteoglycans from bone, cornea, sclera and tendon, which among them show identity in enzyme-linked immunosorbent assay and similar peptide patterns. Proteoglycans from the two groups, however, show partial immunological cross-reactivity.

Amino Acids↗

PubMed1985

The integrity of cervical collagen during pregnancy and labor.

The concentration of collagen in the human uterine cervix decreases during pregnancy. This change contributes to the ripening process, which facilitates cervical dilatation during labor. It has been suggested that cervical collagen is partly degraded at term and after delivery. In order to evaluate this hypothesis we investigated the proportion of intact to degraded collagen after solubilization of about 86% of the collagen with pepsin in acetic acid. Total collagen was quantitated as hydroxyproline whereas intact collagen was estimated by SDS-gel electrophoresis. The proportion of intact collagen to total collagen did not change during pregnancy and delivery. Furthermore it was similar to that of intact standard collagen. Thus, even if the concentration of collagen is diminished during pregnancy as a result of increased collagenolytic activity, there is no indication of a qualitative change of the remaining collagen.

Adult↗

PubMed1985

Proteoglycans from cultures of fibroblast from the human uterine cervix.

A small dermatan sulphate proteoglycan has been isolated from the human uterine cervix. The concentration of this proteoglycan has been shown to decrease during pregnancy. To study the mechanism behind this change fibroblasts from the human uterine cervix were established in culture. Their synthesis and secretion of proteoglycans were investigated by incubating the monolayer with 35SO4 and (3H) leucine for 48 h. Two dermatan sulphate proteoglycans and one heparan sulphate proteoglycan were isolated from the medium. The cell layer contained the same proteoglycans although in different proportions. The major dermatan sulphate proteoglycan accounting for 55% of the total macromolecular 35S was small with a molecular weight (MW) of 100,000 daltons. The side chains were calculated to be approximately 1-3 per protein core and included more than 50% iduronic acid containing disaccharides. This small dermatan sulphate proteoglycan is very similar to that isolated from the intact human uterine cervix. However, when cells were established in culture an additional larger dermatan sulphate proteoglycan with an MW of 400,000 daltons was synthesized. This proteoglycan was substituted for 4-8 polysaccharide side chains rich in glucuronic acid. 24% of the total macromolecular 35S were found in this proteoglycan.

Adult↗

PubMed1984

Biosynthesis and secretion of dermatan sulphate proteoglycans in cultures of human skin fibroblasts.

Fibroblasts in culture were incubated with [3H]leucine and [35S]sulphate for 1-24 h. A large glucuronic acid-rich and a small iduronic acid-rich dermatan sulphate proteoglycan were isolated with the use of isopycnic density-gradient centrifugation, ion-exchange and gel chromatography. After 3 h the accumulation in the cell layer of the small proteoglycan reached a steady state, whereas the large one continued to increase, albeit more slowly. In the medium both proteoglycans accumulated 'linearly', although the large one appeared somewhat later than the small one. The composition of the polysaccharide chains and the size of the protein cores did not vary during the experiment. The two proteoglycans were synthesized at approximately similar rates, but were distributed differently in the culture. The small proteoglycan was mainly confined to the medium, whereas the large one was found in the medium as well as in a cell-associated pool. There was an intracellular accumulation of iduronic acid-rich dermatan sulphate as free polysaccharides.

Cells, Cultured↗

PubMed1984

Biosynthesis of dermatan sulfate. II. Substrate specificity of the C-5 uronosyl epimerase.

Epimerization of D-glucuronosyl residues to L-iduronosyl ones during biosynthesis of dermatan sulfate involves an abstraction of the C-5 hydrogen of the target sugar residue. After inversion, a hydrogen from the medium is reinserted into the uronosyl residue. In the present study, microsomal enzyme prepared from cultured embryonic skin fibroblasts was incubated with dermatan or chondroitin in the presence of 3H2O of high specific activity. Incubation resulted in incorporation of tritium on C-5 of uronosyl residues of the substrates. The rate of the reaction was highest for dermatan. Incubation of the products with chondroitinase ABC released essentially all the tritium. Dermatan sulfate and chondroitin sulfate were inactive as substrates, which indicates that epimerization takes place before sulfation. Analyses of the product obtained after incubation of chondroitin in 3H2O-containing medium for different incubation times showed that tritium accumulated first in L-iduronosyl residues. Later, tritium was also found in D-glucuronosyl residues. The reverse situation was observed when dermatan was used as substrate. After extended incubation times, the ratio of D-[3H]glucuronic acid to L-[3H]iduronic acid in both dermatan and chondroitin reached a value of 85/15, which may reflect the equilibrium value. Digestion of labeled chondroitin with chondroitinase AC and oxidation of labeled dermatan with periodate showed that after 96 h of incubation with the epimerase and 3H2O, most of the uronic acid residues had been involved in the reaction. Both products were composed of long blocks of D-glucuronic acid-containing disaccharides interrupted by a few L-iduronic acid-containing disaccharides arranged singly of in clusters of two to three. Reincubation of the 3H-labeled products originating from dermatan or chondroitin with the epimerase resulted in release of tritium, which was linear with time and with increasing protein concentration.

Carbohydrate Epimerases↗

PubMed1984

Equilibration of [3H]glucosamine and [35S]sulfate with intracellular pools of UDP-N-acetylhexosamine and 3'-phosphoadenosine-5'-phosphosulfate (PAPS) in cultured fibroblasts.

Nucleotides and sugar nucleotides were extracted from cultures of human fibroblasts with perchloric acid, separated by isotachophoresis, and quantified by uv absorption analysis at 254 nm. ATP (936 pmol/micrograms DNA) was, as expected, the dominating nucleotide pool. The energy charge was estimated to 0.9. The UDP-N-acetylhexosamine pool was also a very prominent compound (596 pmol/micrograms DNA). After incubation of fibroblasts with [3H]glucosamine, more than 95% of the acid-soluble radioactivity was found in the UDP-N-acetylhexosamine pool. Incubation with [35S]sulfate resulted in the incorporation of [35S]sulfate into 3'-phosphoadenosine-5'-phosphosulfate (PAPS). The latter could, however, only be measured as radioactivity, as the amount was too small to be quantified as total mass. Pulse-labeling of fibroblasts with [35S]sulfate and [3H]glucosamine from 5 min to 16 h showed that [35S]PAPS was equilibrated in less than 10 min, while [3H]glucosamine required a longer time, 2-4 h, to attain a steady state with UDP-N-acetylhexosamine. [14C]Glucose required approximately the same time as [3H]glucosamine to reach steady state with UDP-acetylhexosamine, which suggests that the reason for the long equilibration time is the slow turnover of this pool.

Adenine Nucleotides↗

PubMed1984

Binding of transferrin to the core protein of fibroblast proteoheparan sulfate.

Cell-surface-associated proteoheparan sulfate from confluent human skin fibroblasts appears to consist of two disulfide-bonded polypeptides of Mr approximately equal to 90,000. The transferrin receptor, a ubiquitous cell-surface component of proliferating cells, also consists of two subunits of Mr 90,000 linked by S--S bonds. Radiolabeled proteoheparan sulfate mixed with holotransferrin or apotransferrin at pH 4-5 followed by rabbit anti-human transferrin was adsorbed onto protein A-Sepharose to approximately equal to 80-90%. At pH 7.5 apotransferrin bound approximately equal to 40% of the proteoglycan, whereas approximately equal to 80% was bound to holotransferrin. Trypsin digestion of the proteoglycan markedly lowered its ability to bind transferrin. However, binding was essentially unaffected by heparan-sulfate lyase treatment and after reduction and alkylation. Over 90% of the 3H activity of an L-[3H]leucine-labeled proteoglycan was recovered by immunoprecipitation (transferrin.antitransferrin) of a heparan-sulfate lyase digest of the proteoglycan. The immunoprecipitated core protein had an apparent Mr of 150,000 before reduction and Mr of 90,000 after reduction of disulfide bonds. The core protein of the proteoglycan was recognized by the monoclonal antibody B3/25, which is known to be receptor specific. The present findings suggest that the core polypeptides of proteoheparan sulfate and the transferrin receptor may be identical or closely similar.

Blood Proteins↗

PubMed1983

Proteoheparan sulfate from human skin fibroblasts. Evidence for self-interaction via the heparan sulfate side chains.

We have studied the affinity between fibroblast proteoheparan sulfate (medium- and cell surface-derived species) and heparan sulfate-agaroses by affinity chromatography. The evidence for an interaction between the heparan sulfate side chains of the proteoglycans and the immobilized heparan sulfate are as follows: (a) the individual side chains released from the proteoglycan by papain bind to the affinity matrix, (b) the bound proteoglycans are desorbed by a solution of cognate heparan sulfate chains, and (c) the core protein obtained by heparan sulfate-lyase digestion of the proteoglycan does not bind to the affinity matrix. The proteoglycans interact only with one subtype of heparan sulfate. The binding of free heparan sulfate chains to the affinity matrix is completely abolished by heparan sulfate oligosaccharides provided they are composed of both iduronate- and glucuronate-containing disaccharide sequences.

Animals↗

PubMed1983

Ripening of the human uterine cervix related to changes in collagen, glycosaminoglycans, and collagenolytic activity.

Connective tissue in biopsy specimens taken from the lower part of the uterine cervix in 40 pregnant women at various gestational ages was compared to that in similar biopsy specimens from 15 nonpregnant women. The concentrations of collagen, sulfated glycosaminoglycans, and hyaluronic acid decreased during pregnancy. At the gestational age of 10 weeks, the collagen concentration was 70%, and at term 30%, of that in the nonpregnant cervix. After delivery, no further decrease was observed. The extractability of collagen increased during pregnancy, as well as during labor. Also, the water concentration increased. An increase in the collagenolytic activity was observed with advancing gestational age. The 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gin-D-Arg hydrolytic activity (collagenase) and the concentration of leukocyte elastase increased gradually by a factor of 10. The physiologic importance of the collagen was also demonstrated, since the cervical dilatation time during spontaneous labor was long in women with high concentrations of collagen and short in women with low concentrations of collagen.

Adult↗

PubMed1983

Proteoheparan sulfate from human skin fibroblasts. Isolation and structural characterization.

Fibroblasts in culture were incubated with [35S]sulfate/[3H]glucosamine or [35S]sulfate/[3H]leucine. Proteoglycans were isolated from the medium and a 4 M guanidinium chloride extract of the cell layer or from a trypsin digest of the cells and an extract of the cell residue. Proteoglycans were isolated by density gradient centrifugation, gel permeation, and ion exchange chromatography after digesting contaminating proteogalactosaminoglycans with chondroitinase ABC. Gel chromatography suggests that the cell-derived protoheparan sulfate had an Mr = 350,000 whereas the trypsin-released and the medium-derived counterparts both had an Mr = 140,000. Reduction and alkylation of the cell-derived proteoglycan gave rise to a component with Mr = 140,000, whilst the medium-derived form was not affected. Degradation of cell-associated proteoheparan sulfate by trypsin followed by papain or alkali suggest that the core protein consists of three types of regions, heparan sulfate-containing regions of Mr = 140,000, oligosaccharide-containing regions, and nonglycosylated peptide regions containing most of the [3H]leucine. The heparan sulfates of the cell- and medium-derived proteoglycans were similar in size distribution and charge density and with regard to the proportions and arrangements of various building blocks.

Cells, Cultured↗

PubMed1983

Isolation and characterization of dermatan sulphate proteoglycan from human uterine cervix.

Proteoglycans were extracted from human uterine cervix with 4 M-guanidinium chloride in the presence of proteinase inhibitors. They were purified by density-gradient centrifugation in 4 M-guanidinium chloride/CsCl (starting density 1.32 g/ml) followed by DEAE-cellulose and Sepharose chromatography. Only one polydisperse proteoglycan was found. s020,w was 2.1S and the weight-average molecular weight was 73 000 (sedimentation-equilibrium centrifugation) to 110 500 (light-scattering). The core protein was monodisperse, with an apparent molecular weight of 47 000. The proteoglycan contained about 30% protein and probably two or three glycosaminoglycan side chains per molecule. High contents of aspartate, glutamate and leucine were found. The glycan moiety of the proteoglycan was exclusively dermatan sulphate, with a co-polymeric structure with approximately equal quantities of iduronic acid- and glucuronic acid-containing disaccharides.

Aged↗

PubMed1983

New assay for uronosyl 5-epimerases.

Simple assays have been developed for the two uronosyl 5-epimerases which participate in the biosynthesis of heparin and dermatan sulfate (heparosan N-sulfate D-glucuronosyl 5-epimerase and chondroitin D-glucuronosyl 5-epimerase, respectively). Following previously published procedures, substrates labeled with tritium in the C-5 positions of the D-glucuronosyl and L-iduronosyl residues were prepared enzymatically by incubation of O-desulfated heparin and dermatan with 3H2O and crude epimerase preparations from bovine liver and human skin fibroblasts, respectively. In the new assays, 3H2O generated from these substrates during the epimerase reactions was quantitated by the method of Pollard et al. (Anal. Biochem. (1981) 110, 424-430). In this procedure, 3H2O in the aqueous reaction mixture is extracted into a toluene-based organic phase containing 25% isoamyl alcohol, while the polysaccharide substrate remains in the aqueous phase and does not generate scintillations. This procedure is much simpler than that used previously which involves distillation of each reaction mixture and quantitation of the radioactivity in the distillate. The new assays have been validated by the demonstration that conditions of linearity with time and enzyme concentration can be established for both epimerase reactions. Assays of this type should be useful in the study of any enzymatic reaction where 3H2O is formed from a 3H-labeled substrate and the unreacted substrate is not appreciably soluble in the organic phase.

Animals↗

PubMed1983

Dermatan sulphate and mucin glycopeptides from the human uterine cervix.

High molecular weight glycopeptides and glycosaminoglycans were isolated from the human uterine cervix. The major part of the material (82%) was derived from cervical mucins. The remainder contained hyaluronic acid (3%), heparan sulphate (2%) and dermatan sulphate (13%). Chondroitin sulphate and keratan sulphate were not present, but chondroitin sulphate-like segments were included in the dermatan sulphate. The composition of the cervix apart from the mucus-filled crypts is similar to that of other fibrous connective tissues.

Adult↗

PubMed1983

Increased postpartum collagenolytic activity in cervical connective tissue from women treated with prostaglandin E2.

Cervical biopsies obtained from 7 patients immediately following parturition induced by intracervical application of 0.5 mg prostaglandin E2 (PGE2) in viscous gel were compared with similar biopsies from 11 spontaneously delivered women. A DNP-peptide hydrolytic activity (collagenase) was significantly increased in cervical tissue from the PGE2-induced patients compared with controls. In patients with prompt clinical response, the increase was nearly twofold. No differences were found in the concentrations of water, sulfated glycosaminoglycans, hyaluronic acid, hydroxyproline or leukocyte elastase. Thus, PGE2-induced cervical priming seems to be associated with an increased collagenolytic activity.

Adult↗

PubMed1983

Biochemical changes in human cervical connective tissue after local application of prostaglandin E2.

Intracervical application of prostaglandin E2 in the late first trimester induces (1) softening of the cervix tissue; (2) increase in sulfated glycosaminoglycans (18 +/- 12%, mean +/- SEM); (3) no change in hyaluronic acid and water; (4) decrease in pepsin-extractable collagen, and (5) apparent decrease in collagenase. A high activity of collagenase in combination with a replacement of collagen with sulfated glycosaminoglycans may be of importance for the ripening process.

Body Water↗

PubMed1983

Human cervical connective tissue and its reaction to prostaglandin E2.

A survey of connective tissue in general and human cervical connective in particular is presented. It is concluded that about 90% of the human cervix consists of fibrous connective tissue which, according to the composition of the collagen and the proteoglycans, is very similar to that in skin and sclera. The high activity of collagenase probably gives the cervical connective tissue from pregnant women its potential to ripen following prostaglandin treatment. Electron microscopical examination has revealed an increased amount of amorphous substance after such treatment. This may be caused partly by degraded collagen fibers and partly by newly synthesized proteoglycans.

Animals↗

PubMed1982

Self-association of scleral proteodermatan sulfate. Evidence for interaction via the dermatan sulfate side chains.

Previous studies on scleral proteoglycans (proteodermatan sulfate) using light scattering and ultracentrifugation techniques have shown that the molecules form aggregates in 0.15 M NaCl (Cöster, L., Fransson, L.-A., Sheehan, J. K., Nieduszynski, I. A., and Phelps, C. F. (1981) Biochem. J., 197, 483-490). Aggregation was not promoted by hyaluronate but addition of free scleral dermatan sulfate chains enhanced multimerization. To investigate the possibility that scleral proteoglycans interact via their dermatan sulfate side chains, we have adopted an affinity chromatography procedure where binding of proteoglycans to various dermatan sulfate-agaroses may be studied. The evidence for an interaction between the side chains of the macromolecules and the immobilized dermatan sulfates are as follows: (a) the dermatan sulfate chains released from the proteoglycan by proteolysis display affinity for dermatan sulfate-agarose, (b) a significant proportion of the [3H]acetylated proteoglycans that were bound to the dermatan sulfate gel can be displaced by eluting with a solution of dermatan sulfate chains, (c) selective periodate oxidation of L-iduronate in the dermatan sulfate chains of the proteoglycans abolishes the affinity, (d) the core protein prepared by chondroitinase ABC digestion of the proteoglycan does not bind to dermatan sulfate-agarose, and (e) binding is retained after reduction-alkylation of the protein core. Furthermore, free [3H]dermatan sulfate chains co-elute with the proteoglycan upon gel filtration in 0.2 M NaCl.

Animals↗

PubMed1982

Biosynthesis of dermatan sulphate. Assay and properties of the uronosyl C-5 epimerase.

During biosynthesis of dermatan sulphate D-glucuronate (GlcA) residues are converted to L-iduronate (IdoA) residues via the reaction [Formula: see text]. The reaction occurs on the polymer level and is catalysed by a C-5 uronosyl epimerase. The reversible release of the C-5 hydrogen was utilized as a measure of the enzyme activity with 5-3H-labelled chondroitin as a substrate. 3H released during incubation was distilled and quantified by liquid-scintillation counting. The epimerase has a low pH optimum (5.6) and requires divalent cations, Mn2+ being the most efficient for activity. The Km for chondroitin is 1.2 x 10(-4) M. The epimerase is largely associated with the microsomal fractions (90%). Two-thirds of the activity can be solubilized by detergents. Microsomes from cultured fibroblasts contain two different uronosyl epimerases, one for the biosynthesis of heparan sulphate and one for that of dermatan sulphate. The two epimerases have different cofactor and pH requirements.

Animals↗