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S Gustafson

Publications and source records attributed to S Gustafson.

At least 37 records · Page 2Linked to original sources
PubMed1994

Receptors for hyaluronan on corneal endothelial cells.

Previous investigations suggest that the corneal endothelium has specific binding sites for hyaluronan (HYA). In the present study, biochemical and immunological techniques were used to characterize these binding sites and to compare them with the liver endothelial cell (LEC) HYA receptor. Affinity chromatography of solubilised, 125I-labelled corneal endothelial cell surface proteins on immobilised HYA proved that there were molecules that were strongly bound to the polysaccharide. A part of these molecules formed a 100-kDa band when analysed by autoradiography after SDS polyacrylamide electrophoresis (PAGE). A specific antibody against the rat LEC HYA receptor was used for immunohistochemical studies of monkey and human corneas. There was a specific staining of the corneal endothelium of both species, and hyaluronan treatment before isolation of the human eyes reduced the staining intensity. Hyaluronidase treatment of the tissue sections before receptor staining strikingly increased the specific staining of the corneal endothelial cells (CEC). Immunoblotting of human corneal proteins, separated by SDS-PAGE, showed staining at 200, 150-160 and 55 kDa. Uptake experiments of tritiated HYA in cultured monkey CEC showed only a slight increase in cell associated radioactivity over 2-6 hr. The results make it unlikely that the corneal endothelial receptor, like its liver endothelial counterpart, is actively involved in receptor-mediated endocytosis. Our studies suggest that CEC carry receptors for HYA that are immunologically similar to the LEC receptors. CEC receptors might act as binding structures increasing the concentration of HYA close to the CEC as a protection of these vulnerable cells from physicochemical damage.

Animals↗
PubMed1994

Labelling of high molecular weight hyaluronan with 125I-tyrosine: studies in vitro and in vivo in the rat.

Studies on the metabolism of the polysaccharide hyaluronan has previously been hampered by the lack of radioactive hyaluronan of high molecular weight (MW) and high specific activity. In the present study 125I-tyrosine (T)-labelled hyaluronan was produced after CNBr-activation of the polysaccharide. A specific activity of approximately 0.1 MBq microgram-1 was achieved using 100 micrograms of 0.5 x 10(6) Da hyaluronan labelled for 2 h with 18 MBq 125I. The 125I-T-hyaluronan kept a high MW-profile upon gel filtration chromatography and was found to be cleared from the circulation with the kinetics and organ distribution reported for biosynthetically labelled hyaluronan of high MW. The 125I-labelled polysaccharide is also taken up by liver endothelial cells both in vivo and in vitro, indicating that the labelling does not interfere with the binding to specific cell-surface receptors found on these cells. The intracellular degradation is slower than that earlier reported for biosynthetically labelled hyaluronan and seems to be halted at the level of low MW oligo- or mono-saccharides that eventually leave the organism via the urine. Scintigraphic images of rats after intravenous injection of 125I-T-hyaluronan showed rapid uptake in the liver and a redistribution of radioactivity from liver to urine with time. Our results indicate that the 125I-T-hyaluronan is suitable for studies of hyaluronan-metabolism in a number of ways. The gamma emitters 125I and 131I are easy to monitor and can be used also for in vivo 3D-imaging using single photon emission computer tomography.

Animals↗
PubMed1993

The effect of felodipine on the uptake and degradation of acetylated LDL in mouse peritoneal cells and on the distribution of acetylated LDL in macrophage-rich organs of the rat.

The effect of felodipine on lipoprotein metabolism ex vivo and in vivo was investigated. In the ex vivo studies mice were given felodipine (40-125 mumol/kg body weight) or vehicle for one week. Peritoneal macrophages from these animals and controls were isolated and used in binding and degradation studies with human iodinated acetylated LDL (Ac-LDL). Macrophages from felodipine-treated mice showed a significant decrease of binding and degradation of Ac-LDL compared to macrophages from control animals (P < 0.05). The in vivo studies were performed in rats pretreated with felodipine or vehicle. To determine the distribution and plasma turnover of LDL and Ac-LDL, 125I-tyramine cellobiose labelled LDL or Ac-LDL were given i.v. No differences in the removal rate of Ac-LDL or LDL were observed between felodipine-treated or untreated rats. However, an increased uptake of Ac-LDL could be seen in the liver of the felodipine-treated rats. This increased uptake could be ascribed to the parenchymal cells because no differences in uptake could be seen in the liver endothelial cells. However, a significant decreased uptake was seen in the Kupffer cells and in the spleen, a macrophage-rich organ, of the felodipine-treated rats. The present study suggests a possible mechanism behind the antiatherogenic effects of calcium antagonists, a decreased uptake of atherogenic modified lipoproteins by peripheral macrophages and an increased uptake by the liver.

Acetylation↗
PubMed1991

Characterization and purification of the hyaluronan-receptor on liver endothelial cells.

In order to characterize the proteins on liver endothelial cells that bind hyaluronan (HYA), liver endothelial cells were surface-iodinated with 125I, solubilized by Triton X-100 and passed through a column containing HYA coupled to agarose. The column was washed and eluted with HYA-oligosaccharides. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted material, followed by autoradiography, showed a major band with a molecular mass of 100 kDa, that upon reduction gave major bands of 20 and 35 kDa, and minor doublet bands at 60 and 80 kDa. Two-dimensional electrophoresis of liver endothelial cell membrane proteins revealed that the 100 kDa protein has a pI of 6.6-6.8. The protein was purified by preparative SDS-PAGE of liver endothelial cell membrane proteins. The 100 kDa protein was excised from the gel and used for immunization of rabbits. Antiserum from immunized rabbits specifically recognized only the 100 kDa protein on immunoblots of liver endothelial cell membrane proteins separated by SDS-PAGE. The binding of 3H-HYA to liver endothelial cells and liver endothelial cell membranes could be specifically inhibited by Fab-fragments of the antibodies. When we tried to isolate the receptor in large scale by affinity chromatography of proteins from purified liver endothelial cell membranes, the 100 kDa protein could often not be detected on immunoblots or by silver staining following SDS-PAGE of the eluted material. Instead, proteins with molecular masses of 55 and 15 kDa were detected, but the antibodies reacted specifically with these proteins. Thus the 100 kDa protein is apparently susceptible to cleavage into distinct subcomponents.

Animals↗
PubMed1991

Hyaluronan-binding proteins on cultured J 774 macrophages.

Cultivated macrophages of murine cell-line J 774 were found to bind high-molecular-weight (molecular weight average approx. 5.10(6) [3H]hyaluronan (HA) by a saturable mechanism at 4 degrees C. Half-maximal binding was observed at 7-8 microgram/ml (1.4-1.6 nM) and the maximal binding was reached at 30-40 microgram/ml. Scatchard plot analysis revealed that approx. 20,000 molecules could bind to each cell with a Kd of 1.5 nM. The binding could be effectively inhibited by unlabeled HA. Also chondroitin sulphate inhibited the binding, but only to about 50%. At 37 degrees C the J 774 cells took up and degraded the polysaccharide effectively. Affinity chromatography on HA coupled to agarose of solubilized surface-iodinated J 774 cells, revealed that a protein of approx. 60 kDa, when analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis and autoradiography, could be specifically eluted with HA-oligosaccharides. Our results suggest that J 774 macrophages can bind HA by a mechanism compatible with receptor-binding, and carry a 60 kDa HA-binding protein on their surface. This receptor-binding may mediate uptake and degradation of the polysaccharide and influence the levels and turnover of HA in interstitial fluid as well as the release of HA into the bloodstream.

Animals↗
PubMed1990

Metabolism of very low density lipoproteins in rats with isotretinoin (13-cis retinoic acid)-induced hyperlipidemia.

A significant rise in plasma triacylglycerols from the control level of 0.89 mmol/l to 1.88 mmol/l (P less than 0.001) was observed in male Sprague-Dawley rats treated for 11 days with isotretinoin (oral dosing; 10 mg/day). This rise was due to an increased level of plasma very low density lipoproteins (VLDL). When VLDL from untreated rats were labeled with 125I-labeled tyramine-cellobiose and injected intravenously into rats treated for 10 days with isotretinoin (n = 6) and in control rats (n = 6), it was found that the disappearance of radioactivity from the blood was dramatically retarded in the treated animals. The disappearance could be divided into two phases, a rapid (alpha) phase dominated the first 5 min and was followed by a slower (beta) phase. The half-life of the beta-phase increased significantly from 53 +/- 7 min in the controls, to 120 +/- 62 min after isotretinoin. VLDL prepared from isotretinoin-treated animals (n = 6) had about the same half-life in control animals (62 +/- 8 min) as had ordinary VLDL. The elimination of tracer from the blood was mainly due to uptake by the liver. The amount of radioactivity in the liver after 30 min of circulation was significantly reduced from 34 +/- 7% of injected dose in controls to 24 +/- 5% in the isotretinoin group (P = 0.013). The uptake in other organs was less than 3% per organ and was essentially unaffected by the treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Lovastatin is hypertriglyceridemic in Syrian golden hamsters.

The effects of a potent HMG CoA reductase inhibitor, lovastatin, was studied in male Syrian Golden hamsters. Lovastatin (0.1% in food for 6 days) increased hamster serum triglycerides by 12-fold with 2.4-fold increase in serum cholesterol. On continuous treatment serum triglyceride and cholesterol levels gradually decreased to below control values by 12-18 days. When hamsters were fed a mixture of lovastatin and Na-mevalonate no increase in serum triglyceride was observed. [14C]Cholesterol synthesis was increased by 266-fold in livers of hamsters fed lovastatin for 6 days. The increased synthesis of endogenous mevalonate metabolites may be a reason for the decrease in triglyceride levels after 6 days in our studies. The present study suggests that a mevalonate metabolite(s) is necessary for normal triglyceride metabolism in hamsters.

Animals↗
PubMed1988

Apolipoprotein-E-binding proteins of rat liver endothelial cells.

In an attempt to characterise the apolipoprotein-E-binding proteins of rat liver endothelial cells, we prepared membranes from monolayer cultures of liver endothelial cells as an enriched source of membrane receptors. The membranes could specifically bind iodinated very-low-density lipoproteins (VLDL) and the binding could be inhibited effectively by unlabelled VLDL and high-density lipoproteins, but only moderately by low-density lipoproteins. To identify the binding proteins, we performed immunoprecipitation studies of solubilised iodinated liver endothelial cells and cell membranes, respectively, using purified apolipoprotein E and monospecific polyclonal IgG directed towards this apolipoprotein. The antibodies together with the bound apolipoprotein E and iodinated liver endothelial cell proteins were harvested with staphylococcal protein A-Sepharose. The immunoprecipitates were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and after autoradiography of the dried gel, the Mr of the liver endothelial cell proteins bound to apolipoprotein E could be determined. Two protein bands with molecular masses of 55-60 and 110, and a weak band of 170 kDa could be detected from intact cells. These proteins were specifically precipitated only in the presence of divalent cations, and might represent cell-surface receptors for apolipoprotein-E-containing lipoproteins. Additional bands were seen when cell membranes were used, the most prominent ones having molecular masses of 32 and 35 kDa. These proteins could be of intracellular origin, or they may be degradation products of the other apolipoprotein-E-binding proteins.

Actin Cytoskeleton↗
PubMed1987

Parameters affecting the yield of DNA from human blood.

An examination of variables affecting the yield of DNA from blood was undertaken in order to improve sample processing and to evaluate alternative methods of mailing blood samples for DNA analysis. A rapid, high-yield method was developed for the isolation of high-molecular-weight DNA from fresh and frozen blood. In addition, the following observations were made: (1) Of the anticoagulants examined, acid citrate dextrose (ACD) solution B was found to be superior to EDTA and heparin for preserving yields of DNA during incubation at room temperature. If DNA is isolated from frozen blood, high yields of undegraded DNA are achieved after incubation at 23 degrees C for 5 days with ACD solution B. (2) High yields of undegraded DNA are obtained from blood stored with ACD solution B for at least 1 day at 42 degrees C, 5 days at 0 degrees C, or 1 month at -20 degrees C. (3) Three cycles of freezing and thawing may have little if any affect on the yield of DNA. The results indicate that blood for DNA extraction may be mailed in an ambient temperature container and, in many cases, sent by first-class mail rather than by overnight delivery services.

Anticoagulants↗
PubMed1987

Identification of a fibronectin receptor specific for rat liver endothelial cells.

Antibodies raised against the fibronectin receptor of rat hepatocytes recognized one protein (Mr 120 and 135 kDa for unreduced and reduced samples, respectively) in immunoblotting of solubilized rat liver endothelial cells (LEC). The antibodies specifically precipitated a 200-kDa protein together with the 135-kDa component from 125I-labeled LEC. Spreading of LEC on fibronectin, but not on laminin or collagen, was inhibited by monovalent Fab fragments of the antibodies, implicating that the 135/200-kDa complex is a specific fibronectin receptor. The results indicate that LEC, hepatocytes, and fibroblasts of rat carry different fibronectin receptors, suggesting that the interaction of fibronectin with these cells may have different functional roles.

Animals↗
PubMed1987

Lipoprotein composition of human suction-blister interstitial fluid.

Interstitial fluid (IF) was obtained in 27 apparently healthy subjects (12 males, 15 females) by applying mild suction (200-250 mm Hg) on the skin either on the midvolar forearm or on the paraumbilical region of the abdomen. The IF concentrations of lipids and apolipoproteins (apo) were studied and compared with those of serum (S). The mean ratio between interstitial fluid and serum (IF/S ratio) varied from 0.14 for forearm apoE to 0.29 for apoA-II on the abdomen. This ratio was consistently lower for apoE, C-II, C-III, and B than for apoA-I and A-II, and significantly lower on the arm than on the abdomen for all apolipoproteins studied. The IF/S ratios showed marked variations among individuals. However, interstitial fluid apolipoprotein concentrations at different blister sites were highly correlated within each individual. Studies with agarose gel electrophoresis and density gradient ultracentrifugation revealed that large triglyceride-rich particles were virtually lacking in interstitial fluid and that the relation between the low density lipoproteins (LDL) and high density lipoproteins (HDL) was shifted towards a greater proportion of HDL. The lipoprotein distribution in the HDL range of interstitial fluid differed from that of serum showing one maximum at a density of about 1.070 g/ml (serum HDL2 about 1.090 g/ml) and one at a density of 1.130-1.140 g/ml (serum HDL3, 1.110-1.120 g/ml). The former subfraction contained most of the lipoprotein-bound apoE while the latter contained the major part of apoA-I and apoA-II. Studies of the lipoproteins of interstitial fluid may add to our understanding of the development of atherosclerosis and xanthomatosis and may also provide valuable information on the permeability of the capillary membrane in normo- and pathophysiological states.

Adult↗
PubMed1986

HDL from type III dysbetalipoproteinaemic patients show decreased capacity to inhibit VLDL uptake in rat liver endothelial cells.

The saturable uptake and degradation of 125I-labelled human very low density lipoproteins in cultured rat liver endothelial cells could be effectively inhibited by high density lipoproteins (HDL) from normal subjects. Up to eight times more HDL (in relation to cholesterol content) was needed from patients with hyperlipoproteinaemia (HLP) type III to give the same inhibition. The HDL apolipoprotein (apo) E concentrations that were needed to give the same inhibition as normal HDL apo E were between 3 and 50 times higher in HLP type III. Our results suggest that the lipoprotein abnormality in HLP type III not only affects chylomicron remnant metabolism but also the composition and function of HDL.

Adult↗
PubMed1985

Serum and interstitial fluid apolipoprotein E levels in the healthy and in hyperlipoproteinemia type III as studied by radioimmunoassay.

In order to study the relationships between serum lipoprotein lipid concentrations and the concentrations of apo E in serum and interstitial fluid, we have developed a specific, sensitive and rapid radioimmunoassay for this apolipoprotein. Studies of the interstitial fluid lipoproteins and of the gradient between the lipoprotein concentrations in interstitial fluid and serum may add to our understanding of the development of atherosclerosis and xanthomatosis. Serum, interstitial fluid, lipoproteins or standards were incubated with 125I-labelled apo E and rabbit antiserum against apo E for 90-120 min at room temperature. The immune complexes were harvested with the use of formalin-treated staphylococci. The displacement curves produced by standard and samples of serum, interstitial fluid and isolated lipoproteins were linear in logit-log plots and had identical slopes. Delipidation did not change the results and the recovery of added apo E to a serum sample was 96 +/- 5% (n = 5). Apo E was found in all major lipoprotein classes and the concentrations of apo E in serum and in interstitial fluid were 36 +/- 19 mg/l and 8 +/- 4 mg/l, respectively, in normals (n = 21) and 305 +/- 125 mg/ml and 20 +/- 9 mg/l, respectively, in patients with HLP type III (n = 11). Highly significant positive correlations were found in HLP type III between the interstitial fluid level of apo E and the corresponding concentrations of cholesterol and triglyceride. Interstitial fluid apo E concentrations were significantly correlated to apo E but not to the lipid levels in serum, indicating that only some subclasses of the serum lipoproteins are transported to the interstitial compartment.

Apolipoproteins E↗
PubMed1985

Uptake and degradation of human very-low-density lipoproteins by rat liver endothelial cells in culture.

Rat liver endothelial cells in primary cultures take up and degrade 125I-labelled human very-low-density lipoproteins (VLDL) in a saturable fashion at physiological triacylglycerol concentrations. The iodinated VLDL are readily taken up by the freshly isolated endothelial cells and degradation products appear in the medium about 30 min after the addition of VLDL to the cultures. Uptake and degradation at 37 degrees C are effectively inhibited by unlabelled human VLDL, low-density lipoproteins (LDL), high-density lipoproteins and lymph chylomicrons, but only modestly by acetylated LDL. Purified apolipoproteins E and C-III:1 also compete with the uptake of iodinated VLDL, but when degradation was studied for longer periods of time, such a competition could not be demonstrated. This may be due to the fact that the added apolipoproteins become associated with the lipoproteins. In binding experiments at 7 degrees C, iodinated apolipoprotein C III:1 bound to the liver endothelial cells in a manner characteristic of receptor binding with a dissociation constant of 0.5 microM. This binding could not only be inhibited by unlabelled apolipoprotein C-III:1 but also by unlabelled apolipoprotein E. The results indicate that rat liver endothelial cells carry receptors for VLDL and that these recognize the apolipoproteins E, C-III and B on the lipoprotein surface. Considering the large endothelial surface and high blood flow through the liver, significant quantities of lipoproteins can be taken up and degraded, thus influencing the levels of circulating lipoproteins in the in vivo situation.

Animals↗
PubMed1984

Increased high density lipoproteins in diabetic children.

Serum lipoprotein lipid and apolipoprotein concentrations were determined in 27 diabetic children (5-18 years old) and 13 matched healthy controls. The serum cholesterol concentrations in the diabetics were slightly higher than in the controls (P less than 0.05) due to a significantly higher level of the high density lipoprotein cholesterol (P less than 0.01). Also the serum concentration of apolipoprotein A-I, the major protein constituent of the high density lipoprotein fraction, was higher in the diabetic children (P = 0.05). There were no significant differences between the groups with regard to the serum triglyceride concentrations or the apolipoprotein C-II and C-III concentrations. Neither the lipoprotein lipid nor the apolipoprotein levels were significantly correlated with variables related to the degree of regulation of the diabetic disease. No obvious explanation, based on the present data, can be given for the increased high density lipoprotein cholesterol concentrations in insulin-treated diabetics in comparison with the healthy children. It is possible, however, that the increased high density lipoprotein cholesterol concentration may be caused by an increased level of insulin in the circulation of insulin-treated diabetic children.

Adolescent↗
PubMed1984

A rapid radioimmunoassay of human apolipoproteins C-II and C-III.

Apolipoprotein (apo) C-II is an activator of lipoprotein lipase, while apo C-III has the ability to inhibit apo C-II activated lipolysis. In order to study further the relationship between lipoprotein lipase mediated hydrolysis and the serum concentrations of apo C-II and apo C-III we have developed radioimmunoassays for these apolipoproteins. Apo C-II and apo C-III were isolated from very low density lipoproteins (VLDL) by ion exchange chromatography. Apo C-II was further purified by affinity-chromatography. The purity of the two proteins was determined to be greater than 98% by polyacrylamide gel electrophoresis and immunodiffusion. The purified apolipoproteins were used as antigens, assay standards and labels. Formalin-treated Staphylococcus aureus Cowan I was used for immunoprecipitation and were shown to give rapid uptake of immune complexes that could easily be harvested by centrifugation. The assays were shown to be sensitive (10 micrograms/l), specific, precise (inter- and intra-assay coefficients of variation below 10%), rapid (completed in less than 6 h) and simple to perform. Delipidation of serum and lipoproteins had no effect on the results, indicating that the immunologically active sites of apo C-II and apo C-III are exposed to the aqueous environment under assay conditions. Serum apo C-II and apo C-III levels of normolipidaemic subjects were approximately 25 mg/l and 110 mg/l, respectively. Highly significant positive correlations were found between VLDL apo C-II and VLDL apo C-III, respectively, and VLDL triglycerides, VLDL cholesterol and total serum TG. There was also a highly significant correlation between the HDL cholesterol concentration and the HDL apo C-III concentration.

Apolipoprotein C-II↗