Maternal body mass index and placental weight: a role for fetal insulin, maternal insulin and leptin.

PURPOSE: Placental weight (PW) has been found to mediate the main effect of maternal BMI on fetal size. Still, the BMI-PW association is poorly understood. Therefore, we aimed to explore potential explanatory variables, including gestational weight gain (GWG), early- and late-pregnancy circulating levels of maternal glucose, insulin, leptin, adiponectin, triglycerides, LDL-C, and HDL-C, and fetal insulin. METHODS: We included two studies of pregnant women from Oslo University Hospital, Norway: the prospective STORK (n = 263) and the cross-sectional 4-vessel method study (4-vessel; n = 165). We used multiple linear regression for data analyses. A non-linear BMI-PW association was observed, which leveled off from BMI25. Therefore, BMI <25 and ≥25 were analyzed separately (n = 170/122 and 93/43 for STORK/4-vessel). Confounding variables included maternal age, parity, and gestational age. RESULTS: PW increased significantly per kg m-2 only among BMI <25 (univariate model's std.ß[p] = 0.233 [0.002] vs. 0.074[0.48]/0.296[0.001] vs. -0.030[0.85] for BMI <25 vs. ≥25 in STORK/4-vessel). Maternal early- but not late-pregnancy insulin and term fetal insulin were associated with PW. The estimated effect of early pregnancy insulin was similar between the BMI groups but statistically significant only among BMI <25 (std.ß[p] = 0.182[0.016] vs. 0.203[0.07] for BMI <25 vs. ≥25). Late pregnancy leptin was inversely associated with PW with a 1.3/1.7-fold greater effect among BMI ≥25 than BMI <25 in the STORK/4-vessel. CONCLUSIONS: The BMI-PW association was non-linear: an association was observed for BMI <25 but not for BMI ≥25. Leptin may be involved in the non-linear association through a placental-adipose tissue interplay. Maternal early pregnancy insulin and fetal insulin at term were associated with PW.

Differences in bone mineral density, markers of bone turnover and extracellular matrix and daily life muscular activity among patients with recent motor-incomplete versus motor-complete spinal cord injury.

Spinal cord injury (SCI) leads to severe bone loss, but the associated mechanisms are poorly described in incomplete SCI individuals. The purpose of the study is to compare alterations in bone mineral density (BMD) and serum biomarkers of bone turnover in recent motor-incomplete to -complete SCI men, as well as to describe their physical activity and spasticity. We studied 31 men with acute SCI. Whole-body DXA scans, serum biomarkers and self-reported activity and spasticity were examined 1 and/or 3 and 12 months after the injury. We observed a decrease in proximal femur BMD (p < 0.02) in both the groups. Serum phosphate and carboxy-terminal-collagen crosslinks were significantly lower in motor-incomplete versus complete SCI men, whereas albumin-corrected Ca(2+) (p = 0.02) were lower only 3 months after injury. When data from all 31 SCI participants were pooled, we observed increased serum matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of MMP-2 (TIMP-2) (p < 0.02) whereas TIMP-1 decreased (p = 0.03). BMD correlated positively with self-reported activity (r = 0.59, p = 0.04) and negatively with spasticity (r = 0.74, p = 0.02) 12 months after injury. As a summary, men with motor-incomplete SCI developed significant proximal femur bone loss 12 months after injury and exhibited increased bone resorption throughout the first year after the injury. Compared with complete SCI men, incomplete SCI men show attenuated bone resorption. Our pooled data show increased turnover of extracellular matrix after injury and that increased exercise before and after injury correlated with reduced bone loss.

Type of carbohydrate in feed affects the expression of small leucine-rich proteoglycans (SLRPs), glycosaminoglycans (GAGs) and interleukins in skeletal muscle of Atlantic cod (Gadus morhua L.).

Aquaculture requires feed that ensures rapid growth and healthy fish. Higher inclusion of plant ingredients is desirable, as marine resources are limited. In this study we investigated the effects of higher starch inclusion in feed on muscular extracellular matrix and interleukin expression in farmed cod. Starch was replaced by complex fibers in the low-starch diet to keep total carbohydrate inclusion similar. Blood glucose and fructosamine levels were elevated in the high-starch group. The group fed a high-starch diet showed up-regulation on mRNA level of proteoglycans biglycan and decorin. ELISA confirmed the real-time PCR results on protein level for biglycan and also showed increase of lumican. For decorin the protein levels were decreased in the high-starch group, in contrast to real-time PCR results. Disaccharide analyses using HPLC showed reduction of glycosaminoglycans. Further, there was up-regulation of interleukin-1ß and -10 on mRNA level in muscle. This study shows that the muscular extracellular matrix composition is affected by diet, and that a high-starch diet results in increased expression of pro-inflammatory genes similar to diabetes in humans.

Biocompatibility and pathways of initial complement pathway activation with Phisio- and PMEA-coated cardiopulmonary bypass circuits during open-heart surgery.

A randomized open-heart surgery study comprising 30 patients was undertaken to compare the biocompatibility of Phisio-(phosphorylcholine) and PMEA-(poly-2-methoxyethyl acrylate) coated cardiopulmonary bypass (CPB) circuits and to assess the initial complement pathway activation during open-heart surgery. Blood samples were obtained at five time points, from the start of surgery to 24 hours postoperatively. The following analyses were performed: haemoglobin, lactate dehydrogenase, leukocyte and platelet counts, myeloperoxidase and neutrophil-activating peptide-2, thrombin-anti-thrombin complexes, syndecan-1 and the complement activation products C1rs-C1-inhibitor complexes, C4bc, C3bc, C3bBbP and the terminal complement complex (TCC). No significant inter-group difference was found in any parameters, except for the concentration of TCC which was moderately lower in the PMEA group at termination of CPB. Complement activation during open-heart surgery was mainly mediated through the alternative pathway. In conclusion, PMEA- and Phisio-coated circuits displayed similar biocompatibility with respect to inflammatory and haemostatic responses during and after open-heart surgery.

Reducing added sugar intake in Norway by replacing sugar sweetened beverages with beverages containing intense sweeteners - a risk benefit assessment.

A risk benefit assessment in Norway on the intake of added sugar, intense sweeteners and benzoic acid from beverages, and the influence of changing from sugar sweetened to diet beverages was performed. National dietary surveys were used in the exposure assessment, and the content of added sugar and food additives were calculated based on actual contents used in beverages and sales volumes provided by the manufactures. The daily intake of sugar, intense sweeteners and benzoic acid were estimated for children (1- to 13-years-old) and adults according to the current intake level and a substitution scenario where it was assumed that all consumed beverages contained intense sweeteners. The change from sugar sweetened to diet beverages reduced the total intake of added sugar for all age groups but especially for adolescent. This change did not result in intake of intense sweeteners from beverages above the respective ADIs. However, the intake of acesulfame K approached ADI for small children and the total intake of benzoic acid was increased to above ADI for most age groups. The highest intake of benzoic acid was observed for 1- to 2-year-old children, and benzoic acid intake in Norwegian children is therefore considered to be of special concern.

Differentiation-associated changes in the expression of chondroitin sulfate proteoglycan in induced U-937 cells.

A monoblastic cell line U-937 (clone 4), was induced to differentiate along the monocytoid lineage by 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA), and vitamin D3 (VD3). By immunochemical and morphological criteria the cells were found to differentiate into macrophage-like cells in the presence of all three inducers. The expression of proteoglycans was investigated in control cultures and in cells differentiated in the presence of both TPA, RA, and VD3. The cells were labeled with [35S]sulfate and cell and medium-associated 35S-macromolecules were either solubilized in sodium dodecyl sulfate or subjected to proteolytic digestion. By use of chondroitinase ABC digestions and deaminative cleavage at pH 1.5 it was demonstrated that all cell cultures incorporated [35S]sulfate exclusively into chondroitin sulfate proteoglycan (CSPG). The expression of CSPG was found to decrease with differentiation to 60% in the presence of TPA, 67% in RA, and 40% in VD3 of control cultures on a cellular basis. The CSPG synthesized was consistently recovered from the medium fractions, whereas free glycosaminoglycan (GAG) chains were found in the cell fraction in all the cell cultures. GAG chains from both control and TPA-, RA-, and VD3-induced cultures were found to be exclusively of the chondroitin 4-sulfate type. However, the CSPGs from RA- and VD3-treated cells were found to differ in molecular size from those of control and TPA-induced cultures, as judged by Sepharose CL-6B gel chromatography. This difference in macromolecular properties following the induced differentiation of the monoblastic cells into macrophage-like cells was found to reside in expression of CSPGs (in the presence of RA and VD3) with smaller GAG chains. Control cells and TPA-induced cells synthesized CSPGs with GAG chains of approximate Mr of 30,000, contrasted by approximate Mr of 17,000 and 16,000 in RA- and VD3-induced cells, respectively. Accordingly, all three agents used in this study were found to induce differentiation of the U-937-4 cells and a decrease in the expression of CSPG, but only RA and VD3 were found to influence the structure of the proteoglycans synthesized.

Proteoglycans in haemopoietic cells.

Proteoglycans are produced by all types of haemopoietic cells including mature cells and the undifferentiated stem cells. The proteinase-resistant secretory granule proteoglycan (serglycin; Ref. 14), is the most prevalent and best characterised of these proteoglycans. Although its complete pattern of distribution in the haemopoietic system is unknown, serglycin has been identified in the mast cells, basophils and NK cells, in which secretion is regulated, and in HL-60 cells and a monocytoid cell line (Kolset, S.O., unpublished data) in which secretion is constitutive. Proteinase-resistant proteoglycans have been detected in human T-lymphocytes and murine stem cells (FDCP-mix) and the core proteins may be closely related to serglycin. A variety of glycosaminoglycan chains are assembled on the serglycin protein and it is likely that this class of proteoglycan can carry out a wide variety of functions in haemopoietic cells including the regulation of immune responses, inflammatory reactions and blood coagulation. There is strong evidence that in mast cells, NK cells and platelets, the proteoglycans are complexed to basic proteins (including enzymes and cytolytic agents) and amines in secretory granules and such complexes may dissociate following secretion from the cell. The stability of the complexes may be regulated by the ambient pH which may be acidic in the granules and neutral or above in the external medium. However, proteinase-proteoglycan complexes in mast cell granules seem to remain stable after secretion and it has been proposed that the proteoglycan regulates activity of proteinases released into the pericellular domain. The functions of proteoglycans which are constitutively secreted from cells are less clear. If cells have no requirement for storage of basic proteins why do they utilise the same design of proteoglycan as cells which accumulate secretory material prior to regulated release? We should stress that the so-called constitutive secretory pathway has been identified in haemopoietic cells in culture, which are usually maintained and grown in the presence of mitogenic factors (e.g., IL-2, IL-3). the cells are therefore activated and it has not been established that continuous proteoglycan secretion occurs in quiescent cells circulating in the peripheral blood. It is possible that lymphocytes, monocytes and macrophages, in which the constitutive secretion pathway operates in vitro, may store proteoglycan in vivo unless stimulated by mitogens or other activating agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Decorin and a large dermatan sulfate proteoglycan in bovine striated muscle.

Proteoglycans were extracted and isolated from adult bovine muscle tissue by dissociative extraction followed by density gradient centrifugation, gel chromatography and ion-exchange chromatography. Two proteoglycans were characterized; one of large molecular size (PG-L) and one of small molecular size (PG-S). The recovery of PG-L and PG-S was 33% and 67%, respectively. By cellulose acetate electrophoresis before and after treatment with chondroitinase AC and ABC both samples were shown to carry predominantly dermatan sulfate chains. The large proteoglycan was recognized with an antibody against a large dermatan sulfate proteoglycan from bovine sclera, whereas the small was recognized by an antibody against decorin from bovine sclera. Chondroitinase ABC treatment of PG-S followed by SDS-PAGE showed a core protein with a molecular weight of 45 kDa, which also reacted with the decorin antibody. Amino-acid analysis of both PG-L and PG-S revealed an amino-acid composition closely similar, although not identical, to the large dermatan sulfate proteoglycan from bovine sclera and decorin, respectively. Immunohistochemical analyses of muscle tissue sections showed that decorin and the large dermatan sulfate proteoglycan are present in the perimysium layers of muscle tissue, although although with a somewhat different pattern of distribution. Decorin was, in addition, found in the endomysium.

Stimulation of serglycin and CD44 mRNA expression in endothelial cells exposed to TNF-alpha and IL-1alpha.

Serglycin is a widely distributed proteoglycan, previously assumed to be hematopoietic cell specific. However, the results presented show that serglycin mRNA is expressed outside the hematopoietic cell system. High levels of serglycin mRNA were detected in endothelial cells and smooth muscle cells, whereas low levels were detected in skin fibroblasts. To further analyze the importance of serglycin in endothelial cells, the expression of serglycin mRNA was measured following activation of an endothelial cell line derived from human umbilical cord vein (HUV-EC-C), by the proinflammatory cytokines TNF-alpha and IL-1alpha. The level of serglycin mRNA increased in a time- and dose-dependent way. TNF-alpha (7 ng/ml) was the most potent inducer, increasing the level of serglycin mRNA 2.5 times after 24 h of stimulation. Serglycin has been shown to be a ligand for CD44, a membrane protein expressed in endothelial cells. Following stimulation of the endothelial cells, the level of CD44 mRNA also increased. Again, TNF-alpha (7 ng/ml) turned out to be the most potent inducer, increasing the level of CD44 mRNA 5.5 times after 24 h of stimulation. Both TNF-alpha and IL-1alpha stimulation of the endothelial cells resulted in an increase in the total incorporation of [(35)S]sulfate into macromolecules, which probably indicates an increase in the total production of proteoglycans. A stimulation of endothelial cells by proinflammatory agents resulted in an increase in both serglycin and CD44 mRNA expression, indicating that serglycin, as well as CD44, may participate in the inflammatory process of leukocyte migration.

Internalization and stepwise degradation of heparan sulfate proteoglycans in rat hepatocytes.

Intracellular transport and degradation of membrane anchored heparan sulfate proteoglycans (HSPGs) were studied in cultured rat hepatocytes labeled with [35S]sulfate and [3H]glucosamine. Pulse chase experiments showed that membrane anchored HSPGs were constitutively transported to the cell surface after completion of polymerization and modification of the glycosaminoglycan chains in the Golgi apparatus. The intact HSPGs had a relatively short residence time at the cell surface and in non-degrading compartments (T(1/2) approximately 2-3 h), while [35S]sulfate labeled degradation products were found in lysosomes, and to a lesser extent in late endosomes. These degradation products which were free heparan sulfate chains with little or no protein covalently attached, were approximately half the size of the original glycosaminoglycan chains and were the only degradation intermediate found in the course of HSPG catabolism in these cells. In cells incubated in the presence of the microtubule perturbant vinblastine, or in the presence of the vacuolar ATPase inhibitor bafilomycin A1, and in cells incubated at 19 degrees C, the endocytosed HSPGs were retained in endosomes and no degradation products were detected. Disruption of lysosomes with glycyl-phenylalanine 2-naphthylamide (GPN) revealed a GPN resistant degradative compartment with both intact and partially degraded HSPGs. This compartment probably corresponds to late endosomes. Treatment of hepatocytes with the thiol protease inhibitor leupeptin inhibited the final degradation of the protein moiety of the HSPGs. The protein portion seems to be degraded completely before the glycosaminoglycan chains are cleaved. The degradation of the glycosaminoglycan chains is rapid and complete with one observable intermediate.

Decreasing the metastatic potential in cancers--targeting the heparan sulfate proteoglycans.

The heterogeneity of proteoglycans (PG)s contributes to their functional diversity. Many functions depend on their ability to bind and modulate the activity of components of the extracellular matrix (ECM). The ability of PGs to interact with other molecules, such as growth factors, is largely determined by the fine structure of the glycosaminoglycan (GAG) chains. Tumorigenesis is associated with changes in the PG synthesis. Heparan sulfate (HS) PGs are involved in several aspects of cancer biology including tumor progression, angiogenesis, and metastasis. PGs can have both tumor promoting and tumor suppressing activities depending on the protein core, the GAG attached, molecules they associate with, localization, the tumor subtype, stages, and degree of tumor differentiation. Perlecan is an angiogenic factor involved in tumor invasiveness. The C-terminal domain V of perlecan, named endorepellin, has however been shown to inhibit angiogenesis. Another angiogenic factor is endostatin, the COOH-terminal domain of the part-time PG collagen XVIII. Glypicans and syndecans may promote local cancer cell growth in some cancer tissues, but inhibit tissue invasion and metastasis in others. The GAG hyaluronan (HA) promotes cancer growth by providing a loose matrix for migrating tumor cells and mediates adhesion of cancer cells. HSPG degrading enzymes like heparanase, heparitinase, and other enzymes such as hyaluronidase and MMP are also important in tumor metastasis. Several different treatment strategies that target PGs have been developed. They have the potential to be effective in reducing tumor growth and inhibit the formation of metastases. PGs are also valuable tumor markers in several cancers.

Macrophages secrete matrix metalloproteinase 9 covalently linked to the core protein of chondroitin sulphate proteoglycans.

Matrix metalloproteinases (MMPs) secreted from the leukemic macrophage cell-line THP-1 have been investigated. Under serum-free conditions, this cell-line synthesizes and secretes proMMP-9, which was detected in the culture medium as a monomer of 92 kDa, and in dimeric forms, including a homodimer of approximately 225 kDa. In addition, a new heterodimer complex is described, in which proMMP-9 is covalently linked to the core protein of chondroitin sulphate proteoglycan (CSPG) through one or more disulphide bridges. After SDS-PAGE electrophoresis, at least two forms of this complex were detected, a large form in the stacking gel and a smaller form with an estimated size of 300 kDa. When the CS chains were removed by chondroitin ABC lyase treatment, heterodimers of proMMP-9/CSPG core protein of approximately 145, 127 and 109 kDa were found, based on zymography and Western blots. Since as much as 10-15 % of the total proMMP-9 secreted from THP-1 cells was covalently linked to CSPG, this association may have important implications for transport, targetting and regulation of the enzyme activity.

Multiplication and death-type of leukemia cell lines exposed to very long-chain polyunsaturated fatty acids.

Polyunsaturated fatty acids (PUFA) may reduce cell multiplication in cultures of normal, as well as transformed, white blood cells. We assessed the sensitivity of 14 different leukemia cell lines to PUFA by measuring cell number after 3 days of incubation. Ten of the examined cell lines were sensitive to 30, 60 and/or 120 microM of arachidonic, eicosapentaenoic and docosahexaenoic acid, whereas four cell lines were resistant. The sensitivity to PUFA was not associated with any particular cell lineage, clinical origin or specific mRNA pattern of bcl-2 and c-myc. Effects on cell viability were assessed by studying cell membrane integrity, DNA fragmentation and cell morphology. The sensitive cell lines Raji and Ramos died by necrosis and apoptosis, respectively, during incubation with eicosapentaenoic acid, whereas the viability of the resistant U-698 cell line was unaffected. The effects of EPA on Raji cells, was counteracted by vitamin E, indicating that lipid peroxidation was involved. However, apoptosis induced by eicosapentaenoic acid in Ramos cells, was unaffected by vitamin E, as well as eicosanoid synthesis inhibitors. In conclusion, our results indicate that a majority of leukemia cell lines are sensitive to PUFA. This sensitivity may be caused by induction of apoptosis or necrosis by very long-chain polyunsaturated fatty acids.

Binding of metastatic colon carcinoma cells to liver macrophages.

The liver is frequently colonized by metastatic tumor cells despite its dense population of macrophages (Kupffer cells). We have studied the interactions between metastatic colon carcinoma cells (DHD) and syngeneic Kupffer cells under different experimental conditions in vitro. In an adhesion assay the binding of DHD cells to Kupffer cell monolayers was shown to be time and temperature dependent, reaching a maximum level after about 90 min of incubation at 37 degrees C. In contrast, only a low level of binding could be observed at 4 degrees C. The level of binding could be increased by pretreatment of the Kupffer cells with phorbol 12-myristate 13-acetate. A firm interaction between the two cell types was shown to be dependent on the presence of calcium- and trypsin-sensitive structures on the surface of the Kupffer cells. Pretreatment of the macrophages with the cytoskeletal inhibitors colchicine and cytochalasin B was also found to reduce significantly the binding of tumor cells. This binding was also inhibited to a large extent by D-mannose and N-acetyl-D-galactosamine. The Kupffer cells were not cytotoxic against the colon carcinoma cells.

Serglycin-binding proteins in activated macrophages and platelets.

The major proteoglycan in macrophages and platelets is the chondroitin sulphate proteoglycan serglycin. To study the biological role of serglycin, its binding to secreted and cell-associated proteins from macrophages and blood platelets was examined. Affinity chromatography with serglycin-Sepharose and chondroitin sulphate-Sepharose was used to isolate proteoglycin-binding proteins from macrophages and platelets. Antibodies against human macrophage inflammatory protein-1 alpha (MIP-1 alpha) precipitated a 14-kDa 35S-methionine-labeled protein among the chondroitin sulfate binding proteins secreted from the macrophage-like U937 cells after stimulation. Two proteins from murine macrophage J774 cells with molecular masses of approximately 10 and 14 kDa were precipitated by an antiserum against the murine MIP-1 alpha. Protein sequencing of fragments obtained by trypsin digestion of a 14-kDa chondroitin sulfate-binding protein from cell extracts of stimulated U937 cells revealed 100% homology with lysozyme, a bacteriolytic enzyme. Fragment of one other protein with approximate molecular mass of 8 kDa showed high homology with bone morphogenetic protein. Inhibition studies showed that chondroitin 6-sulfate inhibited the bacteriolytic activity of lysozyme in a competitive manner more efficiently than heparin and chondroitin 4-sulphate. Amino-terminal sequencing of two proteins from platelet extracts that bound to serglycin-Sepharose revealed that they corresponded to multimeric forms of human platelet factor 4 (PE4). Chondroitin sulfate-Sepharose was shown to be equally efficient in retaining PF4 from platelet extracts as serglycin-Sepharose indicating that the glycosaminoglycan chains mediate the binding to PF4 in the intact proteoglycan molecule. Competition experiments showed that serglycin was as efficient as heparin sulfate in blocking the binding of [3H] chondrotin sulfate to PF4, whereas heparin was one order of magnitude more efficient. Affinity measurements using fluoresceinamine-labeled glycosaminoglycans showed that the affinity of heparin for PF4 is on the order of 30 nM, whereas chondroitin sulfate has an affinity of 260 nM. Both PF4, MIP-1 alpha, and lysozyme play important role in different types of inflammatory reactions. The interaction with serglycin may indicate that this proteoglycan is involved in the regulation of the inflammatory response.

The influence of cellular ATP levels on receptor-mediated endocytosis and degradation of asialo-glycoproteins in suspended hepatocytes.

Receptor-mediated endocytosis in suspended hepatocytes was studied in conjunction with ATP levels of the cells, which were decreased by the use of metabolic inhibitors. The receptor system studied was the asialo-glycoprotein receptor, and multiple aspects of the endocytic pathway were examined: binding of ligand, internalization, intracellular transport and proteolysis. Moderate concentrations of the inhibitors (e.g. 30 microM rotenone or 200 microM iodoacetamide) produced only a transient decline in the ATP levels of the cells. Two to four times higher concentrations reduced the ATP levels to about 1/10 of control cells. At low levels of ATP (less than 30% of controls) the uptake ceased completely after 10-20 min. Moderate reductions brought about by rotenone reduced the uptake roughly in proportion to the ATP levels; iodoacetamide and sodium fluoride had little influence on the energy production by the cells, but the rate of asialo-glycoprotein uptake was reduced to a small fraction of controls. The effect of rotenone on the rate of uptake was mainly due to a lower rate of internalization of occupied receptors; the half-time for internalization of surface-bound ligand was increased from 2.9 to 6.2 min in the presence of 42 microM rotenone. The binding capacity of the cell surface was also somewhat lower. There was no degradation of the asialo-glycoproteins which were taken up by cells treated with high concentrations of rotenone or iodoacetamide. This was shown to be due to a low rate of transport of the endocytosed protein into those endosomes (at density 1.15 g/ml in a sucrose gradient) which were delivering their contents to the lysosomes; coincidentally, there was an accumulation of ligand in light endosomes (density 1.11 g/ml), in which the ligand appears immediately after endocytosis.

Omega-3 fatty acids--nutritional aspects.

Omega-3 fatty acids contain a double bond in the third position from the methyl group. The very long-chain (20 or 22 carbon atoms) omega-3 fatty acids are mostly found in fatty fish and fish oils. The omega-3 fatty acids are essential and may act as precursors for eicosanoids, altering membrane fluidity or binding to transcription factors. Dietary intake of omega-3 fatty acids reduces plasma concentration of triglycerides, probably by decreasing hepatic secretion of very low density lipoprotein (VLDL) and by increasing catabolism of chylomicrons. In addition, lipid peroxidation of omega-3 fatty acids may take place, with good and bad consequences. As the number of double bonds is high, the omega-3 fatty acids may easily react with oxygen radicals. We performed studies where 5 g/day of very long-chain omega-3 fatty acids was given as a supplement for four months along with vitamin E, whereas control groups received similar amounts of other oils. The unsaturation index was higher in fatty acids of LDL from individuals exposed to omega-3 fatty acids, and the amounts of cholesteryl esters and total lipids were lower compared with control LDL, whereas similar electrophoretic mobility and apolipoprotein B structure were observed. There was a decrease in the melting temperature of cholesteryl esters in omega-3 fatty acid-enriched LDL, but no change in the susceptibility of LDL to Cu2+ catalyzed lipid peroxidation, as measured by changes in amounts of lipid peroxides or in the uptake of LDL in macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Sulfated glycosaminoglycans and collagen in two bovine muscles (M. Semitendinosus and M. Psoas major) differing in texture.

M. semitendinosus (ST) and M. psoas major (PM) were used as models for tough and tender meat to study a possible role of sulfated glycosaminoglycans (GAGs) for muscle tenderness. The difference in texture was confirmed by Warner Bratzler shear force measurements. No significant difference in total amount of GAGs in the muscles was found. In contrast, a significant difference in the ratio of GAG/collagen was found between the two muscles. After separation of the GAGs by density gradient ultracentrifugation and ion-exchange chromatography, dermatan sulfate (DS), keratan sulfate (KS), chondroitin sulfate (CS), and heparan sulfate (HS) were identified by cellulose acetate electrophoresis after use of specific enzymes and chemical methods. The content of DS was higher in the tougher muscle (ST) than in PM, and the difference in DS content was statistically significant. Furthermore, a significant difference in the GAG composition pattern of the two muscles was found. The yield of GAGs extracted from the muscles was 77% for ST and 87% for PM. The residue after extraction was further analyzed and found to contain mainly HS. Immunohistochemical studies using antibodies against CS/DS showed a staining pattern of the perimysium of ST different from that of PM.

Cultivation of mouse macrophages in vitro on different protein substrates.

Macrophages were isolated from the peritoneal cavity of mice and cultured in vitro under serum-free conditions on glass coverslips or glass coverslips coated with either collagen, fibronectin or fibrin. The recovery of greater than 95% pure populations of macrophages was highest on fibronectin and fibrin coats when compared to controls on glass coverslips. The cultivation of macrophages on fibronectin and fibrin coats induced a large degree of cell spreading, normally regarded as a parameter of stimulation in cultured macrophages. However, the ability of macrophages to lyse tumour cells and to release lysosomal enzymes was not found to differ to any considerable extent in cells cultured on the different substrates. Culturing macrophages on protein substrates may be used to study macrophage function in vitro, particularly under serum-free conditions.

Sulfur-containing macromolecules in cultured monocyte-like cells.

The monocytic cell line U-937 was cultured in vitro in the absence or presence of phorbol myristate acetate, and agent known to induce differentiation of these cells along the monocyte/macrophage lineage. The cells were fixed, prepared for, and subjected to transmission scanning electron microscopy. The intracellular structures of the cells were compared with those of mastocytoma cells. The latter cell type, known to produce heparin, was shown to contain numerous electron dense granules; by X-ray micro-analyses shown to contain significant amounts of sulfur. In contrast, neither control nor PMA-treated U-937 cells contained such granules. Both control and PMA-treated U-937 cells were pulsed with 35S-sulfate for 60 min and chased. The amount of 35S-proteoglycan in the medium of both cell cultures was found to increase in a time-dependent manner, suggesting that these products are destined for release and not intracellular storage under in vitro conditions.