Surface-associated heparin inhibits zymosan-induced activation of the human alternative complement pathway by augmenting the regulatory action of the control proteins on particle-bound C3b.

Discrimination by the human alternative pathway between activating and nonactivating particles occurs after deposition of C3b by the continuous low-grade interaction of the alternative pathway components in biologic fluids and is dependent on the modulation by surface constituents of the interaction of bound C3b with the control proteins, beta 1H, and C3b inactivator (C3bINA). When heparin glycosaminoglycan was coupled to activating particles, such as zymosan or Sepharose, by cyanogen bromide activation, their capacity to activate the human alternative pathway was inhibited. The loss of alternative pathway-activating capacity was directly correlated to the number of heparin molecules bound/zymosan particle, whether the ratio was varied by increasing the amounts of heparin in the initial coupling reactions or by treating a fully inhibited particle with incremental concentrations of heparinase. Analysis by linear regression of the inhibitory effect of each procedure (r = 0.97, r = 0.98, respectively) for adjusting the number of heparin molecules/particle revealed that the dose-response relationships were identical and that complete inhibition occurred with greater than 12 X 10(8) molecules of heparin/zymosan particle. The coupling of heparin to zymosan did not impair the uptake of C3b from the fluid-phase interaction of C3, B, and D, and did not alter the capacity of bound C3b to associate with B so as to permit its inactivation by D. Although the regulatory proteins present in normal serum chelated with EDTA or presented as a combination of purified C3bINA and beta 1H were relatively inefficient in inactivating C3b function on an activating particle of the alternative pathway such as zymosan or zymosan-cyanogen bromide, the control proteins rapidly inactivated C3b on a nonactivating particle wuch as a sheep erythrocyte or zymosan with coupled heparin. The increased numbers of C3b sites susceptible to inactivation by C3bINA in the presence of beta 1H were significantly correlated to the number of molecules of heparin/particle. By linear regression analysis of the correlation (r = 0.99) the number of heparin molecules/particle required to promote total inactivation of bound C3b by purified control proteins was 13.8 X 10(6). This molecular analysis suggests that the action of heparin coupled to an activating particle of the alternative pathway is to promote the interaction between particle-bound C3b and the regulatory proteins, thereby preventing particle-associated amplified C3 cleavage. It is noteworthy that both surface constituents known to maintain a particle as a nonactivator of the alternative pathway, sialic acid and N-sulfated mucopolysaccharide, act by facilitating the inactivation by regulatory proteins of the function of particle-bound C3b.

Cell surface heparan sulfate mediates some adhesive responses to glycosaminoglycan-binding matrices, including fibronectin.

Proteins with affinities for specific glycosaminoglycans (GAC's) were used as probes for testing the potential of cell surface GAG's to mediate cell adhesive responses to extracellular matrices (ECM). Plasma fibronectin (FN) and proteins that bind hyaluronate (cartilage proteo-glycan core and link proteins) or heparan sulfate (platelet factor 4 [PF4]) were adsorbed to inert substrata to evaluate attachment and spreading of several 3T3 cell lines. Cells failed to attach to hyaluronate-binding substrata. The rates of attachment on PF4 were identical to those on FN; however, PF4 stimulated formation of broad convex lamellae but not tapered cell processes fibers during the spreading response. PF4-mediated responses were blocked by treating the PF4-adsorbed substratum with heparin (but not chondroitin sulfate), or alternatively the cells with Flavobacter heparinum heparinase (but not chondroitinase ABC). Heparinase treatment did not inhibit cell attachment to FN but did inhibit spreading. Cells spread on PF4 or FN contained similar Ca2+-independent cell-substratum adhesions, as revealed by EGTA-mediated retraction of their substratum-bound processes. Microtubular networks reorganized in cells on PF4 but failed to extend into the broadly spread lamellae, where fine microfilament bundles had developed. Stress fibers, common on FN, failed to develop on PF4. These experiments indicate that (a) heparan sulfate proteoglycans are critical mediators of cell adhesion and heparan sulfate-dependent adhesion via PF4 is comparable in some, but not all, ways to FN-mediated adhesion, (b) the uncharacterized and heparan sulfate-independent "cell surface" receptor for FN permits some but not all aspects of adhesion, and (c) physiologically compatible and complete adhesion of fibroblasts requires binding of extracellular matrix FN to both the unidentified "cell surface" receptor and heparan sulfate proteoglycans.

Differentiated microdomains on the luminal surface of the capillary endothelium. II. Partial characterization of their anionic sites.

To investigate the chemical nature of the cationic ferritin (CF)-binding sites of the differentiated microdomains of the capillary endothelium, the vasculature of the mouse pancreas and intestinal mucosa was perfused in situ with neuraminidase, hyaluronidase, chondroitinase ABC, heparinase, and three proteases: trypsin, papain, and pronase. Proteases of broad specificity removed all anionic sites, suggesting that the latter are contributed by acid glycoproteins or proteoglycans. Neuraminidase, hyaluronidase, and chondroitinase ABC reduced the density of CF-binding sites on the plasmalemma proper, but had no effect on either coated pits or fenestral diaphragms. Heparinase removed CF-binding sites from fenestral diaphragms and had no effect on coated pits. Taken together, these results indicate that the anionic sites of the fenestral diaphragms are contributed primarily by heparan sulfate and/or heparin, whereas those of the plasmalemma proper are of mixed chemical nature. The membranes and diaphragms of plasmalemmal vesicles and transendothelial channels do not bind CF in control specimens; this condition is not affected by the enzymic treatments mentioned above.

Galactosyl transferase of a Golgi fraction from cultured neoplastic mast cells.

Suspension cultures of neoplastic mouse mast cells were used to obtain large quantities of a homogeneous cell population as starting material for cell fractionation. A Golgi fraction was prepared by slight modification of established techniques and identified by electron microscopy. Assay of galactosyl transferase activity using ovalbumin, desialylated degalactosylated orosomucoid, and N-acetylglucosamine as galactose acceptors showed that the Golgi fraction was enriched in specific activity over the homogenate. The Golgi galactosyl transferase was examined in detail. Acceptor concentrations for optimal galactose incorporation were determined, and substrate inhibition effects were shown with higher concentrations of all three acceptors. Manganese was shown to be necessary for galactose incorporation. A higher concentration of manganese afforded some protection from substrate inhibition by acceptors, but at the same time was itself inhibitory. All three acceptors competed with one another for galactose incorporation, indicating that a single enzyme catalyzed the transfer of galactose for all acceptors.

Alterations in alveolar basement membranes during postnatal lung growth.

We studied the ultrastructural characteristics of alveolar basement membranes (ABM) and capillary basement membranes (CBM) in rat lungs at birth, at 8-10 d of age, during alveolar formation, and at 6-10 wk of age, after most alveoli have formed. We also measured in vitro lung proteoglycan and heparan sulfate synthesis at each age. We noted three major age-related changes in pulmonary basement membranes. (a) Discontinuities in the ABM through which basilar cytoplasmic foot processes extend are present beneath alveolar type-2 cells but not alveolar type-1 cells. These discontinuities are most prevalent at birth but also exist in the adult. (b) Discontinuities are also present in CBM at the two earliest time points but are maximal at 8 d of age rather than at birth. Fusions between ABM and CBM are often absent at 8 d of age, but CBM and CBM/ABM fusions were complete in the adult. (c) Heparan sulfate proteoglycans identified with ruthenium red and selective enzyme degradation are distributed equally on epithelial and interstitial sides of the ABM lamina densa at birth, but decrease on the interstitial side with age. In vitro proteoglycan and heparan sulfate accumulation at birth was two times that at 8 d and five times that in the adult. Discontinuities in ABM allow epithelial-mesenchymal interactions that may influence type-2 cells cytodifferentiation. Discontinuities in CBM suggest that capillary proliferation and neovascularization are associated with alveolar formation at 8 d. When CBM becomes complete and forms junctions with ABM, lung neovascularization likely ends as does the ability to form new alveoli.

The synthesis of glycosaminoglycans by cultures of corneal stromal cells from patients with keratoconus.

Keratoconus is a disease that results in thinning and ectasia of the central cornea. Cultures of corneal stromal cells from patients with keratoconus were established and the synthesis of glycosaminoglycans compared with the synthesis of glycosaminoglycans by normal human corneal stromal cells in culture. Keratoconus and normal control cell cultures were incubated with sodium [(35)S]sulfate and [(3)H]glucosamine for 4 h. After incubation, the labeled glycosaminoglycans were isolated from the medium fractions and cells. Keratoconus and normal control cultures synthesized similar amounts of sulfated glycosaminoglycans independent of the age of donors and(or) the number of subcultures. In contrast to normal control cultures, most of the newly synthesized glycosaminoglycans produced by keratoconus cells were found in the growth medium and much less were in the cell layer. Treatment with glycosaminoglycan-degrading enzymes followed by paper chromatography showed that keratoconus cells, as normal control cells, produced hyaluronic acid and various sulfated glycosaminoglycans. The production of cell layer-related heparan sulfate was markedly reduced in keratoconus cultures. Because heparan sulfate has been shown to be associated with cell surfaces, the decreased heparan sulfate content could reflect changes at this location.

Involvement of cell surface heparin sulfate in the binding of lipoprotein lipase to cultured bovine endothelial cells.

It has been postulated that lipoprotein lipase, an enzyme important in the uptake of fatty acids into tissues, is bound to the vascular endothelial cell surface and that this binding occurs through attachment to heparinlike glycosaminoglycans. Furthermore, it is thought that heparin releases the enzyme from its attachment to the endothelium into the circulation. These hypotheses have never been tested directly in cell systems in vitro. In the present study we have directly evaluated the interaction of lipoprotein lipase, purified from bovine skim milk with monolayer cultures of endothelial cells, isolated from bovine pulmonary artery. Endothelial cells in primary culture had no intrinsic lipoprotein lipase activity but were able to bind lipoprotein lipase quantitatively. The binding reached equilibrium and was saturable at 0.24 nmol of lipoprotein lipase/mg of cell protein. The concentration of lipoprotein lipase at half-maximal binding was 0.52 microM. Bound lipoprotein lipase could be detached from cultured cells by increasing concentrations of heparin, and at and above 0.6 microgram/ml of heparin, 90% of the cell-bound lipoprotein lipase activity was released. Heparan sulfate and dermatan sulfate released the enzyme to a lesser extent and chondroitin sulfate caused little, if any, release of lipoprotein lipase. The release of lipoprotein lipase with heparin was not associated with a release of [3S]glycosaminoglycans from 35S-prelabeled cells. Reductions of lipoprotein lipase binding to endothelial cells and of cell surface-associated [3S]glycosaminoglycans in 35S-prelabeled cells occurred in parallel both when cells were pretreated with crude Flavobacterium heparinum enzyme before lipoprotein lipase binding and when cells were treated with this enzyme after lipoprotein lipase binding. The removal of heparan sulfate from the cell surface by purified heparinase totally inhibited the binding of lipoprotein lipase by endothelial cells, but the removal of chondroitin sulfate by chondroitin ABC lyase had no effect on this binding. These results provide direct evidence for lipoprotein lipase attachment to endothelial cells through heparan sulfate on the cell surface, and provide evidence for the release of lipoprotein lipase by heparin through a detachment from this binding site.

Isolation and characterization of heparin from human lung.

Heparin as measured by azure A metachromasia and anticoagulant activity has been extracted with 1 M NaCl from (35)S-labeled human lung fragments or dispersed human lung cells enriched for mast cells. The (35)S-labeled metachromatic material in the 3 M NaCl eluate from Dowex-1 chromatography of the extract from lung fragments exhibited an average mol wt of 20,000 by Sepharose 4B gel filtration. The (35)S-labeled metachromatic material with the charge characteristics of commercial porcine heparin on DEAE cellulose chromatography was entirely heparin by the criteria of resistance to degradation by chondroitin ABC lyase and complete degradation by purified heparinase. Antithrombin affinity chromatography of purified heparin with an anticoagulant activity of 137 U/mg, revealed that the one-third that was bound and eluted had a 273 U/mg sp act, whereas the unbound activity was 31 U/mg. Thus, the previously observed heterogeneity of commercial porcine heparin for binding to human antithrombin was also observed with human heparin. The mast cell-enriched human lung cell preparations yielded [(35)S]mucopolysaccharides with an average mol wt of 60,000 by Sepharose 4B gel filtration. Approximately 30% of this fraction was degraded by chondroitin ABC lyase, and the residual 70% was degraded by purified heparinase. When the chondroitin ABC lyase-resistant fraction was subjected to alkali degradation the average mol wt was reduced to 20,000. The calculated human lung mast cell heparin content of 2.4-7.8 mug/10(6) cells gave a ratio to histamine on a weight basis similar to that of intact lung fragments, thereby implying that heparin in the lung fragments was largely restricted to the mast cells.

Structural characterization of the early indoleacetic acid-inducible genes, PS-IAA4/5 and PS-IAA6, of pea (Pisum sativum L.).

Two early auxin-inducible genes (PS-IAA4/5 and PSIAA6) from pea were cloned using previously isolated complementary DNA sequences. They are present in single copy per haploid genome, and are members of a large divergent multigene family that encodes similar proteins. The genes were structurally characterized and sequence analysis of their 5'-flanking regions revealed the presence of several highly conserved sequences found in various auxin-regulated genes from other plant species. Their coding regions are interrupted by three and two introns, respectively. Introns two and three of PS-IAA4/5 and introns one and two of PS-IAA6 are located in identical positions. These genes encode proteins of 189 (21,036 Da) and 179 (20,330 Da) residues that are 46% identical. They also share a significant degree of identity (42 to 80%) with other proteins encoded by auxin regulated genes in soybean, mungbean and Arabidopsis thaliana. All proteins contain four conserved domains ranging in size from 9 to 43 amino acids. Their most prominent feature is the presence of a highly charged N terminus consisting of two clusters of acidic residues separated by a cluster of basic amino acids.

Structure and metabolism of proteoglycans and glycosaminoglycans.

Proteoglycans and glycosaminoglycans have the common structural characteristics of linear polysaccharide chains consisting of a hexosamine alternating with another sugar. They play an important role in skin as part of the support matrix of connective tissue, and may be related to cell-cell, and cell-matrix interactions. In general the polysaccharide chains are covalently linked to protein and may contain varying amounts of sulfate resulting in a strong negative charge. Biosynthesis consists of the formation of the protein core followed by the sequential addition of sugars and sulfate to the nonreducing ends of growing chains. The synthetic process is highly organized with the final polysaccharide polymerization and sulfation taking place in the Golgi. Degradation of the proteoglycans is less well understood but probably involves endoglycosidases, exoglycosidases, and proteases which work in concert to degrade these substances.

Assessment of abdominal fat content by computed tomography.

Computed tomography (CT) produces thin cross-sectional radiographs that may prove very useful in body composition research. CT images of the abdomen allow computerized measurement of total fat area, and also enable the differentiation of subcutaneous fat from intraabdominal fat. The preset investigation examines whether a single CT scan of the abdomen provides an accurate indication of overall abdominal adiposity. Graphs of measurements from seven sequential scans of the abdomen in eight patients showed that rankings of total abdominal area, total fat area, subcutaneous and intraabdominal fat area are relatively consistent no matter which abdominal level is chosen. Correlations of 0.89 to 0.99 between single scans and the average values for all scans show that a single CT image contains the same information on adiposity as a series of scans. These results suggest that future CT studies of body composition can limit radiation exposure by using single scans at different anatomical sites. If only a single scan at one site can be obtained, the level of the umbilicus may be the most useful, because it contains the largest percentage of fat in the body, and best allows differentiation of intraabdominal from subcutaneous fat.

Glycosaminoglycans of bovine aorta endothelial cells: identification and localization by use of a platelet factor 4-fluorescein probe.

We utilized platelet factor 4 (PF4) conjugated to fluorescein to stain the proteoglycans of permeabilized fixed bovine aorta endothelial cells in monolayer culture. Treatment of the monolayers with chondroitin ABC lyase and/or a preparation from Flavobacterium heparinum was used to remove chondroitin sulfate and/or heparan sulfate before staining, with resultant separate identification and partial localization of these glycosaminoglycans. When PF4-fluorescein was utilized with untreated control monolayers, fairly uniform reticular, perinuclear, and cell surface fluorescence was seen. After treatment with chondroitin ABC lyase, fluorescence was retained only on the cell surface. In contrast, treatment with the F. heparinum preparation resulted in the loss of all cell surface fluorescence. Use of both glycosaminoglycan lyases together resulted in loss of essentially all the fluorescence. The cell surface heparan sulfate observed by fluorescence after removal of cell surface chondroitin sulfate appeared to be unevenly distributed, with a heavier accumulation at one pole of each cell. This technique offers a specific method for identification and partial localization of cell surface heparan sulfate.

Synthesis of glycosaminoglycans by cultures of normal human corneal endothelial and stromal cells.

Monolayer cultures of normal human corneal endothelial and stromal cells were incubated with [35S]sulfate and [3H]glucosamine for 4 hr. The labeled glycosaminoglycans resulted from this incubation were isolated from the cell layer and the growth medium and further characterized. Both endothelial and stromal cell cultures synthesized a variety of sulfated glycosaminoglycans, with chondroitin 6-sulfate as the major product. Chondroitin 4-sulfate, dermatan sulfate, and heparan sulfate were present in smaller amounts. Keratan sulfate was produced only in minimal amounts. Both cell types also synthesized hyaluronic acid. The hyaluronic acid production in stromal cell strains derived from donors of different ages was similar. The demonstration that the endothelial cell strain derived from a 1-day-old baby contained more hyaluronic acid than cultures from older donors suggests a possible age-related phenomenon as seen in developing tissues.

Alteration in plasma proteins and platelet functions with aging and cigarette smoking in healthy men.

Blood samples were obtained on four different occasions from 18 cigarette smoking and 34 non-smoking healthy men (age 40-69) and analyzed to assess age- and smoking-associated changes in plasma proteins, blood coagulation and platelet functions. Collagen-induced platelet aggregation was significantly increased with aging in non-smokers. Significant changes in chronic smokers were increases in platelet count and fibrinogen in plasma; elevation of platelet factor-3 (PF-3) activity in platelet-poor plasma (PPP); increase in serum levels of alpha 1-antitrypsin, orosomucoid, haptoglobin and properdin factor B; and shortening of the lag period of collagen-induced platelet aggregation. Filtration of PPP through Millipore filters removed PF-3 membranes. The differences in PF-3 activities in filtered plasma were no longer significant between smokers and non-smokers. Results suggest that chronic smokers have higher levels of acute phase proteins reflecting underlying inflammatory processes, and higher levels of PF-3 activity in plasma due to liberation of PF-3 membranes from platelets.

The effect of penultimate N-acetylgalactosamine 4-sulfate on chondroitin chain elongation.

Previous work has shown that odd-numbered oligosaccharides containing nonreducing terminal, non-sulfated N-acetylgalactosamine (GalNAc) or 6-sulfated GalNAc are excellent acceptors for enzymic addition of glucuronic acid (GlcA). However, the presence of a 4-sulfated GalNAc group blocks further addition. We have now used even-numbered oligosaccharides (a mixture of 4-sulfated, 6-sulfated, and non-sulfated) as acceptors of [3H]GalNAc to investigate the effect of sulfate residues on the GalNAc in the penultimate position. 3H-Labeled oligosaccharides were partially degraded with chondroitin AC lyase. The labeled trisaccharides, consisting of the added [3H]GalNAc and the nonreducing terminal disaccharides of the oligosaccharide acceptors, were then characterized. Both non-sulfated and mono-sulfated 3H-trisaccharides were observed. However, the 3H-trisaccharides were shown by chromatography with prepared standards to be non-sulfated or to be sulfated only at the 6-position. Thus the oligosaccharides containing a 4-sulfated, penultimate GalNAc at the nonreducing end did not serve as acceptors for [3H]GalNAc. Microsomes from mastocytoma cells, which make only chondroitin 4-sulfate, exhibited the same substrate specificity for exogenous oligosaccharide acceptors as did microsomes from chick cartilage which makes chondroitin 6-sulfate.

Biosynthesis of proteochondroitin sulfate: substrate requirements for formation of two independent species.

Formation of two species of [14C]proteochondroitin sulfate has previously been demonstrated with UDP-D-[14C]glucuronic acid and UDP-N-acetylgalactosamine as substrates with a microsomal preparation from chick-embryo epiphyseal cartilage. A large species of [14C]proteoglycan that appeared at an earlier stage of synthesis was excluded on Sepharose CL-2B, indicating that it was larger than proteoglycans found in cartilage matrix. Another newly synthesized, smaller [14C]proteoglycan species also formed was retarded on Sepharose CL-2B, and appeared to be at a later stage of synthesis. A 6-h pulse-chase experiment using UDP-[14C]GlcA and UDP-GalNAc followed by cold UDP-GlcA demonstrates that there was no conversion of the large [14C]proteoglycan to the small [14C]proteoglycan. Sulfation of the newly formed large and small [14C]proteoglycans with adenylyl sulfate 3'-phosphate also did not alter their chromatographic patterns, indicating that sulfation did not trigger any post-synthetic size modification. Synthesis with lower concentrations of the sugar nucleotides resulted in a disproportionate diminution in formation of the large proteoglycan. The apparent Km values for UDP-GlcA for the formation of large and small proteoglycans were 0.055 and 0.015 mM, respectively. Concentration requirements for UDP-GalNAc also showed a similar 4-fold difference. These results indicate that, even though the large proteoglycan was at an earlier stage of synthesis, it was not a precursor to the small proteoglycan, and that these proteoglycans represent two separately synthesized species.

The influence of cigarette smoking and age on bone loss in men.

Hand X-rays were examined among 341 healthy male participants (age 40-80 yr) of the Normative Aging Study who had two successive X-rays 3 to 5 yr apart. Bone density was estimated at midshaft of the second metacarpal as percent cortical area. As expected, cross-sectional analysis revealed a decrease in percent cortical area with age. Current smokers tended to have slightly lower percent cortical areas than "never" smokers. When participants were followed longitudinally over a 3 to 5 year period, a trend toward greater bone loss with increasing age was generally observed in both smoking status groups, although smokers' rate of loss after age 55 yr deviated slightly from this pattern. Current smokers under age 55 yr consistently showed greater bone loss than never smokers.