Interferon-gamma inhibits 35S incorporation in heparan sulfate synthesized by human skin fibroblasts

Glycosaminoglycans synthesized by human skin fibroblasts were simultaneously radiolabelled with D-[1-(3H)]glucosamine and Na2(35)SO4. Considering 3H incorporation, we found that IFNgamma increased the production of glycosaminoglycan synthesis, including hyaluronic acid, heparan and chondroitin/dermatan sulfate. In contrast, the production of heparan and chondroitin/dermatan sulfate was slightly decreased on the basis of the 35S signal. Furthermore, when heparan sulfate was treated with nitrous acid, the release of free 35S was greater in control than in treated cells, although the 3H patterns of depolymerization with this agent were similar. These data demonstrate that IFNgamma inhibits the incorporation of sulfate from extracellular medium into heparan sulfate.     

Relationship of transformation, cell density, and growth control to the cellular distribution of newly synthesized glycosaminoglycan

Mouse 3T3 cells and their Simian Virus 40-transformed derivatives (3T3SV) were used to assess the relationship of transfromation, cell density, and growth control to the cellular distribution of newly synthesized glycosaminoglycan (GAG). Glucosamine- and galactosamine-containing GAG were labeled equivalently by [3H=A1-glucose regardless of culture type, allowing incorporation into the various GAG to be compared under all conditions studied. Three components of each culture type were examined: the cells, which contain the bulk of newly synthesized GAG and are enriched in chondroitin sulfate and heparan sulfate; cell surface materials released by trypsin, which contain predominantly hyaluronic acid; and the media , which contain predominantly hyaluronic acid and undersulfated chondroitin sulfate. Increased cell density and viral transformation reduce incorporation into GAG relative to the incorporation into other polysaccharides. Transformation, however, does not substantially alter the type or distribution of newly synthesized GAG; the relative amounts and cellular distributions were very similar in 3T3 and 3T3SV cultures growing at similar rates at low densities. On the other hand, increased cell density as well as density-dependent growth inhibition modified the type and distribution of newly synthesized GAG. At high cell densities both cell types showed reduced incorporation into hyaluronate and an increase in cellular GAG due to enhanced labeling of chondroitin sulfate and heparan sulfate. These changes were more marked in confluent 3T3 cultures which also differed in showing substantially more GAG label in the medium and in chondroitin-6-sulfate and heparan sulfate at the cell surface. Since cell density and possibly density-dependent inhibition of growth but not viral transformation are major factors controlling the cellular distribution and type of newly synthesized GAG, differences due to GAG's in the culture behavior of normal and transformed cells may occur only at high cell density. The density-induced GAG alterations most likely involved are increased condroitin-6-sulfate and heparan sulfate and decreased hyaluronic acid at the cell surface.     

Effect of irradiation with a low-intensity diode laser on the metabolism of equine articular cartilage in vitro

OBJECTIVE: To determine whether irradiation with a low-intensity diode laser, which produces radiation at a wavelength of 810 nm, will induce nonthermal enhancement of chondrocyte metabolism. SAMPLE POPULATION: 144 grossly normal articular cartilage explants aseptically harvested from the femoral condyles of 6 adult horses. PROCEDURE: Treated cartilage explants were irradiated with a diode laser at 1 of 7 fluence levels that ranged from 8 to 1,600 J/cm2. Explants were incubated for 24 or 72 hours, labeled for 24 hours with [35S]Na2SO4, and assayed for newly synthesized sulfated glycosaminoglycan (GAG; measured incorporation of 35SO4) and endogenous GAG, chondroitin 6-sulfate (CS), and keratan sulfate (KS) content, using a dimethylmethylene blue assay. Laser-induced temperature changes were measured during irradiation with a diode laser and a neodymium:yttrium aluminum garnet (Nd:YAG) laser, which produces radiation at a wavelength of 1,064 nm, using conditions that were reported in previous studies to increase explant metabolism. RESULTS: After incubation for 24 or 72 hours, rate of 33SO4 uptake or endogenous GAG, CS, or KS content in irradiated explants was not significantly different than in nonirradiated explants. Cartilage temperature increased < 4.75 C during diode laser application. Cartilage temperature increased 5 to 12 C during Nd:YAG laser application. CONCLUSIONS: Minimal thermal increases in cartilage explants with use of a low-intensity diode laser resulted in no change in proteoglycan metabolism of chondrocytes. An increase in tissue temperature over a narrow range with use of a Nd:YAG laser may have contributed to the metabolic alteration of chondrocytes reported in previous studies.     

Apolipoprotein E containing high density lipoprotein stimulates endothelial production of heparan sulfate rich in biologically active heparin-like domains. A potential mechanism for the anti-atherogenic actions of vascular apolipoprotein e

Reduced heparin and heparan sulfate (HS) proteoglycans (PG) have been observed in both inflammation and atherosclerosis. Methods to increase endogenous heparin and heparan sulfate are not known. We found that incubation of endothelial cells with 500-1,000 micrograms/ml high density lipoprotein (HDL) increased 35SO4 incorporation into PG by 1.5-2.5-fold. A major portion of this increase was in HS and was the result of increased synthesis. Total PG core proteins were not altered by HDL; however, the ratio of 35SO4 to [3H]glucosamine was increased by HDL, suggesting increased sulfation of glycosaminoglycans. In addition, HDL increased the amount of highly sulfated heparin-like HS in the subendothelial matrix. HS from HDL-treated cells bound 40 +/- 5% more 125I-antithrombin III (requires 3-O sulfated HS) and 49 +/- 3% fewer monocytes. Moreover, the HS isolated from HDL-treated cells inhibited smooth muscle cell proliferation (by 83 +/- 5%) better than control HS (56 +/- 6%) and heparin (42 +/- 6%). HDL isolated from apolipoprotein E (apoE)-null mice did not stimulate HS production unless apoE was added. ApoE also stimulated HS production in the absence of HDL. ApoE did not increase 35SO4 incorporation in macrophages and fibroblasts, suggesting that this is an endothelial cell-specific process. Receptor-associated protein inhibited apoE-mediated stimulation of HS only at higher (20 micrograms/ml) doses, suggesting the involvement of a receptor-associated protein-sensitive pathway in mediating apoE actions. In summary, our data identify a novel mechanism by which apoE and apoE-containing HDL can be anti-atherogenic. Identification of specific apoE peptides that stimulate endothelial heparin/HS production may have important therapeutic applications.     

Biosynthesis of glycosaminolgycans during corneal development

Glycosaminoglycan biosynthesis was studied in developing chick corneas, with particular attention paid to keratan sulfate I, the major glycosaminoglycan of this tissue. This polysaccharide is unique to the cornea and may be required for the development and maintenance of corneal transparency. Corneas from 5-to 20-day chick embryos were labeled in vitro with D-[6- 3H] glyhucosamine and H(2)35SO(4)35SO(4) and the amount of label in each glycosaminoglycan was determined. The data indicate that, contrary to previous suggestions, keratan sulfate biosynthesis in the cornea begins at the time of fibroblast invasion of the primary stroma, at least 8 days prior to the onset of corneal transparency, which occurs on Day 14 of the development in the chick. The rate of incorporation of radioactivity into keratan sulfates, on a dry weight basis, increases rapidly after Day 6 and levels off on Day l4. The proportion of 3H and 35S in keratan sulfate reaches nearly maximal levels as early as Day 9. In contrast, the proportion of radioactivity in corneal heparan sulfates declines rapidly after Day 5. However, the rate of incorporation of radioactivity into heparan sulfates, on a dry weight basis, increases or remains the same during early development. On and after Day 14, keratan sulfates appear to become more highly sulfated. Moreover, the ratios of 4-sulfated to 6-sulfated chondroitin sulfates increase during development, reaching a maximum on Day 14. These changing patterns of glycosaminoglycan biosynthesis during corneal development may play an important role in corneal morphogenesis and the achievement of corneal transparency     

Exogenous heparin induces an increase in glycosaminoglycans of the chick embryo chorioallantoic membrane: its possible role in the regulation of angiogenic processes

In this study heparin (HE) was injected into the allantoic sac of chick embryo eggs at 5, 9, 14 days of incubation. 48 h after injection glycosaminoglycan (GAG) concentration was measured in the chorioallantoic membrane (CAM) in order to verify if HE-related CAM angiogenic activity previously demonstrated [Ribatti et al: Acta Anat 1987; 130:257-263] might be correlated with changes in GAG concentration. The results showed that HE inoculation induced an increase of 3H-glucosamine incorporation into total GAGs in comparison to control CAMs. Furthermore, HE altered the balance between the GAG classes, and in particular it produced a significant increase in the accumulation of hyaluronic acid and heparan sulfate between 7 and 11 days of incubation in comparison to control CAMs.     

Use of immunoplot analysis for the identification of immunodominant non-variant antigens of Trypanosoma brucei rhodesiense

BACKGROUND: Common elements in many different types of amyloid may have important roles in amyloidogenesis. The proteinaceous tissue deposits have a common appearance in polarized light and other similar features. The present investigation describes for the first time the relation between beta 2-microglobulin (beta 2-M)-type amyloidosis and colocalized materials, as demonstrated using specific antibodies and hyaluronan-binding protein. EXPERIMENTAL DESIGN: Amyloid-rich carpal tunnel synovium was obtained surgically from 28 patients who were being treated by maintenance hemodialysis. Serial sections were examined using a hyaluronan (hyaluronic acid)-binding protein and antibodies against heparan sulfate-glycosaminoglycan, chondroitin sulfate-proteoglycan, dermatan sulfate-proteoglycan, alpha 1-antichymotrypsin, alpha 1-antitrypsin, inter-alpha-trypsin inhibitor, haptoglobin, and ubiquitin. RESULTS: Accumulation of hyaluronan was of three types, namely, localization around beta 2-M deposits, colocalization with deposition of beta 2-M itself and localization at a small distance from beta 2-M deposits. Immunostaining for heparan sulfate glycosaminoglycan was demonstrated at the sites of beta 2-M plaques. Chondroitin sulfate-proteoglycan did not show specific patterns of immunostaining, resembling hyaluronan rather than heparan sulfate. The other materials tested, alpha 1-antichymotrypsin, alpha 1-antitrypsin, inter-alpha-trypsin, haptoglobin and ubiquitin, were not immunostained at sites of beta 2-M plaques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed that the molecular weight of heparan sulfate-glycosaminoglycan was 16,000. CONCLUSIONS: These results suggest that HS has an important role in hemodialysis-associated amyloidosis as it does in other types of amyloidosis. Moreover, accumulation of hyaluronan may be an indication of inflammation of the carpal synovium.     

Activation of the autophagy, c-FOS and ubiquitin expression, and nucleolar alterations in Schwann cells precede demyelination in tellurium-induced neuropathy

The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth factor-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8-fold less tightly than heparin (Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of fibroblast growth factor-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400-fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.     

Comparative acute toxicity and primary irritancy of the ethylidene and vinyl isomers of norbornene

Perlecan and aggrecan are proteoglycans that receive primarily heparan sulfate and chondroitin sulfate side chains, respectively. Their large multidomained core proteins have little or no homology to each other and their glycosaminoglycan (GAG) attachment sites are restricted to certain domains only. We examined the involvement of the non-GAG bearing domains in designating the GAG type added to the GAG attachment domain by preparing cDNA constructs that expressed perlecan/aggrecan chimeras as recombinant products in COS-7 cells and then determining the size and GAG composition of the recombinant products. The results showed that domain I of perlecan receives primarily (73-81%) heparan sulfate when coupled with domain II and III of perlecan, but when coupled with the G3 domain of aggrecan, it receives primarily (59-63%) chondroitin sulfate. Furthermore, the chondroitin sulfate attachment region of aggrecan received GAG side chains more readily when coupled to the G3 domain of aggrecan than when coupled to domains II and III of perlecan. The GAG side chains on all these recombinant products were small and similar in size. These findings indicate that the utilization of attachment sites for heparan and chondroitin sulfate or the sulfation of these GAGs can be influenced, in part, by non-GAG bearing domains.     

Glycosaminoglycan binding properties of annexin IV, V, and VI

We have previously demonstrated that annexin IV, one of the calcium/phospholipid-binding annexin family proteins, binds to glycosaminoglycans (GAGs) in a calcium-dependent manner (Kojima, K., Yamamoto, K., Irimura, T., Osawa, T., Ogawa, H., and Matsumoto, I. (1996) J. Biol. Chem. 271, 7679-7685). In this study, we investigated the GAG binding specificities of annexins IV, V, and VI by affinity chromatography and solid phase assays. Annexin IV was found to bind in a calcium-dependent manner to all the GAG columns tested. Annexin V bound to heparin and heparan sulfate columns but not to chondroitin sulfate columns. Annexin VI was adsorbed to heparin and heparan sulfate columns in a calcium-independent manner, and to chondroitin sulfate columns in a calcium-dependent manner. An N-terminal half fragment (A6NH) and a C-terminal half fragment (A6CH) of annexin VI, each containing four units, were prepared by digestion with V8 protease and examined for GAG binding activities. A6NH bound to heparin in the presence of calcium but not to chondroitin sulfate C, whereas A6CH bound to heparin calcium-independently and to chondroitin sulfate C calcium-dependently. The results showed that annexin IV, V, and VI have different GAG binding properties. Some annexins have been reported to be detected not only in the cytoplasm but also on the cell surface or in extracellular components. The findings suggest that the some annexins function as recognition elements for GAGs in extracellular space.     

Opportunistic fungal pneumonia

Epithelial cells are important components of the thymus microenvironment and are involved in thymocyte differentiation. The production and secretion of sulfated glycosaminoglycans by these cells grown in culture were investigated using labeling with radioactive 35S-Na2SO4 and 3H-glucosamine. The major glycosaminoglycans synthesized by these cells are heparan sulfate and hyaluronic acid. The structure of the heparan sulfate was investigated by the pattern of degradation products formed by deaminative cleavage with nitrous acid. The ratio 35S-sulfate/ H-glucosamine is high in the segments of the heparan sulfate released during the deaminative cleavage with nitrous acid but low in the resistant portion of the molecule. Thus, the heparan sulfate synthesized by the thymic epithelial cells contains a highly sulfated region. Digestion with heparitinase reveals that this highly sulfated region is a heparin-like segment of the molecule. The heparan sulfate is rapidly incorporated into the cell surface but its secretion to the extracellular medium requires a longer incubation period. Finally, heparin was used to mimic the possible effect of this heparan sulfate with a highly sulfated region, as ascertained by its ability to modulate thymocyte adhesion to thymic epithelial cells. Since heparin actually enhanced thymocyte adhesion, it is suggested that the heparan sulfate described herein, secreted by the thymic epithelium, may play a role upon intrathymic heterotypic cellular interactions.     

Ubiquitin profile in inflammatory leukocytes and binding of ubiquitin to murine AA amyloid: immunocytochemical and immunogold electron microscopic studies

Lysosomes in activated murine monocytoid cells have been implicated in AA amyloid formation. The pathophysiology of this process is not well understood. Previous studies into the nature of the relationship between ubiquitin (UB), possessing intrinsic amyloid enhancing factor (AEF) activity; serum amyloid A (SAA), the precursor protein of AA amyloid; and activated monocytoid cells have indicated a temporal and spatial relationship between these proteins and tissue AA amyloid deposits. To extend these findings, we have examined murine peritoneal leukocytes and splenic tissues during the early amyloid deposition phase by immunocytochemical and immunogold electron microscopic methods using monospecific anti-ubiquitin and anti-mouse AA amyloid antibodies. We show here enrichment of endosome-lysosome-like (EL) vesicles in the activated monocytoid cells with UB and SAA, and the presence of UB-bound AA amyloid fibrils in the EL vesicles, perikarya, and interstitial spaces. The importance of these findings is emphasized by the fact that activated monocytoid cells, containing UB in the EL vesicles, sequester and eventually localize SAA in their EL vesicles, and that UB binds to the EL-contained AA amyloid fibrils. These findings may also have functional consequences for studies on the role of EL and UB in amyloidogenesis.     

Neurotrophic growth factors stimulate glycosaminoglycan synthesis in identified retinal cell populations in vitro

Glycosaminoglycans (GAG) are known to participate in central nervous system processes such as development, cell migration, and neurite outgrowth, but little is known with respect to their regulation through soluble neurotrophic factors. In the present study, we have addressed this issue using cell culture models of three distinct cell populations derived from young rat retinas, namely, purified M uller glia, pigmented epithelium, and neurons respectively. Cultures were maintained in chemically defined media in the presence or absence of either basic fibroblast or epidermal growth factor. In control glial and epithelial cultures, hyaluronic acid dominated the soluble GAG pool, with lesser contributions from dermatan sulfate, chondroitin sulfate, and heparan sulfate (in decreasing order). Retinal neuronal GAG were almost exclusively chondroitin sulfate (approximately 90%). Treatment of glial and epithelial cultures with either factor led to dose-dependent increases in especially hyaluronic acid synthesis (a maximum 6-fold increase relative to control levels), with smaller but consistent changes in chondroitin sulfate. Similar treatment of retinal neurons did not lead to any changes in GAG synthesis. These data indicate that glia and pigment epithelia are the principal sources of GAG components in retina at least in vitro, and that endogenous neurotrophic growth factors can greatly modify GAG synthesis in these two retinal cell populations. Such data suggest that a delicate balance may exist between growth factor availability and glycoconjugate metabolism in vivo, participating in normal or pathological states of the retina.     

Formation of promutagenic methylation damage in tissue-DNA of mice treated with antischistosomal agents

BACKGROUND: Cultured bovine corneal endothelial cells (CEC) synthesize heparan sulfate and dermatan sulfate containing proteoglycans and distribute them between different compartments. METHODS AND RESULTS: [35S]sulfate labelled proteoglycans are found associated with the cell layer, secreted into the culture medium and deposited into the underlaying extracellular matrix. In the presence of basic fibroblast growth factor (bFGF)-a strong mitogen for CEC-subconfluent cells incorporate [35S]sulfate into the sulfated proteoglycans at a rate three times higher as compared with the proteoglycans of CEC in the absence of bFGF. The enhanced proteoglycan synthesis is accompanied with a shift in the proteoglycan distribution pattern. While in control cells the cell-associated heparan sulfate accounts for about 30% of the total glycosaminoglycans under the influence of bFGF the HS percentage increases to approximately 60%. CONCLUSIONS: CEC synthesize and deposit endogenous bFGF into the extracellular matrix. Heparitinase treatment of the extracellular matrix releases bFGF activity which is able to stimulate the 35S incorporation into proteoglycans in a comparable manner as exogenous bFGF but does not influence the proteoglycan distribution pattern. Pretreatment of the matrix-bound bFGF activity with polyclonal antibodies against bFGF abolishes its stimulating activity.     

Clinical resistance to topoisomerase-targeted drugs

The objectives of this study were to determine whether dietary manganese deficiency alters total glycosaminoglycan (GAG) concentration and composition and glycosyltransferase activity in rat aortas. Sprague-Dawley rats were fed either a manganese-deficient or a manganese-sufficient diet. Arterial GAGs were isolated and quantified by measuring uronic acid content. The individual GAGs were separated and quantified with cellulose acetate electrophoresis. The activity of the enzyme galactosyltransferase I was measured using a 100,000 g particulate fraction and 4-methylumbelliferylxyloside (Xyl-MU) as an acceptor. There was a significant decrease (p < or = 0.05) in uronic acid content in the manganese-deficient (1.18 +/- 0.08 mg/g) rat aortas as compared with the manganese-sufficient (1.59 +/- 0.10 mg/g) ones. Chondroitin sulfate and heparan sulfate concentrations were decreased by 38% (p < or = 0.01) and 36% (p < or = 0.05), respectively, in the manganese-deficient rat aortas. The incorporation of UDP-galactose to acceptors by the manganese-deficient rat aorta preparations was increased by 28% as compared to the manganese-sufficient preparations. These results indicate that manganese is involved in arterial GAG metabolism by affecting the enzyme galactosyltransferase and that changes in GAG concentration and composition with manganese deficiency may ultimately affect arterial wall integrity and subsequently cardiovascular health. This is the first work to demonstrate that manganese nutrition is important in arterial GAG metabolism.     

Morphological and biochemical features affecting the antithrombotic properties of the aorta in adult rabbits and rabbit pups

We hypothesised that there are important physiologic differences in arterial wall structure and function with respect to antithrombotic activity in the very young (pre-puberty) compared to adults. Electron microscopy, gel electrophoresis, and activity assays were used to examine differences in aorta structure and function comparing prepubertal rabbits (pups) to adult rabbits. Differences in endothelial function, extracellular matrix structure, proteoglycan (PG) distribution and glycosaminoglycan (GAG) content and function were shown. In both intima and media, total PG, chondroitin sulfate (CS) PG and heparan sulfate (HS) PG content were significantly increased in pups compared to adult rabbits. These findings corresponded to increased concentrations by mass analyses of CS GAG and DS GAG in aortas from pups. There was also a significant increase in antithrombin activity in pups due to HS GAG. In conclusion, differences in both structure and antithrombin activity of aortas from pups compared to adult rabbits suggest that young arteries may have greater antithrombotic potential that is, at least in part, related to increased HS GAG.     

A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.     

Cell surface proteoglycans are necessary for A27L protein-mediated cell fusion: identification of the N-terminal region of A27L protein as the glycosaminoglycan-binding domain

We previously showed that vaccinia virus infection of BSC40 cells was blocked by soluble heparin, suggesting that cell surface heparan sulfate mediates vaccinia virus binding (C.-S. Chung, J.-C. Hsiao, Y. -S. Chang, and W. Chang, J. Virol. 72:1577-1585, 1998). In this study, we extended our previous work and demonstrated that soluble A27L protein bound to heparan sulfate on cells and interfered with vaccinia virus infection at a postbinding step. In addition, we investigated the structure of A27L protein that provides for its binding to heparan sulfate on cells. A mutant of A27L protein, named D-A27L, devoid of a cluster of 12 amino acids rich in basic residues, was constructed. In contrast to the soluble A27L protein, purified D-A27L protein was inactive in all of our assays, including binding to heparin in vitro, binding to heparan sulfate on cells, and the ability to block virus infection. These data demonstrated that the N-terminal region acts as a glycosaminoglycan (GAG)-binding domain critical for A27L protein binding to cells. Previously A27L protein was thought to be involved in fusion of virus-infected cells induced by acid treatment. When we investigated whether cell surface GAGs also participate in A27L-dependent fusion, our results indicated that soluble A27L protein blocked cell fusion, whereas D-A27L protein did not. Taken together, the results therefore demonstrated that A27L-mediated cell fusion is triggered by its interaction with cell surface GAGs through the N-terminal domain.     

UV-B radiometry and dosimetry for solar measurements

Glycosaminoglycan administration has favourable effects on morphological and functional renal abnormalities in different models. The possibility that exogenous glycosaminoglycans modulate glomerular matrix synthesis was explored in both primary and SV40-MES13 murine mesangial cell cultures. On both cell types, both low-molecular-weight heparin and different glycosaminoglycans showed dose-dependent inhibition of proliferation and increase of 35SO4(2)-uptake. After 36 h the cell compartment contained a spectrum of 35S-molecules of less than 200 kDa; under heparin treatment, the two main 35SO4(2)-components (high and medium MW) increased by 16 and 37% respectively. Susceptibility to glycosidases revealed that heparin promotes the expression of heparan sulphate and increases that of chondroitin sulphate. Moreover, heparin modifies the expression of decorin and biglycan, involved in adhesion and fibrillogenesis, while not affecting perlecan. The extracellular matrix modulation in renal cells, for which the sulphation type and ratio of heparin are crucial, may thus explain the beneficial renal effects of heparin.     

Inhibitory effect of pentoxifylline on HLA-DR expression and glycosaminoglycan synthesis by retrobulbar fibroblasts

OBJECTIVE: Glycosaminoglycan (GAG) production by retro-ocular fibroblasts (REF) is increased in patients with thyroid-associated ophthalmopathy (TAO). Various cytokines stimulate REFs to proliferate and elaborate GAG, free oxygen radicals as well as induce HLA-DR expression on these cells. Pentoxifyllin (Ptx) regulates the production of several cytokines including tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1) and, interferon gamma (IFN-gamma). We wished in this study to determine whether Ptx modified the spontaneous and cytokine-induced GAG synthesis by REF and IFN-gamma induced HLA-DR expression. DESIGN: REF derived from extraocular muscles of healthy subjects were cultured without and with cytokines (IFN-gamma, TNF alpha and IL-1) and the effect of Ptx on the production of GAG by REF and HLA-DR expression was determined. MEASUREMENTS: Glycosaminoglycan was measured by incorporation of (3H) glycosamine into GAG. HLA-DR expression was analyzed by fluorescence activated cell sorter. RESULTS: Both spontaneous and cytokine induced GAG synthesis by REF was inhibited by Ptx (100, 500 and 1000 mg/l, respectively). IFN-gamma (50, 100 and 500 U/ml) induced a dose-dependent increase in the expression of HLA-DR molecules by REF. Ptx, which was not toxic to REF, inhibited HLA-DR expression on those cells dose-dependently. CONCLUSIONS: Our in vitro results suggest that Ptx reduces cytokine-induced GAG production and HLA-DR expression by REF. It thus has potential as a therapeutic agent which regulates the function of lymphocytes infiltrating the retro-orbital tissues, and which are instrumental in TAO.