Specific intracellular hyaluronic acid binding to isolated rat hepatocytes is membrane-associated.

Intact isolated rat hepatocytes show a small amount of specific 125I-labeled hyaluronic acid (HA) binding. However, in the presence of digitonin, a very large increase in the specific binding of 125I-HA is observed. Chondroitin sulfate, heparin and dextran sulfate were as effective as unlabeled HA in competing for 125I-HA binding to permeabilized hepatocytes, indicating that the binding sites may have a general specificity for glycosaminoglycans. After rat hepatocytes had been homogenized in a hypotonic buffer, more than 98% of the 125I-HA binding activity could be pelleted by centrifugation at 100,000 x g for 1 h. Mild alkaline treatment of hepatocyte membranes did not release 125I-HA binding activity, suggesting that the HA binding site is an integral membrane molecule. Furthermore, trypsin treatment of deoxycholate-extracted membranes destroyed the binding activity, as assessed by a dot-blot assay. This suggests that a protein component in the membrane is necessary for 125I-HA binding activity. Rat fibrinogen could be a possible candidate for the HA binding activity because HA binds specifically to human fibrinogen (LeBoeuf et al. (1986) J. Biol. Chem. 261, 12 586). Also, fibrinogen can be found in a quasi-crystalline form in rat hepatocytes and could be pelleted with the membranes. Rat fibrinogen was not responsible for the 125I-HA binding activity, since (1) purified rat fibrinogen did not bind to 125I-HA, and (2) immunoprecipitation of rat fibrinogen from hepatocyte extracts did not decrease the 125I-HA binding of these extracts. We conclude that the internal HA binding sites are membrane- or cytoskeleton-associated proteins and are neither cytosolic proteins nor fibrinogen.

Transcriptional regulation of the human transforming growth factor-alpha gene.

We and others have previously reported that transforming growth factor-alpha (TGF alpha) expression is hormonally responsive and its expression is coregulated with that of its receptor [the epidermal growth factor (EGF) receptor]. The 5'-flanking region of the TGF alpha gene was characterized to determine whether it could confer hormone responsiveness to a reporter gene (luciferase) in human mammary carcinoma cells (MDA468). This segment of the gene is GC rich and contains an element strikingly similar to the core element of the EGF receptor gene that has been shown to mediate both basal and hormone-stimulated expression of the EGF receptor. We now report that a 313-basepair (bp) proximal element of the TGF alpha 5'-flanking region (-373 to -59 relative to the TGF alpha translation start codon) is capable of conferring responses to phorbol ester and EGF. This gene segment does not contain the EGF receptor gene homolog or potential AP-2-binding sites, suggesting that these elements are not necessary for basal and EGF- or phorbol ester-responsive TGF alpha gene expression. This 313-bp proximal element also confers proper transcriptional initiation to the chimeric TGF alpha-luciferase reporter construct, indicating it is the TGF alpha promoter. A 1.1-kilobase segment of the TGF alpha 5'-flanking region also confers retinoic acid, thyroid hormone, and glucocorticoid responsiveness despite the absence of recognizable steroid hormone receptor-binding sites. These hormones stimulate reporter expression 1.5- to 2-fold in a dose-dependent manner. Extension of the 5'-flanking region to -3500 results in marked suppression of reporter gene expression. These results indicate that the TGF alpha gene 5'-flanking sequence contains the elements responsible for hormonal responsiveness of this gene and that these elements are distinct from those that regulate the expression of the EGF receptor gene.

Cattle plague in Shangri-La: observations on a severe outbreak of rinderpest in northern Pakistan 1994-1995.

Between April 1994 and November 1995 the most severe epidemic of rinderpest reported in the world for over a decade affected domestic livestock in the Northern Areas of Pakistan. As many as 40,000 cattle and yaks died, more by some estimates, and mortality rates may have exceeded 80 per cent in these species in several villages. This report describes some of the clinicopathological and epidemiological features peculiar to the outbreak, including laboratory-confirmed rinderpest in a goat, and the difficulties encountered before the disease was eradicated. It also describes the human costs and emphasises the need to accelerate the global eradication of this most eradicable disease.

Endocytosis of hyaluronic acid by rat liver endothelial cells. Evidence for receptor recycling.

Hyaluronic acid (HA) is cleared from the blood by liver endothelial cells through receptor-mediated endocytosis [Eriksson, Fraser, Laurent, Pertoft & Smedsrod (1983) Exp. Cell Res. 144, 223-238]. We have measured the capacity of cultured rat liver endothelial cells to endocytose and degrade 125I-HA (Mr approximately 44,000) at 37 degrees C. Endocytosis was linear for 3 h and then reached a plateau. The rate of endocytosis was concentration-dependent and reached a maximum of 250 molecules/s per cell. Endocytosis of 125I-HA was inhibited more than 92% by a 150-fold excess of non-radiolabelled HA. HA, chondroitin sulphate and heparin effectively competed for endocytosis of 125I-HA, whereas glucuronic acid, N-acetylglucosamine, DNA, RNA, polygalacturonic acid and dextran did not compete. In the absence of cycloheximide, endothelial cells processed 13 times more 125I-HA in 6 h than their total (cell-surface and intracellular) specific HA-binding capacity. This result was not due to degradation and rapid replacement of receptors, because, even in the presence of cycloheximide, these cells processed 6 times more HA than their total receptor content in 6 h. Also, in the presence of cycloheximide, no decrease in 125I-HA-binding capacity was seen in cells processing or not processing HA for 6 h, indicating that receptors are not degraded after the endocytosis of HA. During endocytosis of HA at 37 degrees C, at least 65% of the intracellular HA receptors became occupied with HA within 30 min. This indicates that the intracellular HA receptors (75% of the total) function during continuous endocytosis. Hyperosmolarity inhibits endocytosis and receptor recycling in the asialoglycoprotein and low-density-lipoprotein receptor systems by disrupting the coated-pit pathway [Heuser & Anderson (1987) J. Cell Biol. 105, 230a; Oka & Weigel (1988) J. Cell. Biochem. 36, 169-183]. Hyperosmolarity inhibited 125I-HA endocytosis in liver endothelial cells by more than 90%, suggesting use of a coated-pit pathway by this HA receptor. We conclude that liver endothelial cell HA receptors are recycled during the continuous endocytosis and processing of HA.

Peptide analogs to a fibronectin receptor inhibit attachment of Staphylococcus aureus to fibronectin-containing substrates.

Binding of cells of Staphylococcus aureus to fibronectin has been proposed as a mechanism of bacterial adhesion to host tissues. In this study, we have attempted to define the role of a recently identified fibronectin receptor in the adhesion of staphylococcal cells to fibronectin-containing substrates by using different receptor analogs as potential inhibitors of bacterial adherence. The results showed that synthetic peptides D1, D2, and D3, corresponding to variations of a repeated unit in the fibronectin-binding domain of the receptor, and ZZ-FR, a chimeric protein containing the fibronectin-binding domain of the receptor with the D1, D2, and D3 sequences, inhibited the attachment of staphylococcal cells to microtiter wells coated with intact fibronectin or with the 29-kilodalton amino-terminal fragment of fibronectin. The chimeric protein ZZ-FR also partially inhibited the adherence of staphylococci to human plasma clots formed in vitro but had no effect on bacterial adhesion to clots formed from fibronectin-depleted plasma. These data confirm previous reports suggesting that fibronectin may serve as a substrate for adhesion of staphylococcal cells and indicate that bacterial adhesion is mediated by the identified fibronectin receptor. Furthermore, analogs to the fibronectin receptor can be used to inhibit the adhesion of bacterial cells to these model substrates, and these analogs may be of clinical use.

Affinity and distribution of surface and intracellular hyaluronic acid receptors in isolated rat liver endothelial cells.

125I-Hyaluronic acid (HA) uniquely modified only at the reducing end (Raja, R.H., LeBoeuf, R. D., Stone, G.W., and Weigel, P.H. (1984) Anal. Biochem. 139, 168-177) binds specifically to rat liver endothelial cells in suspension or in culture. About 67-85% of the HA binding sites in isolated cells in suspension and 50% in cultured cells were intracellular, since they were exposed after permeabilizing cells with digitonin. Specific 125I-HA binding at 4 degrees C varied from 60 to 80% for intact cells and from 70 to 90% for permeabilized cells. Freshly isolated permeabilized cells bound about 500,000 HA molecules/cell at saturation. Within 5 h of culture, however, total HA binding decreased to 250,000 molecules/cells and then remained constant for at least 36 h. Surface HA receptor activity was essentially the same on cultured cells or cells in suspension (approximately 10(5)/cell). Cultured cells had 1.8 x 10(5) fewer intracellular receptors/cell. The affinities of surface and intracellular receptors of cells in culture and in suspension were essentially the same. The average Kd, determined by equilibrium binding studies, was 5.8 +/- 2.8 x 10(-8) M (n = 12). Dissociation of bound 125I-HA from permeable cultured cells was rapid (t1/2 = 30.9 min;kappa off = 3.7 x 10(-4) s-1). A variety of carbohydrates had essentially identical effects on 125I-HA binding to surface or total cellular receptors in cells in culture or in suspension. Chondroitin sulfate and heparin competed almost as effectively as unlabeled HA for 125I-HA binding at 4 degrees C. Other saccharides including polygalacturonic acid, dextran, glucuronic acid, and N-acetylglucosamine competed poorly or not at all. We conclude that (i) the 125I-HA binding sites within liver endothelial cells are HA receptors, identical in affinity and specificity to those on the cell surface; (ii) the distribution of cellular HA receptors is similar to other receptor systems with about 50-80% being intracellular; (iii) the liver endothelial cell HA receptor recognizes several glycosaminoglycans; and (iv) the liver endothelial receptor is different in function and characteristics than the fibroblast HA receptor.

Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes.

125I-HA, prepared by chemical modification at the reducing sugar, specifically binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilization of hepatocytes with the nonionic detergent digitonin results in increased specific 125I-HA binding (45-65%) and a very large increase in the number of specific 125I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspension at 4 degrees C indicates a Kd = 1.8 x 10(-7) M and 1.3 x 10(6) molecules of HA (Mr approximately 30,000) bound per cell at saturation. Hepatocytes in primary culture for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decreases to approximately 6.2 x 10(5). Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength above causes an approximately 4-fold increase. The divalent cation chelator EGTA does not prevent binding nor does it release 125I-HA bound in the presence of 2 mM CaCl2, although higher divalent cation concentrations stimulate 125I-HA binding. Ten millimolar CaCl2 or MnCl2 increases HA binding 3-6-fold compared to EGTA-treated cells. Ten millimolar MgCl2, SrCl2, or BaCl2 increased HA binding by 2-fold. The specific binding of 125I-HA to digitonin-treated hepatocytes at 4 degrees C increased greater than 10-fold at pH 5.0 as compared to pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation of alkylamine and 125I-radiolabeled derivatives of hyaluronic acid uniquely modified at the reducing end.

Present procedures to obtain radiolabeled hyaluronic acid derivatives are limited to low-specific-activity isotopes and small amounts of material, and often involve multiple points of chemical modification within the polymer. A synthesis has been developed which affords large quantities of a unique, chemically modified derivative of hyaluronic acid containing a single hydroxyphenyl group at the reducing end, which can be radioiodinated to high specific activity. Very little alteration in oligosaccharide structure is expected since only the terminal reducing sugar is modified. Oligosaccharides of hyaluronic acid, which have no free amino groups, were first converted to alkylamine derivatives to allow subsequent reaction with the Bolton-Hunter reagent, N-succinimidyl-3(4-hydroxyphenyl)propionate. Synthesis of the hyaluronate-amine was achieved by (i) reduction of the terminal reducing sugar with sodium borohydride, (ii) controlled sodium periodate oxidation to generate an aldehyde group only at the reduced end, and (iii) coupling this aldehyde to an alpha,omega- alkyldiamine (e.g., 1,6- hexanediamine ) in the presence of sodium cyanoborohydride. Purified hyaluronate-amine oligosaccharides were then reacted with the Bolton-Hunter reagent, and the hydroxyphenyl derivative thus obtained was radioiodinated with Na125I. Specific activities up to 8 X 10(9) cpm/nmol oligosaccharide can be obtained. This approach yields a uniquely modified, highly radioactive probe which will be useful in studies of cellular and extracellular matrix interactions with hyaluronic acid. In addition, the uniquely modified alkylamine derivative of hyaluronic acid has been used to prepare affinity chromatography media and synthetic cell culture surfaces.

Preparation and use of synthetic cell culture surfaces. A new reagent for the covalent immobilization of proteins and glycoproteins on a nonionic inert matrix.

To study cell interactions with external molecules immobilized on a chemically defined nonionic, inert matrix, we have prepared flat polyacrylamide matrices containing covalently attached carbohydrate or protein. A new acrylamide derivative, containing a terminal 1,2-dihydroxy group, was synthesized and then copolymerized with acrylamide and bisacrylamide to make 20% polyacrylamide matrices, which could be oxidized with NaIO4 to generate reactive aldehyde groups. Molecules containing a free amine (e.g. proteins or glycopeptides) can be coupled to the aldehyde-activated matrix by formation of a Schiff base and reduction with NaCNBH3 to form a stable -CH2-NH-bond. Unreacted aldehyde groups are reduced to hydroxyl groups with NaBH4. In order to immobilize polysaccharides on the activated surfaces, these molecules are first modified to contain a free amine. We have described a procedure to convert purified hyaluronic acid oligosaccharides to a reactive alkylamine derivative uniquely modified at the reducing end (Raja, R. H., LeBoeuf, R. D., Stone, G. W., and Weigel, P. H. (1984) Anal. Biochem. 139, 168-177). The covalent attachment of [3H]hyaluronate-amine, [14C]ethanolamine, or 125I-bovine serum albumin, to activated surfaces was complete within 5-24 h. The amount immobilized was directly proportional to the amine concentration and to the aldehyde content of the matrix and inversely proportional to the molecular weight of the amine. About 90% of the available aldehyde groups reacted with ethanolamine, whereas less than 0.01% reacted with albumin. Molecules larger than 3300 Da were excluded from the interior of the matrix and could therefore only be attached to the surface of the matrix. These synthetic surfaces can be used in long term culture experiments to study cellular interactions with virtually any type of immobilized molecule.

Human fibrinogen specifically binds hyaluronic acid.

Fibrin and hyaluronic acid (HA) are macromolecules whose concentrations are elevated at the same time in the extracellular space of damaged tissues. We have investigated whether HA can bind to fibrinogen using solid phase and soluble assays. Purified human fibrinogen specifically bound to HA-Sepharose to a greater extent (greater than 5-fold) than did alpha 1-acid glycoprotein, DNaseI, ovalbumin, haptoglobin, or lysozyme. Fibrinogen did not bind to ethanolamine-Sepharose, a control chromatographic support. Treatment of HA-Sepharose containing bound 125I-fibrinogen with ovine testicular hyaluronidase released 44% of the 125I radioactivity, indicating that fibrinogen was specifically bound to HA. Moreover, 125I-fibrinogen bound to HA-Sepharose could be displaced by free HA but not by either of the monosaccharide components of this polymer, glucuronic acid, or N-acetylglucosamine. Chondroitin sulfate and polygalacturonic acid competed only weakly for bound 125I-fibrinogen. Bound 125I-fibrinogen was also not released by high concentrations of NaCl (up to 4 M), indicating that the interaction is not simply ionic. The apparent affinity of fibrinogen for HA covaried with the molecular weight of the HA. Small HA oligosaccharides (Mr = 3900) were only 50% as effective as larger HA (Mr = 8 X 10(5)) in eluting bound 125I-fibrinogen from HA-Sepharose. The optimal oligosaccharide size for displacement of bound 125I-fibrinogen was greater than or equal to 200 monosaccharides. Additionally, the amount of 125I-fibrinogen bound to HA-Sepharose was directly related to the size of the HA-amine linked to the affinity support. The affinity constant for fibrinogen binding to 125I-HA (approximately 150 monosaccharides) is estimated to be at least 2 X 10(7) M-1. These results demonstrate for the first time a specific, reversible binding between HA and fibrinogen.

Human immunodeficiency virus 1 Tat stimulates transcription of the transforming growth factor alpha gene in an epidermal growth factor-dependent manner.

The human immunodeficiency virus 1 (HIV-1) tat gene encodes a protein of critical importance for viral transcription. In addition, Tat has been shown capable of entering cells, stimulating cell proliferation, and altering host cell gene expression. We examined the effect of Tat on the expression of the transforming growth factor alpha (TGF-alpha) gene in MDA468 human breast carcinoma cells. We showed that these cells were capable of supporting the activation of the HIV-1 long terminal repeat by Tat. Then, in cotransfection assays, in which the TGF-alpha promoter was linked to a luciferase reporter gene and the tat gene was expressed under the control of the SV40 early promoter, we showed that tat gene expression increased TGF-alpha-luciferase reporter function but only in cells stimulated with epidermal growth factor (EGF). The effects of tat and EGF were dose dependent. To confirm these cotransfection data, Tat was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) and purified on glutathione-agarose. GST-Tat was introduced into the MDA468 cells either in the presence of chloroquine or by scrape loading. The biological activity of GST-Tat was tested on cells that had been stablely transfected with the HIV-1 long terminal repeat linked to luciferase as a reporter. GST-Tat was then introduced into the cells, and the level of TGF-alpha mRNA was determined.(ABSTRACT TRUNCATED AT 250 WORDS)