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Biomedical subjects

E G Hayman

Publications and source records attributed to E G Hayman.

At least 19 recordsLinked to original sources
PubMed1999

Recombinant glycoproteins that inhibit complement activation and also bind the selectin adhesion molecules.

Soluble human complement receptor type 1 (sCR1, TP10) has been expressed in Chinese hamster ovary (CHO) DUKX-B11 cells and shown to inhibit the classical and alternative complement pathways in vitro and in vivo. A truncated version of sCR1 lacking the long homologous repeat-A domain (LHR-A) containing the C4b binding site has similarly been expressed and designated sCR1[desLHR-A]. sCR1[desLHR-A] was shown to be a selective inhibitor of the alternative complement pathway in vitro and to function in vivo. In this study, sCR1 and sCR1[desLHR-A] were expressed in CHO LEC11 cells with an active alpha(1,3)-fucosyltransferase, which makes possible the biosynthesis of the sialyl-Lewisx (sLex) tetrasaccharide (NeuNAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc) during post-translational glycosylation. The resulting glycoproteins, designated sCR1sLex and sCR1[desLHR-A]sLex, respectively, retained the complement regulatory activities of their DUKX B11 counterparts, which lack alpha(1-3)-fucose. Carbohydrate analysis of purified sCR1sLex and sCR1[desLHR-A]sLex indicated an average incorporation of 10 and 8 mol of sLex/mol of glycoprotein, respectively. sLex is a carbohydrate ligand for the selectin adhesion molecules. sCR1sLex was shown to specifically bind CHO cells expressing cell surface E-selectin. sCR1[desLHR-A]sLex inhibited the binding of the monocytic cell line U937 to human aortic endothelial cells, which had been activated with tumor necrosis factor-alpha to up-regulate the expression of E-selectin. sCR1sLex inhibited the binding of U937 cells to surface-adsorbed P-selectin-IgG. sCR1sLex and sCR1[desLHR-A]sLex have thus demonstrated both complement regulatory activity and the capacity to bind selectins and to inhibit selectin-mediated cell adhesion in vitro.

Animals↗
PubMed1996

A tightly regulated high level expression vector that utilizes a thermosensitive lac repressor: production of the human T cell receptor V beta 5.3 in Escherichia coli.

A series of vectors has been constructed to express the human T cell receptor V beta 5.3 under the control of the hybrid trc promoter in Escherichia coli. Transcriptional induction of the trc promoter was achieved chemically by using isopropyl beta-D-thiogalactopyranoside (IPTG) in a bacterial strain that harbors the lacIq gene, or thermally by using the mutant lacIts gene that encodes a temperature-sensitive lac repressor [Bukrinsky et al. (1988) Multicopy expression vector based on temperature-regulated lac repressor: expression of human immunodeficiency virus env gene in Escherichia coli. Gene 70, 415-417]. Several of the plasmids tested also contain the E. coli heat-stable enterotoxin II (STII) signal sequence for protein secretion. In addition, the gene 10 leader sequence from bacteriophage T7 and a minicistron localized upstream of the V beta 5.3 coding sequence were tested for their potential effect on protein production. These elements increased protein yield two-fold when transcription was induced by IPTG, but had no detectable effect on protein yield when transcription was induced thermally. The highest protein yield was obtained when V beta 5.3 was expressed either from plasmid pKB containing the STII signal in strain LJ24, or from plasmid pKBi that lacks the signal sequence, in the protease deficient strain SG21173 (lon, htpR. clp). Both plasmids contain the lacIts gene, the trc promoter, the two transcription terminators of the rrnB operon, and a tetracycline selection marker. Production of V beta 5.3 using pKBi-V beta 5.3 in strain SG21173 in a 5-liter fermenter under controlled growth conditions yielded over 25 mg V beta 5.3/liter culture. Conversion of the lacIts to the lacIqts gene yielded vector pKBiq-V beta 5.3 which exhibits complete repression of the trc promoter at 30 degrees C. This stringent regulation of the thermally inducible trc promoter, the elimination of IPTG, the inclusion of the tetracycline resistance gene, and the high level of protein yield should render this expression system broadly useful for the high level production of heterologous proteins in E. coli, for both basic research and human therapeutic applications.

Amino Acid Sequence↗
PubMed1996

A soluble deletion mutant of the human complement receptor type 1, which lacks the C4b binding site, is a selective inhibitor of the alternative complement pathway.

The human complement receptor type 1 (CR1, CD35), is a single-chain glycoprotein consisting of 30 repeating homologous protein domains known as short consensus repeats (SCR) followed by transmembrane and cytoplasmic domains. The SCR themselves, considered in groups of seven, form long homologous repeats (LHR) which have been designated LHR-A, -B, -C, and -D for the most common human allotype of CR1. A soluble deletion mutant of CR1 which lacks the first seven N-terminal SCR (LHR-A) as well as the transmembrane and cytoplasmic domains was produced and characterized. The resulting protein, designated sCR1[desLHR-A], lacks the C4b binding site found in LHR-A, but retains the two C3b binding sites found in LHR-B and -C, respectively. The functional activities of sCR1[desLHR-A] were quantitatively compared in vitro to those of soluble complement receptor type 1 (sCR1) which has been shown to retain all known functions of the native cell surface receptor. sCR1[desLHR-A] and sCR1 competed equally for the binding of dimeric C3b to erythrocyte CR1. sCR1[desLHR-A] and sCR1 were similar in their capacity to serve as a cofactor in the factor I-mediated degradation of the C3b and C4b alpha chains. sCR1[desLHR-A] and sCR1 were comparable in their capacity to inhibit erythrocyte lysis and anaphylatoxin production mediated by the alternative complement pathway. sCR1[desLHR-A], however, was significantly less effective an inhibitor of erythrocyte lysis and anaphylatoxin production than sCR1 under conditions which allow classical pathway activation. These results demonstrate sCR1[desLHR-A] to be a selective inhibitor of the alternative complement pathway in vitro.

Binding, Competitive↗
PubMed1996

Extended in vivo half-life of human soluble complement receptor type 1 fused to a serum albumin-binding receptor.

A new approach has been used to extend the T(1/2) of human soluble complement receptor type 1 (sCR1) in rats. The albumin-binding domains B2A3 (BA) and B1A2B2A3 (BABA) from Streptococcal protein G were fused to the carboxyl terminus of sCR1, and the recombinant genes were expressed and amplified in Chinese hamster ovary cells. Western blot analysis and surface plasmon resonance measurements demonstrated the binding of rat serum albumin to both sCR1-BA and sCR1-BABA but not to sCR1. The in vitro complement inhibitory activity of the fusion proteins was shown to be similar to that of sCR1, indicating that neither the albumin-binding domains nor the presence of bovine serum albumin interfere with sCR1 function. Pharmacokinetic analysis showed that the T(1/2) of the distribution phase (T(1/2alpha)) was 3.3, 20.0 and 6.0 min for sCR1, sCR1-BA and sCR1-BABA, respectively. The T(1/2) of the elimination phase (T(1/2beta)) was 103, 297 and 170 min for sCR1, sCR1-BA and sCR1-BABA, respectively. The plasma elimination of sCR1-BA and sCR1-BABA was significantly (P < .05) prolonged as compared to sCR1. The proteins showed similar tissue distribution; at 4-hr postdosing, the highest levels of 125I-radioactivity per gram of tissue were localized in the urine, blood, liver, stomach, and small intestine.

Animals↗
PubMed1986

Interaction of vitronectin with collagen.

Purified human plasma vitronectin was demonstrated to bind to type I collagen immobilized on plastic as measured by enzyme-linked immunosorbent assay and by binding of 125I-radiolabeled vitronectin to a collagen-coated plastic surface. Vitronectin did not bind to immobilized laminin, fibronectin, or albumin in these assays. Vitronectin showed similar interaction with all types of collagen (I, II, III, IV, V, and VI) tested. Collagen unfolded by heat treatment bound vitronectin less efficiently than native collagen. Vitronectin-coated colloidal gold particles bound to type I collagen fibrils as shown by electron microscopy. Salt concentrations higher than physiological interfered with the binding of vitronectin to collagen, suggesting an ionic interaction between the two proteins. Binding studies conducted in the presence of plasma showed that purified vitronectin added to plasma bound to immobilized collagen, whereas the endogenous plasma vitronectin bound to collagen less well. Although fibronectin did not interfere with the binding of vitronectin to native collagen, vitronectin inhibited the binding of fibronectin to collagen. These results show that vitronectin has a collagen-binding site(s) which, unlike that of fibronectin, preferentially recognizes triple-helical collagen and that the binding between vitronectin and collagen has characteristics compatible with the occurrence of such an interaction in vivo.

Blood Proteins↗
PubMed1985

Complete amino acid sequence of human vitronectin deduced from cDNA. Similarity of cell attachment sites in vitronectin and fibronectin.

cDNA clones for vitronectin, a cell adhesion-promoting plasma and tissue protein, were isolated from a lambda gt11 library containing cDNA inserts made from human liver mRNA. The library was screened with anti-vitronectin antibodies and the positive clones were further identified with synthetic oligonucleotide probes deduced from the partial amino acid sequence of vitronectin. Nucleotide sequence analysis showed that the largest insert was 1545 bp long and contained the whole sequence corresponding to plasma vitronectin. It showed that vitronectin contains the entire 44-amino acid somatomedin B peptide at its NH2 terminus and, near its COOH terminus, a 34-amino acid glycosaminoglycan binding site in which half of the amino acids are basic residues. Three potential carbohydrate attachment sites are present in the sequence. An Arg-Gly-Asp sequence, which has previously been shown to be the cell attachment site in fibronectin, was found in vitronectin immediately after the NH2-terminal somatomedin B sequence. No other homologies with fibronectin were found. The Arg-Gly-Asp sequence appears to constitute the cell attachment site of vitronectin, since it is in the region where we have previously localized the cell attachment site, its presence correlate with cell attachment activity among the insert-coded polypeptides, and because previous results have shown that synthetic peptides containing the Arg-Gly-Asp sequence inhibit the cell attachment function of vitronectin. The discovery of an Arg-Gly-Asp cell attachment site in a protein with a known cell attachment function emphasizes the general importance of this sequence in cell recognition.

Amino Acid Sequence↗
PubMed1985

The cell attachment determinant in fibronectin.

Fibronectin possesses a domain that interacts with cell surfaces. The ability of fibronectin to promote cell attachment can be duplicated with a short amino acid sequence, glycyl-L-arginyl-glycyl-L-aspartyl-L-serine, taken from that domain. The tripeptide Arg-Gly-Asp appears to be irreplaceable for maintenance of the activity of this peptide, whereas the serine residue can be replaced with some, but apparently not all, possible residues. This recognition sequence, or a closely related sequence, is present in a number of proteins other than fibronectin that interact with cells. These proteins include collagens, fibrinogen, thrombin, a bacterial surface protein, and two viral proteins, as well as discoidin-I, a protein implicated in the aggregation of Dictyostelium discoideum. A similar sequence is also repeated in some, but not all, fibronectin molecules, making it possible that some fibronectin molecules have more than a single cell attachment site. Synthetic peptides constructed from sequences taken from several of these other proteins have also been shown to promote cell attachment. The tripeptide sequence may, therefore, constitute an ancient cellular recognition mechanism common to many proteins.

Amino Acid Sequence↗
PubMed1985

Vitronectin--a major cell attachment-promoting protein in fetal bovine serum.

Bovine serum is a constituent of most media used for the culture of animal cells. The adhesion-promoting properties of serum are generally attributed to fibronectin, yet there have been frequent reports of other adhesion-promoting molecules in bovine serum. Using a technique in which adhesive proteins are visualized after separation by SDS-PAGE, we graphically confirm the presence of a second cell attachment protein in bovine serum and present the evidence that this molecule is the bovine equivalent of vitronectin. The molecular size of this protein is in the same range as the size of the adhesive human plasma protein, vitronectin. The bovine protein also shared with human vitronectin an affinity for glass, and it could be purified by a combination of glass bead and ion exchange chromatography. The isolated bovine protein had varying proportions of an 80 and a 65 kD polypeptide. It showed immunological cross-reactivity with anti-human vitronectin and with anti-human somatomedin B. Somatomedin B is a serum peptide which has a NH2-terminal sequence identical to that of human vitronectin. The identity of the bovine protein as vitronectin was established by showing that its NH2-terminal amino acid sequence is strongly homologous with those of human vitronectin and somatomedin B. Quantitation of the adhesive activities of fibronectin and vitronectin in bovine plasma and fresh serum showed that more activity is associated with vitronectin than with fibronectin. The preponderance of vitronectin was particularly clear in fetal bovine serum intended for cell culture. In various batches, cell attachment activity attributable to vitronectin was 8-16-fold greater than that of fibronectin, making vitronectin the main adhesive protein in routine cell culture media.

Amino Acid Sequence↗
PubMed1985

Detachment of cells from culture substrate by soluble fibronectin peptides.

The synthetic cell attachment-promoting peptides from fibronectin (Pierschbacher, M. D., and E. Ruoslahti, 1984, Nature (Lond.)., 309:30-33) were found to detach cultured cells from the substratum when added to the culture in a soluble form. Peptides ranging in length from tetrapeptide to heptapeptide and containing the active L-arginyl-glycyl-L-aspartic acid (Arg-Gly-Asp) sequence had the detaching activity, whereas a series of different peptides with chemically similar structures had no detectable effect on any of the test cells. The Arg-Gly-Asp-containing peptides caused detachment of various cell lines of different species and histogenetic origin. Studies with defined substrates showed that the active peptides could inhibit the attachment of cells to vitronectin in addition to fibronectin, indicating that vitronectin is recognized by cells through a similar mechanism as fibronectin. The peptides did not inhibit the attachment of cells to collagen. However, cells cultured on collagen-coated plastic for 24-36 h, as well as cells with demonstrable type I or type VI collagen in their matrix, were susceptible to the detaching effect of the peptides. These results indicate that the recognition mechanism(s) by which cells bind to fibronectinand vitronectin plays a major role in the substratum attachment of cells and that collagens may not be directly involved in cell-substratum adhesion. Since vitronectin is abundant in serum, it is probably an important component in mediating the attachment of cultured cells. The independence of the effects of the peptide on the presence of serum and the susceptibility of many different cell types to detachment by the peptide show that the peptides perturb an attachment mechanism that is intrinsic to the cells and fundamentally significant to their adhesion.

Amino Acid Sequence↗
PubMed1985

Effects of ricin A chain conjugates of monoclonal antibodies to human alpha-fetoprotein and placental alkaline phosphatase on antigen-producing tumor cells in culture.

Two tumor-associated proteins, alpha-fetoprotein (AFP) and placental alkaline phosphatase (PLAP), were investigated as target proteins for antibody:cytotoxin conjugates. An AFP-producing hepatoma cell line (HepG2) and a PLAP-producing cervical carcinoma cell line (SKGIIIa) were used as target cells. Both cell lines were equally susceptible to the toxic effects of intact ricin. Immunofluorescent studies showed that AFP could be detected at the surface of the HepG2 cells in a speckled distribution, while PLAP was uniformly distributed over the surface of the SKGIIIa cells. The anti-AFP ricin A chain conjugate was not cytotoxic to either cell line at low concentrations and killed both types of cells at high concentrations. The anti-PLAP conjugate at low concentrations was 100-fold more toxic than the anti-AFP conjugate to the PLAP-producing SKGIIIa cells. At high concentrations, it also killed both types of cells. The enhanced toxicity of the anti-PLAP conjugate to the SKGIIIa cells was inhibited by an excess of unconjugated anti-PLAP but not anti-AFP, indicating that the uptake of the conjugate depends on specific cell surface binding to the antigen. The indiscriminate toxicity observed at high concentrations of either conjugate was not inhibited by unconjugated antibody, suggesting that this effect depends on conjugate uptake independent of the identity of the antigen. These results emphasize the importance of the properties of the target antigen to the cytotoxic effects of antibody conjugates as well as the need for caution in experiments using high concentrations of conjugates. They suggest that PLAP may be a suitable target for immunotoxin therapy of human cancer.

Alkaline Phosphatase↗
PubMed1984

Domain structure of vitronectin. Alignment of active sites.

The structure of vitronectin, an adhesive protein isolated from human plasma, was studied by chemical fragmentation. Partial cleavage of vitronectin with cyanogen bromide in 70% formic acid generated four main fragments with masses of 53,000, 43,000, 35,000, and 12,000 daltons arising from both the 75- and 65-kDa vitronectin polypeptides and a 10-kDa fragment arising only from the 75-kDa polypeptide. By varying the reaction conditions, four BrCN cleavage sites and one acid cleavage site could be identified. The latter site gave rise to 40-, 32-, and 26-kDa fragments. The order of these fragments within the vitronectin polypeptides was determined by comparison of the NH2-terminal sequences of the polypeptides and their fragments, by further cleavage of the largest fragments with BrCN or 70% formic acid, and by assaying for heparin-binding and cell-attachment activities. The NH2-terminal sequences of the intact vitronectin polypeptides are the same and identical to a 44-amino acid serum peptide called somatomedin B, indicating that vitronectin may be the precursor of somatomedin B. The cell-attachment site appears to be located within approximately 5 kDa of the NH2 terminus, but it is distinct from the somatomedin B domain. The heparin-binding site is contained in the 12-kDa fragment near the COOH terminus. This fragment was shown to bind to a chondroitin sulfate proteoglycan in addition to heparin. The NH2-terminal amino acid sequence of this glycosaminoglycan-binding fragment is remarkably rich in basic amino acids. The NH2-terminal sequences of this and the other vitronectin fragments showed no homology with the amino acid sequence of the heparin-binding domain of fibronectin or other known sequences from fibronectin. These results show that the biological activities of vitronectin are located in distinct parts of both of the vitronectin polypeptides, which appear to be identical except for the presence of an additional 10-kDa fragment near or at the COOH terminus of the 75-kDa polypeptide.

Amino Acid Sequence↗
PubMed1984

Neurite-promoting factors and extracellular matrix components accumulating in vivo within nerve regeneration chambers.

The outgrowth of neurites from cultured neurons can be induced by the extracellular matrix glycoproteins, fibronectin and laminin, and by polyornithine-binding neurite-promoting factors (NPFs) derived from culture media conditioned by Schwann, or other cultured cells. We have examined the occurrence of fibronectin, laminin and NPFs during peripheral nerve regeneration in vivo. A previously established model of peripheral nerve regeneration was used in which a transected rat sciatic nerve regenerates through a silicone chamber bridging a 10 mm interstump gap. The distribution of fibronectin and laminin during regeneration was assessed by indirect immunofluorescence. Seven days after nerve transection the regenerating structure within the chamber consisted primarily of a fibrous matrix which stained with anti-fibronectin but not anti-laminin. At 14 days, cellular outgrowths from the proximal and distal stumps (along which neurites grow) had entered the fibronectin-containing matrix, consistent with a role of fibronectin in promoting cell migration. Within these outgrowths non-vascular as well as vascular cells stained with anti-fibronectin and anti-laminin. Within the degenerated distal nerve segment, cell characteristic of Bungner bands (rows of Schwann cells along which regenerating neurites extend) stained with anti-fibronectin and laminin. The fluid surrounding the regenerating nerve was found to contain NPF activity for cultured ciliary ganglia neurons which markedly increased during the period of neurite growth into the chamber. In previous studies using this particular neurite-promoting assay, laminin but to a much lesser extent fibronectin also promoted neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Serum spreading factor (vitronectin) is present at the cell surface and in tissues.

Monoclonal antibodies were prepared against a cell attachment-promoting protein, serum spreading factor, which had been partially purified from human serum by chromatography on glass bead columns. The antibodies selected were those that reacted with polypeptides that had cell attachment-promoting activity after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Immunochromatography of human plasma on columns containing the monoclonal antibodies followed by affinity chromatography on heparin-Sepharose yielded material that in sodium dodecyl sulfate/polyacrylamide gel electrophoretic analysis gave polypeptides of molecular mass 65 and 75 kilodaltons. Both polypeptides bound each of three monoclonal antibodies and had cell attachment-promoting activity after transfer to nitrocellulose filters. Immunofluorescent staining of tissues with the monoclonal antibodies revealed a fibrillar pattern that was mostly associated with loose connective tissue and overlapped with fibronectin fibrils. Fetal membrane tissue, which showed strong staining with the antibodies in immunofluorescence, also gave 65- and 75-kilodalton polypeptides with cell attachment-promoting activity after chromatography on columns containing the monoclonal antibodies. One source of the tissue protein may be fibroblastic cells, because cultured human fibroblasts also stained with the monoclonal antibodies. The staining was fibrillar and appeared to be associated with the cell surface extracellular matrix. We propose the name "vitronectin" for the various forms of this protein, on the basis of its binding to glass and its adhesive properties.

Animals↗
PubMed1983

Synthetic peptide with cell attachment activity of fibronectin.

Four synthetic peptides that together constitute the cell attachment domain of fibronectin [Pierschbacher, M.D., Ruoslahti, E., Sundelin, J., Lind, P. & Peterson, P. (1982) J. Biol. Chem. 257, 9593-9597] were constructed and tested for their ability to induce cell attachment and spreading. One of these peptides, consisting of the 30 amino acid residues nearest the COOH terminus of the domain, contained all of the cell attachment activity of the whole domain. Under suitable conditions the peptide was approximately as active as intact fibronectin on a molar basis. The activity could be demonstrated by binding the peptide to polystyrene directly, or via albumin, or by coupling it to agarose beads. This synthetic peptide will be useful in the elucidation of the molecular details of the attachment of cells to fibronectin and could allow manipulation of the adhesive properties of cell culture surfaces and prosthetic materials.

Amino Acid Sequence↗
PubMed1983

Effect of a proteoglycan produced by rat tumor cells on their adhesion to fibronectin-collagen substrata.

In this paper, we demonstrate that a chondroitin sulfate proteoglycan purified from a rat yolk sac tumor alters the adhesion of the tumor cells to substrata containing fibronectin or type I collagen. In the presence of the proteoglycan, these substrata were much less adhesive and did not promote cell spreading. The inhibitory effect of the proteoglycan was reversible and more pronounced during the early stages of cell attachment in vitro. The effect of the proteoglycan was selective in that it depended on the ability of the adhesive substratum to bind the proteoglycan. The proteoglycan did not inhibit the attachment of the cells to type IV collagen, which bound 12 times less proteoglycan than did type I collagen. Similarly, attachment of the cells to fibronectin fragments which did not bind the proteoglycan was not affected by it. The selective effects of the proteoglycan on the adhesion of cells to extracellular matrices suggest that such proteoglycans may promote tumor invasion by reducing interaction of cells with interstitial extracellular matrices while permitting attachment to basement membranes.

Animals↗