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Biomedical subjects

W Borth

Publications and source records attributed to W Borth.

At least 37 records · Page 2Linked to original sources
PubMed1990

Sequence similarity between alpha 2-macroglobulin from the horseshoe crab, Limulus polyphemus, and proteins of the alpha 2-macroglobulin family from mammals.

1. Purified alpha 2-macroglobulin (alpha 2M) from the American horseshoe crab, Limulus polyphemus was cleaved with trypsin and 20 of the tryptic peptides were sequenced and compared with the sequences of human alpha 2M, rat alpha 1M, alpha 2M, and alpha 1-inhibitor 3, and human complement proteins C3 and C4. 2. Ten of the peptides (233 residues), including that containing the thiol ester site, could be aligned unambiguously with stretches in mammalian alpha 2M, with a degree of identity greater than 30%. 3. The 12-residue thiol ester-containing peptide of Limulus alpha 2M showed 67% identity with the same stretch of human alpha 2M.

Amino Acid Sequence↗
PubMed1989

Identification of alpha 2-macroglobulin as a cytokine binding plasma protein. Binding of interleukin-1 beta to "F" alpha 2-macroglobulin.

Treatment of normal human plasma with methylamine resulted in the discovery of an interleukin-1 beta(IL-1 beta) binding protein. The protein was labeled with 125I-IL-1 beta and the relative molecular mass (Mr) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein-IL-1 beta complex had a Mr of approximately 400,000 in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis but became dissociated when exposed to beta-mercaptoethanol. The 125I-IL-1 beta labeled protein complex could be immunoprecipitated from plasma by using an anti-alpha 2-macroglobulin (alpha 2M) antiserum. Similarly, a monoclonal antibody (mAb) specific for electrophoretically fast ("F")alpha 2M was able to adsorb the 125I-IL-1 beta labeled complex from plasma. The mAb was also capable of adsorbing "F" alpha 2M-125I-IL-1 beta complexes from binary reaction mixtures, but failed to adsorb free 125I-IL-1 beta. Experiments carried out with purified plasma alpha 2M established that IL-1 beta became bound to alpha 2M only upon reaction with trypsin or methylamine, which results in the appearance of free thiol groups in alpha 2M ("F" alpha 2M). There was no binding of IL-1 beta to the native form of alpha 2M (electrophoretically slow or "S" alpha 2M), which lacks free thiol groups. Pretreatment of "F" alpha 2M with N-ethylmaleimide or [ethylenebis(oxyethylenenitrilo)] tetraacetic acid prevented complex formation between "F" alpha 2M and IL-1 beta. In contrast, the yield of "F" alpha 2M IL-1 beta complex formation was increased severalfold in the presence of 2.5 mM Zn2+. These findings indicate that "F" alpha 2M interacts with IL-1 beta through a thiol-disulfide exchange reaction. Zn2+ may play a major role in bringing together the reactive domains of the adjoining peptide backbones into proper orientation. The ready complex formation between "F" alpha 2M and the pleiotropic cytokine IL-1 beta suggests a novel biological role for "F" alpha 2M, since "F" alpha 2M-IL-1 beta complexes, but not "F" alpha 2M alone, retained IL-1-like activity in the thymocyte costimulator bioassay.

Biological Factors↗
PubMed1989

IFN-beta 2, B cell differentiation factor 2, or hybridoma growth factor (IL-6) is expressed and released by human epidermal cells and epidermoid carcinoma cell lines.

IL-6, which is also known as IFN-beta 2, hybridoma growth factor, hepatocyte-stimulating factor, and B cell differentiation factor, mediates acute phase responses including fever, has lymphocyte-stimulating capacities, and antiviral activity. IL-6 is produced by monocytes, fibroblasts, certain lymphocytes, and various tumor cells. The present study demonstrates that this multifunctional cytokine is released also by normal human epidermal cells (EC) and human epidermoid carcinoma cell lines (A431, KB). Accordingly, supernatants derived from freshly isolated EC, long term keratinocyte cultures, A431, or KB cells stimulated the proliferation of a hybridoma growth factor/IL-6-dependent plasmacytoma cell line (B9). IL-6 constitutively was produced in the presence of serum proteins. The addition of IL-1 alpha, IL-1 beta, or the tumor promoter PMA significantly enhanced the synthesis and release of EC-derived IL-6 (EC-IL 6). Like monocyte or fibroblast-derived IL-6, EC-IL-6 exhibited Mr microheterogeneity within 21 and 28 kDa. Similarly in Western blotting experiments an antiserum directed against human rIFN-beta 2/IL-6 detected the different Mr forms of EC-IL-6. Moreover, this antiserum was able to block the B9 cell growth-promoting capacity of EC-IL-6 strongly suggesting that this EC-derived mediator is closely related, if not identical with IL-6. This was further confirmed by Northern blot analysis detecting IL-6 specific mRNA both in long term cultured keratinocytes and A431 cells by hybridization with a cDNA fragment encoding for B cell differentiating factor 2/IL-6. Therefore, in addition to the production of other cytokines as previously reported, EC and in particular keratinocytes also synthesize and release IL-6. This further supports the important regulatory role of the epidermis during the pathogenesis of inflammatory, autoimmune, and neoplastic diseases.

Animals↗
PubMed1986

Modulation of antigen-induced T cell proliferation by alpha 2M-trypsin complexes.

Proteinase-complexed alpha 2-macroglobulin (alpha 2M) could be shown to interfere with T cell proliferation in response to antigen presented by autologous antigen-pulsed monocytes (M phi) (antigen-induced M phi-T cell interaction, MTI). Addition of alpha 2M-trypsin (alpha 2M X T) complexes to cultures of T cells and antigen-pulsed M phi led to a dose-dependent decrease of T cell proliferation (up to 91% inhibition of the T cell response), whereas the same concentrations of free (native) alpha 2M had no effect on antigen-induced MTI. The observed interference with MTI could be attributed to residual enzymic activity of the alpha 2M X T complex. Addition of aprotinin, a low Mr protein proteinase inhibitor able to penetrate to the enzyme entrapped within the alpha 2M molecule and thus bind to and inactivate the enzyme's active site, resulted in a reversal of the alpha 2M X T-induced biological effect. Inactivation of the enzyme's active site within alpha 2M X T was monitored by a decrease in the hydrolytic activity of the complex. Kinetic studies (addition of alpha 2M X T 24 to 48 hr after culture onset was shown to be still inhibitory) indicated an effect at the level of the T cell or its mediators, but an overnight incubation of T cells with alpha 2M X T did not alter these cells' capacity to proliferate in response to an antigenic stimulus. An additional effect of alpha 2M X T on the antigen-presenting cell cannot be ruled out at present. However, alpha 2M X T did not alter the percentage of monocytes expressing HLA-DR, -DP, and -DQ or interfere with interleukin 1 release if added to M phi at concentrations that significantly inhibited MTI. Furthermore, incubation of M phi with alpha 2M X T for 1 hr before antigen pulsing had no effect on the M phi antigen presenting capacity.

Antigen-Presenting Cells↗
PubMed1986

Alpha 2-macroglobulin-proteinase complexes as correlated with alpha 1-proteinase inhibitor-elastase complexes in synovial fluids of rheumatoid arthritis patients.

The levels of alpha1-proteinase inhibitor-elastase and alpha 2-macroglobulin (alpha 2M)-proteinase complexes were measured in synovial fluids from arthritis patients by use of specific immunosorbent assays. Both types of proteinase inhibitor-proteinase complexes were significantly correlated with each other as well as with the total neutrophil count in synovial fluids of rheumatoid arthritis patients but were discordant in synovial fluids of patients with osteoarthritis. One synovial fluid sample showed active (inhibitory) alpha 2M as well as active collagenase. We purified alpha 2M from pooled synovial fluids obtained from patients with rheumatoid arthritis. This alpha 2M retained approximately 90% of its proteinase binding (inhibiting) capacity, compared with that of normal plasma alpha 2M. We found no evidence that alpha 2M was inactivated by means other than proteinases.

Arthritis, Rheumatoid↗
PubMed1986

Inactivation of human interleukin-2 (IL-2) by alpha 2-macroglobulin-trypsin complexes.

The loss of the biological activity of interleukin-2 (IL-2, T-cell growth factor) in the presence of alpha 2-macroglobulin-trypsin (alpha 2M.t) complexes has been investigated using an IL-2-dependent cloned 'cytotoxic' murine T-cell line. While reaction mixtures of native alpha 2M, aprotinin or methylamine-treated alpha 2M and IL-2 had no effect on IL-2 activity when incubated at 37 degrees for 5 h, alpha 2M.t (90 nM, [T]:[alpha 2M] = 0.8) inactivated IL-2 at a rate of one-sixth of that of the free enzyme. This effect was abolished by treatment of alpha 2M.t with aprotinin (MW 6500). Soybean trypsin inhibitor coupled to Sepharose 4B was capable of absorbing the IL-2 degrading activity from the trypsin solution. In contrast, alpha 2M.t treated with the solid-phase immobilized soybean trypsin inhibitor continued to inactivate IL-2, but did not degrade a macromolecular substrate (remazol-brilliant blue hide). Thus, IL-2 (MW 15,500) gains access to the active site of the alpha 2M-bound trypsin, resulting in a rapid loss of its biological activity. These observations offer an explanation for the in vitro immunosuppressive effects of alpha 2M.t.

Cell Division↗
PubMed1985

A solid-phase immunosorbent assay to determine the proteinase binding capacity of alpha 2-macroglobulin using 125I-trypsin as indicator proteinase.

Hyperimmune sera against human alpha 2 macroglobulin were raised in rabbits following immunization with 's' alpha 2-macroglobulin over half a year. Immunoglobulins were prepared by DEAE-Sephacel anion exchange chromatography. The immunoglobulin preparations showed a remarkably high and equal titer for 's' and 'f' alpha 2-macroglobulin (plasma alpha 2-macroglobulin fully saturated with pig pancreas trypsin), which amounted to 6.4 X 10(-6) as revealed by passive hemagglutination. Immunoimmobilization experiments revealed that at equilibrium, 's' alpha 2-macroglobulin and both 'f' alpha 2-macroglobulins (27 and 82% saturation of 's' alpha 2-macroglobulin with trypsin) had been bound to the same degree from the fluid phase to the monospecific antibodies that had been adsorbed to polystyrene tubes. Comparison of quantitative gel scans for disappearance of the intact alpha 2-macroglobulin subunit (Mr 182000) with 125I-labeled trypsin binding capacity of immunoimmobilized alpha 2-macroglobulin-trypsin complexes showed conspicuous agreement. Rocket immunoelectrophoresis did not give significant differences between 's' alpha 2-macroglobulin and 'f' alpha 2-macroglobulin. In the fluid phase, a binding ratio of 2.4 mol trypsin/mol alpha 2-macroglobulin was observed. Saturation of solid phase immunoimmobilized 's' alpha 2-macroglobulin with trypsin could be accomplished by incubation with a 100-200-fold molar excess of enzyme for 10 min. The solid-phase experiments showed a binding ratio of 2.0 mol trypsin/mol alpha 2-macroglobulin. The high molar excess of trypsin needed to saturate solid-phase immunoimmobilized alpha 2-macroglobulin, which binds 20% less trypsin than in the liquid phase, is partially explained by an enhancement of the negative cooperativity of trypsin binding to alpha 2-macroglobulin found in the liquid-phase system. Assessment of the trypsin-binding capacity of alpha 2-macroglobulin immunoadsorbed from synovial fluids (n = 19) of patients with seropositive rheumatoid arthritis yielded an inactive alpha 2-macroglobulin of 0-53% when compared to the trypsin-binding capacity of normal plasma alpha 2-macroglobulin.

Antibodies↗
PubMed1984

Experimental synovitis induced by intraarticular administration of alpha 2-macroglobulin-proteinase complexes.

Rheumatoid synovial collagenase was prepared from tissue cultures from rheumatoid arthritis patients, obtained after synovectomy. Alpha 2-macroglobulin was isolated from human plasma and complexed with collagenase or trypsin. Formation of both types of complexes was proven by sodium dodecyl sulfate and rate electrophoresis. Normal rabbits were injected intraarticularly into the right knee, on days 0, 3, and 6, with either alpha 2-macroglobulin-collagenase or alpha 2-macroglobulin-trypsin complexes. Control injections of alpha 2-macroglobulin, trypsin, or rheumatoid synovial collagenase were applied to the left knee joint cavity. Groups of rabbits were killed 18 hours, 1 week, or 3 weeks after the last injection, and cellular exudation into synovial fluid and morphologic alterations of synovium were investigated. Joints injected with alpha 2-macroglobulin showed no synovitis, while joints injected with collagenase showed an experimental synovitis. Alpha 2-macroglobulin-proteinase complexes, however, induced a synovitis, which was more severe than that occurring after injection of proteinases only. In the early stages, synovium showed perivascular accumulation of inflammatory cells, infiltration with neutrophils, proliferation of synovial cells, and exudation of inflammatory cells into synovial fluid. Later stages were characterized by infiltration with mononuclear cells and fibroplasia.

Animals↗
PubMed1984

Association of C1q- and conglutinin-binding immune complexes with high consumption of alpha-2-macroglobulin in synovial fluids of patients with rheumatoid arthritis.

The consumption of alpha-2-macroglobulin (a2M) by proteinases in synovial fluids (SF) was indirectly measured by determining trypsin binding to solid-phase immunoadsorbed SF-a2M. In addition immune complexes (IC) were assessed by the solid-phase C1q-binding assay as well as the solid-phase conglutinin binding assay in rheumatoid (RA; n = 25) and osteoarthritic (OA; n = 9) SF. Elevated levels were found as follows: a2M X proteinase complexes (a2M X P) in 73.5% (RA, n = 20; OA, n = 5), solid-phase C1q-binding IC (C1q-IC) in 52% (13 out of 25 RA-SF) and bovine conglutinin binding IC (KgB-IC) in 54% (13 out of 24 RA-SF). All OA-SF were in either IC assay negative. Consumption of a2M in OA-SF and seronegative RA-SF was not more than 15% of total SF-a2M, showing a mean value of 19.5 and 13.5 micrograms a2M X P/ml SF. Seropositive RA-SF had an a2M X P mean value of 96.4 micrograms/ml SF, which differed significantly (p less than or equal to 0.05) from the former two groups. Similarly, RA-SF which were negative in either IC assay had a low a2M X P mean value (20.0 micrograms/ml SF). This contrasted sharply with the a2M X P means obtained in IC-positive RA-SF, which amounted to 131.5 micrograms a2M X P/ml SF in C1q-IC positive RA-SF and to 110.4 micrograms a2M X P/ml SF in KgB-IC positive RA-SF, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen-Antibody Complex↗
PubMed1983

[Use of the proteinase inhibitor alpha 2-macroglobulin in synovial fluids of arthritis of different origins and its relation to serologic parameters].

Collagen and proteoglycan are major constituents of the articular cartilage. In rheumatic disorders joint damage is attributed to the excessive action of proteoglycan degrading proteinases (P) and specific collagenases, which are released by connective tissue- and inflammatory cells in the synovial compartment. Collagenase is secreted in a latent form, which requires activation by a variety of serine-proteinases. Thus, several different proteinases are involved in pathogenesis of joint disease. alpha 2-macroglobulin was shown to be the major inhibitor of proteinases in complex biological fluids. To assess the utilization of alpha 2M by proteinases in synovial fluids (SF) from different arthritides we have employed a newly introduced solid phase immuno-sorbent assay, which allows concentration of alpha 2M and its proteinase-complexes from biological fluids. Most pronounced utilization of alpha 2M (up to 50% of total SF alpha 2M) was found in marked joint inflammation as judged from RF, immunocomplexes, C3 complement activation and acute phase reactants. Supported by animal experiments, which revealed that alpha 2M.P complexes but not native alpha 2M induce synovitis in rabbits after repeated intra-articular administration, it is suggested that pathophysiological rise of alpha 2M.P may impair cellular functions in inflammatory connective tissue, e.g. synovial tissue.

Antigen-Antibody Complex↗
PubMed1983

[Interaction between fibronectin and aggregated IgG].

The present study shows that plasma fibronectin forms complexes with aggregated human IgG (a model for immune complexes) and that the resulting complexes precipitate spontaneously or after addition of PEG from buffered saline. However, no clearcut interaction was observed in serum or synovial fluid. Two currently-used immune complex assays, the C1q solid phase assay and the conglutinin binding assay in a commercial inhibition variant, were tested as to their reliability in the presence of fibronectin. While the C1q-dependent assay showed low susceptibility to fibronectin, the conglutinin binding assay gave increased immune complex values in the presence of pathological concentrations of fibronectin.

Antigen-Antibody Complex↗
PubMed1983

Comparison of differentiating Dictyostelium discoideum cell types separated by an improved method of density gradient centrifugation.

During the migrating slug stage of development of the cellular slime mold Dictyostelium discoideum, the precursor cells of the mature spores and stalk are known to differ from each other. In order to explore these differences on a variety of levels, we have improved existing methods for the isopyknic purification of the precursor types. Prespore and prestalk amoebae obtained from mechanically dissociated slugs were separated by centrifugation on preformed Percoll density gradients. The isolated precursor cells are greater than 90-95% pure, show good viability and developmental competence, and can be obtained in relatively large amounts. We have performed initial comparisons of the differentiating amoebae with respect to their location within the slug, protein composition, and developmental capabilities. Density centrifugation reveals heterogeneity among posterior slug cells: The majority of these cells have the expected prespore characteristics, but there is also a minor (lighter) density fraction resembling anterior, prestalk amoebae. Thirty polypeptides synthesized preferentially by prespore or prestalk slug cells were detected by two-dimensional gel electrophoresis and fluorography. Some of these proteins are equally cell-specific when their accumulation is measured by silver staining; others are remarkably non-specific when stained. When the isolated precursor populations were allowed to complete development, they differed in terms of developmental territory size and timing, and in ways which suggest differences in cell cohesion and chemotaxis.

Cell Separation↗
PubMed1983

Influence of plasma fibronectin on collagen cleavage by collagenase.

The influence of plasma fibronectin on the cleavage of interstitial collagen types I, II, and III by mammalian collagenase was investigated. To this end, rheumatoid arthritis synovial tissue collagenase was isolated from tissue culture supernatants. A highly active enzyme preparation was obtained. Different dilutions of the collagenase were incubated in tubes coated with [3H]-labeled collagens. Liberation of labeled fragments in the presence (200 micrograms per ml) or absence of fibronectin was measured at different time intervals (1-30 min). A significant inhibition of collagen degradation was observed in case of type I and type III collagens. Analogous experiments were carried out using collagen-coated tubes preincubated with fibronectin at a concentration of 200 micrograms/ml. Here, a strong inhibition (76 percent) of type III collagen digestion by collagenase was observed, while the cleavage of type I or type II collagen was completely unchanged after preincubation with fibronectin. A direct attack of the collagenase preparation on plasma fibronectin could be excluded in control digestion experiments using radioiodinated fibronectin as substrate.

Arthritis, Rheumatoid↗
PubMed1983

Different synthetic profiles and developmental fates of prespore versus prestalk proteins of Dictyostelium.

Depending upon environmental conditions, developing cells of the cellular slime mold Dictyostelium discoideum may enter a slug stage in which the cell mass migrates in response to gradients of light and temperature. This developmental stage has often been used to study the divergent differentiation of the cells that will subsequently form spores and stalk in the mature fruiting body. However, still debated is the extent to which the differentiation evident in slug cells is a precondition for development of the mature cells in fruits. Using two-dimensional gel electrophoresis of polypeptides, we have examined the proteins made by prespore and prestalk cells of migrating slugs and by maturing spore and stalk cells. The data indicate that many of the cell-type specific polypeptides in prespore cells of slugs persist as cell-type specific polypeptides of mature spores. Prestalk slug cells, in contrast, do not contain significant amounts of stalk-specific proteins; these proteins appear only during culmination. The precursor cell types also differ in the times and rates of synthesis of cell-specific proteins: prestalk proteins appear much earlier in development than do the prespore, but never reach the levels of expression that the prespore proteins do later in culmination. These findings may explain the well established ability of prespore cells to regulate their cell type more rapidly than do prestalk cells. There are also implications for our general understanding of what is a 'prestalk' gene product.

Cell Differentiation↗