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T Krakauer

Publications and source records attributed to T Krakauer.

53 records · Page 3Linked to original sources
PubMed1986

Human interleukin 1.

Interleukin 1 (IL-1), a product of stimulated monocytes or macrophages, is defined by its ability to enhance the response of thymocyte to mitogenic stimulation. Numerous studies attribute a multiplicity of other biological functions to IL-1 in immunological and inflammatory reactions. IL-1 also appears to be identical with endogenous pyrogen. Even though this mediator is produced in minute quantities and is active at very low concentrations, greatly complicating its purification, knowledge of its chemical structure and biochemical properties is rapidly increasing. The activity of human IL-1 is associated with multiple molecules of similar size, but subtly different compositions. Structure-function relations, most particularly the question of whether the functional diversity of IL-1 is a reflection of structural diversity or simply of diversity of target-cell responses to a single molecule can now be addressed, as the IL-1 components have been purified to homogeneity, on the one hand, and a precursor molecule, derived from a cloned cDNA sequence, has been obtained.

Animals↗
PubMed1985

Biochemical characterization of interleukin 1 from a human monocytic cell line.

A protein with interleukin 1 (IL-1) activity was obtained from the human acute monocytic leukemia cell line (THP-1) and purified by ultrafiltration, Ultrogel AcA54 chromatography, isoelectric focusing, and discontinuous polyacrylamide gel electrophoresis. Like the pI 6.8 species of IL-1 from human peripheral blood monocytes (PBM), the cell line IL-1 has a molecular weight (MW) of 14,000, a pI of 6.8, is heat labile, and does not bind to concanavalin A-Sepharose. Chemical modification of arginine residues by phenylglyoxal or sulfhydryl groups by N-ethyl maleimide and iodoacetamide completely destroys the activity of IL-1 from THP-1 cells as well as that of the pI 6.8 component from PBM. In contrast, the pI 5.1 component of PBM IL-1 is resistant to heat denaturation and sulfhydryl reagents, although it is totally inactivated by phenylglyoxal. IL-1 from THP-1 cells enhanced the proliferative response of the same subpopulations of PNA- thymocytes as IL-1 from PBM. These observations suggest that IL-1 derived from this cell line is similar to the IL-1 pI 6.8 species produced by human monocytes, and distinct from the pI 5.1 species.

Biological Assay↗
PubMed1985

A macrophage-derived factor that inhibits the production and action of interleukin 2.

The human monocytic leukemia cell line THP-1 produces an immunosuppressive factor that inhibits interleukin 1 (IL-1)-dependent proliferation of mouse thymocytes as well as the mitogenic effects of concanavalin A (Con A) and phytohemagglutinin (PHA) on human peripheral blood mononuclear cells. The mechanism of action of this factor includes interference with both the production of interleukin 2 (IL-2) and its effects on target cells. Thus, the suppressor abrogates the proliferation of an IL-2-dependent cytotoxic T cell line (CTLL), but not of IL-2 independent cells like the L929 fibroblasts or the EL4 T lymphoma and U937 histiocytic lymphoma lines. It also suppresses IL-2 production by human peripheral blood enriched T cells and mouse splenocytes. The mediator has a molecular weight of 60,000-70,000 dalton, as determined by gel filtration chromatography, is heat labile, and is sensitive to trypsin, chymotrypsin, and protease.

Animals↗
PubMed1985

Human interleukin 1 mediates cartilage matrix degradation.

Human monocyte factors mediate cartilage matrix degradation by activation of the resident chondrocytes. In the present work, the cartilage matrix-degrading activity of partially purified human monocyte-derived interleukin 1 has been investigated. Human monocyte or blood mononuclear cell culture supernatants were sequentially purified by phenyl-Sepharose and gel filtration chromatography, or by gel filtration chromatography, isoelectric focusing, or chromatofocusing. Column fractions were simultaneously tested by the standard method that defines interleukin 1 activity--costimulation of mouse thymocyte proliferation--and for matrix macromolecule release from living bovine cartilage explants in organ culture. The two activities showed identical profiles in purification steps that would discriminate according to molecular size, hydrophobicity, and net charge. Moreover, the thermal denaturation profiles of the material purified by chromatofocusing could not distinguish between thymocyte proliferating and matrix degrading activities. These results suggest that interleukin 1 which is present in inflammatory synovial fluids may play an important role in the mediation of cartilage damage in chronic inflammatory arthritides.

Animals↗
PubMed1984

Structural analysis of the charge heterogeneity of human interleukin 1.

Human peripheral blood monocytes stimulated with lipopolysaccharide (LPS) produced two major proteins which enhanced the proliferative response of thymocytes to mitogenic stimulation (IL-1 activity). These proteins were of the same molecular weight (about 14,000) but had different ionization properties (pI 5.1 and 6.8). Treatment with neuraminidase did not alter the pI or the activity of either species, indicating that they did not differ solely in their extent of sialyation. Tunicamycin, at levels which markedly inhibited the incorporation of carbohydrate into proteins secreted by LPS-stimulated macrophages, failed to affect the two forms of IL-1. IL-1 also did not bind to concanavalin A-Sepharose. Thus, carbohydrate groups did not appear to play an important role in the structure or activity of the two species of IL-1. However, the biological activity of these two species of IL-1 was dissociated by treatment with N-ethylmaleimide (NEM), iodoacetamide (IAA), and heat. The protein with a pI of 6.8 was significantly inactivated by NEM, IAA, and heat, whereas the IL-1 with a pI of 5.1 was minimally affected. It appears that the two species of IL-1 differ in the availability of reactive groups, which are most probably sulfhydryls, and, consequently, also in tertiary structure.

Animals↗
PubMed1983

Interleukin 1 production by a human acute monocytic leukemia cell line.

Human interleukin 1 (IL-1) was produced under serum-free conditions by stimulating a human monocytic leukemia cell line (THP-1) with silica or lipopolysaccharide (LPS). The IL-1 from THP-1 cells has a molecular weight of 12,000-20,000, consistent with the low-molecular-weight form of IL-1 from human peripheral blood monocytes. Further characterization by isoelectrofocusing showed one major peak of activity at pI 7 for the THP-1 cell-derived IL-1. In contrast, the low-molecular-weight form of IL-1 from human monocytes has two major species, pI 5 and pI 7. This cloned THP-1 cell line produces levels of IL-1 activity comparable to those obtainable from peripheral blood monocytes. Thus THP-1 cells can serve as a valuable source of relatively homogeneous human IL-1 for further purification and molecular characterization of its role in regulating immune functions.

Animals↗
PubMed1983

Two-dimensional gel comparisons of murine H-2D region-associated antigens of different H-2 haplotypes.

The heterogeneity of histocompatibility antigens encoded within the H-2D region of murine chromosome 17 was investigated in several mouse strains using monoclonal antibodies. Sequential immunoprecipitation and two dimensional gel electrophoresis (2D) were used to determine the number and structural relatedness of antigenically distinct products of the same H-2D region. Strikingly different, haplotype-specific patterns were observed. The three antigenically distinct molecules, Dd, Ld and Rd, all encoded within the Dd region, each showed a unique constellation of spots by 2D analysis, although the Ld and Rd molecules showed more structural similarity to each other than either molecule showed to the Dd molecule. Comparisons of the Ld and Rd molecules using sequential precipitations and a cloned Ld gene suggested that they are not conformation or glycosylation variants of the same polypeptide, implying they have different primary amino acid sequences. Using an anti-Ld antibody, the Ldml molecule was isolated from cells of the Dd region mutant mouse strain B10.D2-H-2dml and was found to have a 2D gel profile clearly distinguishable from the profile of either Ld or Dd molecules isolated from the wild-type strain B10.D2. Of the three antigenically-distinct molecules determined by the Dq region, the 2D gel profile of the Rq molecule was different from that of the Dq and Lq, the latter two molecules being indistinguishable. In contrast to these findings, analysis of the gene products of the Db region failed to resolve either antigenic or molecular heterogeneity and the multiple, antigenically distinct gene products of the Dw16 region were indistinguishable by 2D comparisons. These haplotype specific variations in the structural relatedness and number of antigenically distinct molecules determined by D region genes support the hypothesis that class I MHC genes are in a dynamic evolutionary state of gene expansion and contraction.

Animals↗
PubMed1982

A simple method for electrophoretic analysis of cell surface glycoproteins based on concanavalin A binding.

A simple and sensitive method is described for the detection of cell surface proteins. This method consists of reacting concanavalin A (Con A) with 35S-labeled methionine, detergent solubilized whole cell extracts and immunoprecipitating the Con A-protein complexes with anti-Con A antibody bound to Staphylococcus aureus Cowan I strain (SACI). These reagents are commercially available, stable and easy to standardize. Mouse lymphocytes cell surface proteins purified by this procedure show great similarities to those purified with goat anti-cell surface antibodies.

Animals↗
PubMed1982

DNA-mediated gene transfer: recombination between cotransferred DNA sequences and recovery of recombinants in a plasmid.

Ltk- aprt- mouse L cells were transformed to the tk+ phenotype with 10 ng of the herpes simplex virus-1 thymidine kinase (tk) gene and 20 micrograms of pBR322 or simian virus 40 (SV40) DNA. DNAs from five cloned cell lines show restriction endonuclease fragments that hybridize to both tk and pBR322 or SV40 DNA. In all of the cell lines some of these fragments also contain cellular DNA sequences. The use of carrier DNAs with defined sequences has enabled us to demonstrate that the joining of carrier and selectable gene sequences occurs in mouse cells. In one case we have been able to use the ampicillin resistance marker of pBR322 to "rescue" a recombinant plasmid. An analysis of the junction between pBR322 and tk in this plasmid suggests that a small area of homology (16 of 19 base pairs) might be involved in the recombination process.

Animals↗
PubMed1980

Analysis of the heterogeneity of the mouse H-2K, D, and L gene products.

The detection and partial characterization of the major histocompatibility antigens encoded by the three H-2 loci, K, D, and L were performed by analyzing immunoprecipitates from detergent-solubilized radiolabeled cells on two-dimensional gels. Each locus was found to code for a population of proteins heterogeneous in both charge and m.w. Only part of the heterogeneity was shown to be due to sialylation as demonstrated by shifts in mobility after neuraminidase treatment of antigen preparations. The 2-D gel analysis of the H-2K, H-2D, and H-2L antigens of the d haplotype shows that each constitutes a distinct series of products.

Animals↗
PubMedDate not supplied

Purification to homogeneity of biologically active human interleukin 1.

Human interleukin 1 (IL-1) produced by lipopolysaccharide-stimulated monocytes was purified to homogeneity with retention of biological activity. IL-1 was measured by its ability to enhance the proliferative response of thymocytes to phytohemagglutinin. The purification procedure included hydrophobic affinity chromatography on phenyl Sepharose, gel filtration through Ultrogel AcA54 and preparative isoelectric focusing. Both charged species of IL-1, pI 5.1 and 6.8 have a molecular weight of 14,500 as determined by SDS-polyacrylamide gel electrophoresis. The complete purification resulted in a recovery of approximately 0.01% of IL-1 protein and if protection against losses by denaturation and adsorption in the final purification step was provided by bovine serum albumin, approximately 11% of IL-1 activity can be recovered.

Animals↗