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J A Schmidt

Publications and source records attributed to J A Schmidt.

At least 91 records · Page 5Linked to original sources
PubMed1988

Identification of a high-affinity receptor for interleukin 1 alpha and interleukin 1 beta on cultured human rheumatoid synovial cells.

In this report the binding of recombinant human interleukins 1 alpha and 1 beta (rIL-1 alpha and rIL-1 beta) to primary cultures of human rheumatoid synovial cells is measured and compared to the concentrations of these mediators required for stimulation of PGE2 production by these same cells. The average concentration of IL-1 alpha required for half-maximal stimulation of PGE2 was 4.6 +/- 1.5 pM (+/- SEM) (n = 6), whereas for IL-1 beta half-maximal stimulation was observed at a concentration of 1.3 +/- 0.24 pM (n = 6). Both direct and competitive binding experiments were performed. In direct binding experiments, IL-1 alpha bound with a Kd of 66 pM (n = 1), while IL-1 beta bound with a Kd of 4 pM (n = 2). In competitive binding experiments, IL-1 alpha inhibited binding of 125I-IL-1 alpha with a Ki of 33-36 pM (n = 2) and binding of 125I-IL-1 beta with a Ki of 51-63 pM (n = 2). IL-1 beta inhibited binding of 125I-IL-1 alpha with a Ki of 2-3 pM (n = 2) and binding of 125I-IL-1 beta with a Ki of 7 pM (n = 2). The binding data were best fit by a model specifying a single class of receptors with homogeneous affinity for either IL-1 alpha or IL-1 beta and with an abundance of 3,000-14,000 sites per cell. Autoradiography showed that the vast majority of the synoviocytes within the cultures possessed IL-1 receptors. Comparison of biological response curves with the binding curves indicates that the observed receptors exhibit sufficiently high affinity to mediate the response of human synoviocytes to low picomolar concentrations of IL-1 alpha and IL-1 beta.

Arthritis, Rheumatoid↗
PubMed1987

Expression in Escherichia coli of fully active recombinant human IL 1 beta: comparison with native human IL 1 beta.

Although complementary DNA (cDNA) encoding human interleukin 1 beta (IL 1 beta) have been cloned in several laboratories, there are as yet no data demonstrating that recombinant IL 1 beta (rIL 1 beta) molecules expressed from such cDNA are faithful, fully active replicas of the native protein secreted by human monocytes. To this purpose, cDNA sequences corresponding to the exact NH2-terminus and amino acid sequence of mature, monocyte-derived human IL 1 beta were placed under control of the inducible trp-lac (TAC) fusion promoter and were expressed in E. coli strain JM105. rIL 1 beta was purified to homogeneity by high pressure liquid chromatography (HPLC). Yields of 10 to 20 mg of rIL 1 beta/liter/10(11) cells were obtained. Purified rIL 1 beta was then compared in a number of biochemical and biologic assays to purified native IL 1 beta. Native and rIL 1 beta co-migrated on SDS-polyacrylamide gels as 17.5 kd polypeptides and reacted with specific polyclonal antisera raised to three synthetic peptides of human IL 1 beta in immunoblot experiments. Amino acid sequence analysis of rIL 1 beta showed that the native amino terminus, an ALA residue, was faithfully maintained. Purified native and rIL 1 beta co-chromatographed on reverse-phase HPLC. Specific biologic activities of rIL 1 beta were indistinguishable from those of the native protein in murine thymocyte and human dermal fibroblast proliferation assays, with half-maximal stimulation occurring at concentrations of 25 pM in the murine thymocyte assay and 2 pM in the human dermal fibroblast assay. Similarly, native and rIL 1 beta competed equally well for high affinity IL 1 receptor binding sites, each exhibiting a Ki of 20 pM on MRC-5 human embryonic lung fibroblasts. These observations indicate that E. coli efficiently expresses large quantities of rIL 1 beta, which emulates exactly the properties of the native protein.

Amino Acid Sequence↗
PubMed1987

Identification of a high-affinity receptor for native human interleukin 1 beta and interleukin 1 alpha on normal human lung fibroblasts.

Native human IL-1 beta and IL-1 alpha stimulated prostaglandin E2 secretion by human embryonic lung fibroblasts at half-maximal concentrations of 3 +/- 1.2 pM (+/- SEM) and 10 +/- 2.3 pM, respectively. In contrast to the 20-50-fold lower affinities previously found for IL-1-R on 3T3 cells as well as murine and human lymphoblastoid lines, monoiodo 125I-IL-1 beta bound to normal human fibroblasts with a Kd of 8.4 +/- 4.1 pM in direct binding experiments, and with a Ki of 11.2 +/- 2.8 pM in competitive binding experiments. IL-1 alpha bound to the receptor identified by 125I-IL-1 beta with a Ki of 50 +/- 18 pM. The receptor exhibited homogeneous affinity for IL-1 beta or IL-1 alpha. The receptor did not recognize IL-2, IFN-gamma, tumor necrosis factor alpha, a functionally related monokine, or bovine acidic fibroblast growth factor, a structurally related mediator. Comparison of the biological response curves and binding curves obtained for IL-1 alpha and IL-1 beta showed that they were parallel and that 10-15% occupancy of the estimated 3,000 sites by either species of IL-1 was sufficient to give half-maximal stimulation of prostaglandin E2 secretion. Thus, the amount of apparent signal amplification observed on fibroblasts was considerably lower than the 100-100,000 fold amplification previously reported for lymphoid lines. Crosslinking experiments revealed a major band with a corrected molecular mass of approximately 80 kD and a minor band of approximately 200 kD. Labeling of these bands was blocked by IL-1 beta and IL-1 alpha but not by IL-2, IFN-gamma, or tumor necrosis factor alpha. These results demonstrate that normal human embryonic lung fibroblasts bear IL-1-R of sufficiently high affinity to mediate their biological responsiveness to low picomolar concentrations of IL-1 beta and IL-1 alpha and are consistent with the existence of a single receptor mediating the biological properties of both human IL-1 species.

Binding, Competitive↗
PubMed1987

Is there an interleukin 2 inhibitor in human serum?

Normal mouse serum has been shown to contain an inhibitor of interleukin 2 (IL-2). Here we report that a molecule with similar activity cannot be found in normal human serum (NHS). Although NHS inhibited the IL-2-dependent proliferation of mouse CTLL cells, as expected of an IL-2 inhibitor, it also had inhibitory activity on IL-3-dependent cells and was cytolytic to IL-2-independent mouse cells as measured by a 51Cr release assay, indicating a nonspecific effect. In addition, NHS had no effect on the IL-2-dependent proliferation of human peripheral blood T-cell blasts. Fractionation of NHS by size exclusion HPLC failed to separate cytolytic activity from any putative true IL-2 inhibitor activity. The cytolytic component was not related to immunoglobulin since it had a molecular weight of 50,000 to 60,000 and was not bound by protein-A-Sepharose. However, its molecular weight, heat lability, and trypsin sensitivity suggest it to be a protein.

Blood Proteins↗
PubMed1987

Evaluation of alveolar macrophages in normals and individuals with active pulmonary sarcoidosis for the spontaneous expression of the interleukin-1 beta gene.

We have evaluated the hypothesis that the presence of large numbers of activated helper/inducer T lymphocytes in the lungs of individuals with active pulmonary sarcoidosis is associated with the exaggerated release of interleukin-1 (IL-1) by alveolar macrophages. Evaluation of media from unstimulated cultured sarcoid alveolar macrophages failed to detect IL-1 activity. When parallel cultures of sarcoid and normal alveolar macrophages were stimulated with lipopolysaccharide (LPS), they released similar amounts of IL-1 activity. Using a highly specific polyclonal anti-IL-1 beta antibody and flow cytometry to evaluate cell-associated IL-1 beta, analysis of fresh alveolar macrophages from patients with active sarcoidosis and normal individuals revealed no detectable cell-associated IL-1 beta, but IL-1 beta was present when macrophages from sarcoid patients and normals were stimulated with LPS. Similar observations were made using immunoblot analysis of cell lysates of the same unstimulated and stimulated macrophages. Finally, Northern analysis of alveolar macrophages for IL-1 beta mRNA transcripts demonstrated minimal, but equivalent, amounts of IL-1 beta in both normal and sarcoid macrophages, as compared to the much larger quantities present in LPS-stimulated alveolar macrophages. Thus, while alveolar macrophages of individuals with sarcoidosis are clearly capable of expressing the IL-1 beta gene, these findings suggest that altered expression of the IL-1 beta gene by alveolar macrophages does not play a central role in the exaggerated lung T-cell activation characteristic of sarcoidosis.

Gene Expression Regulation↗
PubMed1986

Control of erythroid differentiation: possible role of the transferrin cycle.

A monoclonal antibody to the chicken transferrin receptor (JS-8) blocked temperature-induced and spontaneous differentiation of avian erythroid cells transformed by ts- and wt-retroviral oncogenes. In cells committed to differentiate, JS-8 caused an arrest at the erythroblast or early reticulocyte stage, followed by premature cell death, whereas proliferation of noncommitted erythroid cells or other hematopoietic cells remained unaffected. JS-8 had no effect on transferrin binding or internalization, but blocked subsequent receptor-recycling resulting in reduced iron uptake. Restoration of high intracellular iron levels neutralized the action of JS-8, whereas an inhibitor of porphyrine biosynthesis (4,6-dioxoheptanoic acid) closely mimicked the effect of JS-8. This suggests that erythroid differentiation might involve coordinate synthesis of erythrocyte proteins subject to regulation by hemin or hemoglobin.

Alpharetrovirus↗
PubMed1986

Purification to homogeneity and amino acid sequence analysis of two anionic species of human interleukin 1.

Two anionic species of human IL-1 have been purified to homogeneity. These molecules were characterized as having pI of 5.4 and 5.2 and molecular weights identical to IL-1/6.8 (17,500). The specific activities of IL-1/5.4 and IL-1/5.2, as measured in the mouse thymocyte co-mitogenic assay, were identical to that of IL-1/6.8, namely 1.2 X 10(7) U/mg, with half-maximal stimulation observed at 2 X 10(-11) M. IL-1/5.4 and IL-1/5.2 were found to be antigenically distinct from IL-1/6.8 in an ELISA. IL-1/5.4 was structurally distinct from IL-1/6.8 based on reverse-phase HPLC or CNBr peptides. Intact IL-1/5.2 and three intact CNBr peptides of IL-1/5.4 were sequenced, with the identification of 74 amino acid residues. These sequences were found to correspond exactly with the amino acid sequence deduced from the IL-1-alpha cDNA reported by March et al.

Amino Acid Sequence↗
PubMed1986

Immunocytochemical detection of interleukin 1 within stimulated human monocytes.

We have used synthetic peptides coupled to KLH to raise high titer antisera to human IL-1 beta, and in the present report show the usefulness of these sera for immunocytochemical analyses of IL-1 production. Using indirect immunofluorescence, we have been able to specifically identify IL-1 within human monocytes and to monitor its accumulation with time. After indirect immunofluorescent staining of LPS- and PHA-stimulated mononuclear cell cultures, intense cytoplasmic fluorescence was observed in 93% of the monocytes, but not in lymphocytes or platelets present in the same preparation. Unstimulated monocytes did not contain immunocytochemically detectable IL-1. When put into culture, however, some of the otherwise unstimulated monocytes subsequently showed a transient accumulation of intracellular IL-1. Monocytes cultured in the presence of LPS and PHA exhibited detectable fluorescence after 2.5 h, and the fluorescent intensity of these cells continued to increase over the course of 21 h. Fluorescent staining was abolished by preincubation of the sera with relevant but not irrelevant peptide, and while preimmune or anti-KLH serum produced no staining, antisera against either the amino terminus or an internal region of IL-1 beta produced identical staining patterns. Immunoblot analyses of lysates from stimulated monocytes showed that the antisera against IL-1 recognize a single intracellular species with an apparent molecular weight (33 kD) similar to that predicted for IL-1 precursor from the nucleotide sequence of IL-1 cDNA. The ability to specifically identify and immunocytochemically localize IL-1 within producing cells should prove extremely useful for studying the in situ production of IL-1 in immune-based and inflammatory diseases.

Cell Adhesion↗
PubMed1986

Primitive series embryonic chick erythrocytes express the transferrin receptor.

A monoclonal antibody specific for the chicken transferrin receptor was used to study receptor expression on circulating red cells from chick embryos of different ages. The use of indirect immunofluorescence with this antibody showed that all circulating immature reticulocytes and primitive series erythrocytes--but not erythrocytes from the definitive series--expressed the receptor. In all cells, the protein was synthesized as a 90-95-kD form. The retention of the transferrin receptor (and another proliferation-dependent cell surface protein) contrasted with the behaviour of a series of other developmentally regulated antigens which are lost during maturation of both primitive and definitive series erythroid cells.

Animals↗
PubMed1986

Monoclonal antibodies to novel erythroid differentiation antigens reveal specific effects of oncogenes on the leukaemic cell phenotype.

A panel of new monoclonal antibodies to antigens on the surface of chick erythroid progenitor cells is described. These are characterised with respect to their binding to different classes of normal haemopoietic cells of both the erythroid and myeloid lineages. Using these antibodies, we have examined the phenotype of avian leukaemic cells transformed by retroviruses carrying defined oncogenes. Our data show that these cells, although similar to the normal haematopoietic precursor from which they are derived, aberrantly express certain markers in an oncogene specific manner.

Animals↗
PubMed1986

Antibodies of predetermined specificity to the major charged species of human interleukin 1.

The development of highly specific antisera to human interleukin 1 (IL-1) has been an elusive goal hampered mainly by the availability of only limited amounts of pure immunogen. To surmount this difficulty, three peptides of the major charged species of IL-1 (pI 6.8) were synthesized and covalently coupled to keyhole limpet hemocyanin (KLH). All three peptide-KLH conjugates raised rabbit heterologous antisera that bound intact pure IL-1 in a dose-dependent and domain-specific manner. Immunoblot analysis of crude concentrated culture supernatants with these antisera showed each of them to be highly specific for mature 18-kDa IL-1. Immunoblot analysis of monocyte lysates revealed a single 33-kDa band consistent with the size of the IL-1 precursor molecule deduced from cloned cDNA. These reagents should prove to be valuable tools in the localization and measurement of IL-1 in cells and fluids and may permit the separate study of individual IL-1 species as well as discrete domains of intact IL-1 molecules.

Animals↗
PubMed1986

Specific bioactivities of monocyte-derived interleukin 1 alpha and interleukin 1 beta are similar to each other on cultured murine thymocytes and on cultured human connective tissue cells.

In this report we compare the bioactivities of pure, human monocyte-derived interleukin 1 (IL-1) alpha and beta in the standard murine thymocyte proliferation assay, a human dermal fibroblast proliferation assay, and in an assay measuring stimulation of prostaglandin E2 (PGE2) release from human rheumatoid synoviocytes. In each case the different species of IL-1 produced saturable stimulation and gave similar dose response curves. Half-maximal stimulation was observed at average IL-1 concentrations of 29 pM in the thymocyte assay, 2 pM in the dermal fibroblast proliferation assay, and 5 pM in the synovial cell assay. Our results show that native, monocyte-derived IL-1 alpha and IL-1 beta are both potent stimulators of connective tissue cells and that the specific bioactivities of these molecules are similar to each other in tests on human connective tissue cells, as well as on murine lymphoid cells.

Adult↗
PubMed1986

Testosterone and epitestosterone metabolism of single hairs in 5 patients with 5 alpha-reductase-deficiency.

5 alpha-reductase activity was investigated in scalp hair roots in 5 patients with 5 alpha-reductase deficiency and in 10 normal volunteers (5 men and 5 women) who served as controls. Metabolism of [3H]testosterone and [3H]epitestosterone was measured under standardized conditions in 1-4 growing (anagen) hair roots. When the mean values of the 5 alpha-reduced metabolites from testosterone (androstanedione and dihydrotestosterone) or from epitestosterone (epidihydrotesterone) were compared in both groups, similar values were observed. However, since most of the hair roots were incubated singly, it could be demonstrated that in two patients 20 respectively 24% of the hair roots showed neither dihydrotestosterone nor epidihydrotestestosterone formation. This did not influence the mean values determined for the whole group perceptibly. Our results support the concept that 5 alpha-reductase deficiency is a genetically heterogenous disorder by demonstrating that the enzyme defect is variously expressed in hair roots of affected individuals.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1985

Identification and characterization of the chicken transferrin receptor.

Among a panel of monoclonal antibodies that recognize chicken haematopoietic-differentiation antigens, one, JS 8, was found to immunoprecipitate a 95000-Mr cell-surface protein from chicken erythroblasts transformed with avian-erythroblastosis virus. This protein was shown, by affinity chromatography on immobilized chicken transferrin (conalbumin), to be the chicken transferrin receptor. Although immunologically unrelated to the human transferrin receptor, biochemical comparison of the chicken transferrin receptor to the human receptor showed similarities with respect to the pattern of biosynthesis, degree of glycosylation, dimerization in the absence of reducing agents and subcellular localization. The present report contrasts with recent ones describing the chicken transferrin receptor isolated from embryonic tissues as a 58000-Mr protein.

Animals↗
PubMed1985

Amino acid sequence analysis of human interleukin 1 (IL-1). Evidence for biochemically distinct forms of IL-1.

The pI-6.8 species of normal human interleukin 1 (IL-1) has been isolated by ion-exchange and reverse-phase high-performance liquid chromatography. The isolated material had a molecular weight of 18,000, and had a specific bioactivity of 1.7 X 10(7) half-maximal U/mg in the murine thymocyte proliferation assay, values similar to those obtained for murine P388D1-derived IL-1 (12), and human IL-1 isolated by a previously published purification protocol (15). Amino-terminal sequence analysis revealed a single N-terminal, and resulted in the identification of 30 of the first 35 amino acid residues. Sequence of three CNBr cleavage fragments of purified IL-1 resulted in the identification of an additional 38 residues. All of the sequences agree exactly with those deduced from complementary DNA (cDNA) by Auron, et al. (18), demonstrating that this cloned cDNA, though considerably different from the cDNA reported for murine IL-1 (12), nevertheless codes for the pI-6.8 species of human IL-1. The evidence also shows that the precursor protein for human IL-1 is largely processed at the N-terminal end. Little or no processing occurs at the carboxy-terminal end. Sequence homology with interferon-inducing factor (26) suggests that the pI-6.8 species of human IL-1 is a member of a gene family. Although equally potent in the murine thymocyte proliferation assay, murine IL-1 and the pI-6.8 species of human IL-1 are structurally distinct. Further study will answer the interesting question as to the relationship of the other charged species of human IL-1 to these distinct IL-1 classes.

Amino Acid Sequence↗
PubMed1985

Effects of inhibitors of glycoprotein processing on the synthesis and biological activity of the erb B oncogene.

Three glycoprotein-processing inhibitors were used to resolve whether correct glycosylation was required for the oncogenic activity of erb B. The two glucosidase-I inhibitors, 1-deoxynojirimycin and 2,5-dihydroxymethyl 3,4-dihydroxypyrrolidine, arrested processing of v-erb B at the immature 68-kd form whereas, in the presence of the alpha-mannosidase-II inhibitor (swainsonine), cells synthesised an abnormally processed 70-kd form of v-erb B. Transport of incorrectly processed v-erb B to the cell surface was, however, unaffected, suggesting that correct processing is not a prerequisite for intracellular routing of v-erb B. Two systems were used to assess whether incorrectly processed erb B could maintain the transformed state. The first asked whether inhibitor treatment would release temperature-sensitive avian erythroblastosis virus (AEV) transformed erythroblasts kept at the viral permissive temperature from the erb B-induced block in differentiation, as seen when cells are normally shifted to the non-permissive temperature. The second tested the ability of AEV-transformed fibroblasts to grow in soft agar. In both systems, all three processing inhibitors did not alter the transformed phenotype suggesting that correct carbohydrate processing is not required for the transforming activity of erb B. In addition, none of the three processing inhibitors were found to have any effect on the normal maturation of bone marrow CFU-E or induced differentiation of temperature-sensitive AEV-transformed erythroblasts.

Alkaloids↗