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A S Stern

Publications and source records attributed to A S Stern.

At least 37 records · Page 2Linked to original sources
PubMed1995

Human IL-12 p40 homodimer binds to the IL-12 receptor but does not mediate biologic activity.

IL-12, a heterodimeric cytokine, consists of two disulfide-linked subunits, p40 and p35. We investigated the role of p40 in ligand binding and signal transduction by expressing this subunit alone in COS cells. Culture media of the transfected COS cells exhibited specific dose-dependent binding to KIT225/K6 cells, a human T cell line that expresses IL-12R. Analysis of the culture media by SDS-PAGE and Western blotting demonstrated the presence of 40-kDa monomers and 80-kDa disulfide-linked homodimers. The two p40 species were purified and identified by N-terminal sequencing and proteolytic peptide mapping. Characterization of the p40 proteins for binding and bioactivity showed that both the p40 monomer and dimer inhibited 125I-labeled IL-12 binding to IL-12R, but the 80-kDa species, having a 50% inhibitory concentration (IC50) of 20 to 70 ng/ml, was at least 20-fold more effective than the monomer. Although neither the monomer nor the dimer stimulated human PHA-blast proliferation, the 80-kDa dimer inhibited IL-12-induced proliferation in a dose-dependent manner with an IC50 of 65 ng/ml. The results suggest that the IL-12 p40 subunit contains the essential epitopes for receptor binding. However, a proper conformation required for high affinity binding is achieved only when p40 is associated with a p35 subunit or another p40 subunit. When p40 is associated with a p35 subunit, the heterodimer acts as an agonist mediating biologic activity. However, when p40 associates with another p40, the homodimer behaves as an antagonist in vitro.

Animals↗
PubMed1995

Mouse interleukin-12 (IL-12) p40 homodimer: a potent IL-12 antagonist.

Interleukin-12 (IL-12) is a cytokine that has regulatory effects on T and natural killer (NK) cells and is composed of two disulfide-bonded subunits, p40 and p35. It was recently reported that supernatants from cultures of mouse IL-12 (moIL-12) p40-transfected COS cells could inhibit IL-12-dependent responses in vitro (Mattner, F., et al., Eur. J. Immunol. 1993. 23: 2202). We have further characterized the nature of the inhibitory substance. Purified mouse p40 produced in a baculovirus expression system was found to consist of two species: the p40 monomer and a disulfide-linked p40 dimer [(p40)2]. The (p40)2 was 25- to 50-fold more active than the p40 monomer in causing specific, dose-dependent inhibition of IL-12-induced mouse concanavalin A (Con A) blast proliferation and could also inhibit IL-12-induced interferon-gamma (IFN-gamma) secretion by mouse splenocytes and IL-12-dependent activation of mouse NK cells. Competitive binding studies on mouse Con A blasts showed that (p40)2 was equally effective as moIL-12 in competing with 125I-labeled moIL-12 ([125I]moIL-12) for binding to mouse Con A blasts. However, in contrast to moIL-12, mouse (p40)2 displayed little ability to compete with 125I-labeled human IL-12 (huIL-12) for binding to high-affinity IL-12 receptors (IL-12R) on human phytohemagglutinin (PHA) blasts and caused little or no inhibition of huIL-12-induced human PHA blast proliferation. Nonetheless, mouse (p40)2 was equally effective as moIL-12 in competing with [125I] huIL-12 for binding to COS cells transfected with the human IL-12R beta subunit and expressing low-affinity IL-12 binding sites. These results suggest that (i) the majority of the structural determinants required for binding of IL-12 to its receptor are contained within the p40 subunit, but p35 is required for signaling, (ii) the p40 subunit of IL-12 interacts with the beta subunit of IL-12R, and (iii) (p40)2 may be a suitable IL-12 antagonist for studying the role of IL-12 in various immune responses in vivo as well as in vitro. Further studies are required to determine whether or not (p40)2 is produced by normal lymphoid cells and is a physiologic regulator of IL-12 activity.

Animals↗
PubMed1994

Improved resolution in triple-resonance spectra by nonlinear sampling in the constant-time domain.

Nonlinear sampling along the constant-time dimension is applied to the constant-time HNCO spectrum of the dimerization domain of Gal4. Nonlinear sampling was used for the nitrogen dimension, while the carbon and proton dimensions were sampled linearly. A conventional ct-HNCO spectrum is compared with a nonlinearly sampled spectrum, where the gain in experiment time obtained from nonlinear sampling is used to increase the resolution in the carbonyl dimension. Nonlinearly sampled data are processed by maximum entropy reconstruction. It is shown that the nonlinearly sampled spectrum has a higher resolution, although it was recorded in less time. The constant intensity of the signal in the constant-time dimension allows for a variety of sampling schedules. A schedule of randomly distributed sampling points yields the best results. This general method can be used to significantly increase the quality of heteronuclear constant-time spectra.

Algorithms↗
PubMed1994

Interleukin 12 exerts a differential effect on the maturation of neonatal and adult human CD45R0- CD4 T cells.

It is now recognized that IL-12 plays a predominant role in protective immunity against intracellular pathogens by promoting the development of T helper type 1 (Th1) responses. We here report the unexpected observations that IL-12 exerts differential effects on the maturation of "native" human CD4 T cells isolated from umbilical cord blood or from the blood of healthy adults. After priming in the presence of IL-12, naive cells of adult donors, defined as CD45R0- CD4+ T cells, acquire a Th1 phenotype whereas neonatal cells develop into effector cells producing high levels of IL-4 in addition to IFN-gamma. This effect of IL-12 on neonatal T cells is direct inasmuch as it is observed on highly purified CD4 T cells, however, it is not inhibited by CD8 T cells and natural killer cells. Unstimulated neonatal T cells which have been preincubated with IL-12 before the priming behave like adult T cells and acquire a Th1 phenotype after stimulation in the presence of IL-12. Given that IL-4 is a potent antagonist of Th1 responses, the finding that IL-12 promotes the maturation of neonatal T cells into IL-4 producers may explain the increased susceptibility of neonates to intracellular pathogens and should be taken into account for the development of vaccines to be used in the perinatal period.

Adult↗
PubMed1993

Application of nonlinear sampling schemes to COSY-type spectra.

Nonlinear sampling along the t1 dimension is applied to COSY-type spectra. The sine dependence of the time domain signals for the cross peaks is matched by a nonlinear sampling scheme that samples most densely around the maximum of the sine function. Data are processed by maximum entropy reconstruction, using a modified implementation of the 'Cambridge' algorithm of Skilling and Bryan. The procedure is demonstrated for P.E. COSY spectra recorded on a cyclic hexapeptide and on a 126-residue domain of the protein villin. The number of t1 values in the nonlinearly sampled experiments was reduced by a factor of four compared to linear sampling. The sensitivity and resolution of the resulting spectra are comparable to those achieved by conventional methods. The method described can thus significantly reduce the measuring time for COSY-type spectra.

Algorithms↗
PubMed1992

IL-12 receptor. I. Characterization of the receptor on phytohemagglutinin-activated human lymphoblasts.

IL-12 is a 75-kDa heterodimeric cytokine composed of disulfide-bonded 35-kDa and 40-kDa subunits. Included among the biologic activities mediated by IL-12 is induction of proliferation of PHA-activated human PBL. The concentration of IL-12 required to stimulate maximum proliferation of PHA-activated lymphoblasts is 50 to 100 pM. In this study, highly purified 125I-labeled IL-12 (7 to 15 microCi/microgram; 50 to 100% bioactive) was used to characterize the receptor for IL-12 on 4-day PHA-activated lymphoblasts. The binding of 125I-labeled IL-12 to PHA-activated lymphoblasts was saturable and specific because the binding of radiolabeled ligand was only inhibited by IL-12 and not by other cytokines. The kinetics of [125I]IL-12 binding to PHA-activated lymphoblasts was rapid at both 4 degrees C and 22 degrees C; reaching equilibrium within 60 min. At 22 degrees C, the rate of dissociation of [125I]IL-12 was slow in the absence of competing IL-12 (t1/2 = 5.9 h) and more rapid in the presence of 25 nM competing IL-12 (t1/2 = 2.5 h). The kinetically derived equilibrium dissociation constant ranged from 10 to 83 pM. Analysis of steady state binding data by the method of Scatchard identified a single binding site with an apparent equilibrium dissociation constant of 100 to 600 pM and 1000 to 9000 sites/lymphoblast. The equilibrium dissociation constant for competing ligands and sites per cell calculated from unlabeled IL-12 competition experiments ranged from 164 to 315 pM and 1067 to 3336, respectively, which is in good agreement with the values determined from steady state binding. The variations in KD and sites per cell were dependent on the individual preparations of lymphoblasts. Although the steady state binding data were consistent with a single class of high affinity binding sites, the kinetic dissociation data indicates a cooperative interaction between receptors on PHA-activated lymphoblasts. Affinity cross-linking of surface bound [125I]IL-12 to PHA-activated lymphoblasts at 4 degrees C identified a major complex of approximately 210 to 280 kDa. Anti-IL-12 antibodies also immunoprecipitated a complex of approximately 210 to 280 kDa that was produced by cross-linking unlabeled IL-12 to 125I-labeled lymphoblast cell-surface proteins. Cleavage of this complex with reducing agent identified one radiolabeled protein of approximately 110 kDa. These data suggest that the IL-12 binding site on PHA-activated lymphoblasts may be composed of a single protein of approximately 110 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)

Cells, Cultured↗
PubMed1992

Molecular characterization of interleukin 12.

Interleukin 12 (IL-12), formerly known as cytotoxic lymphocyte maturation factor and natural killer cell stimulatory factor, is a cytokine secreted by a human B lymphoblastoid (NC-37) cell line when induced in culture with phorbol ester and calcium ionophore. This factor has been purified to homogeneity and shown to synergize with low concentrations of interleukin 2 in causing the induction of lymphokine-activated killer cells. In addition, purified IL-12 stimulated the proliferation of human phytohemagglutinin-activated lymphoblasts by itself and exerted additive effects when used in combination with suboptimal amounts of interleukin 2. The protein is a heterodimer composed of a 40- and a 35-kDa subunit. Amino acid sequence analysis confirmed predicted sequences from the cloned cDNAs of each subunit. Chemical and enzymatic deglycosylation of the heterodimer demonstrated that the 40- and 35-kDa subunits contain 10 and 20% carbohydrate, respectively. Structural analysis of IL-12 using site-specific chemical modification revealed that intact disulfide bonds are essential for bioactivity. The 40-kDa subunit of IL-12 was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by immunoblotting as being present in NC-37 cell supernatant solutions in relatively large amounts uncomplexed to the 35-kDa subunit. Previously it had been shown that the 40-kDa subunit alone does not cause the proliferation of activated human T lymphocytes or enhance the cytolytic activity of human natural killer cells. However, results obtained by site-specific chemical modification suggesting that a tryptophan residue is at or near the active site of IL-12 may imply a direct role of the subunit in interacting with the IL-12 receptor. These data may support the recent proposal (D.P. Gearing and D. Cosman (1991) Cell 66, 9-10) that IL-12 consists of a complex of cytokine and soluble receptor.

Amino Acid Sequence↗
PubMed1991

The role of arginine residues in interleukin 1 receptor binding.

Interleukin 1 (IL-1) is a family of polypeptide cytokines that plays an essential role in modulating immune and inflammatory responses. IL-1 activity is mediated by either of two distinct proteins, IL-1 alpha or IL-1 beta, both of which bind to the same receptor found on T-lymphocytes, fibroblasts and endothelial cells (Type 1 receptor). The effect of specific chemical modification of recombinant IL-1 alpha and IL-1 beta on receptor binding was examined. Modification of the proteins with phenylglyoxal, an arginine-specific reagent, resulted in the loss of Type 1 IL-1 receptor binding activity. The stoichiometry of this modification revealed that a single arginine in either IL-1 alpha or IL-1 beta is responsible for the loss of activity. Cyanogen bromide cleavage of phenylglyoxal modified IL-1 alpha and IL-1 beta, followed by sequencing of the peptides, revealed that arginine-12 in IL-1 alpha and arginine-4 in IL-1 beta, which occupy the same topology in the respective crystallographic structures, are the target of phenylglyoxal. These results suggest that an arginine residue plays an important role in ligand-receptor interaction.

Amino Acid Sequence↗
PubMed1991

IL-12: monoclonal antibodies specific for the 40-kDa subunit block receptor binding and biologic activity on activated human lymphoblasts.

IL-12, formerly known as cytotoxic lymphocyte maturation factor, is a cytokine that stimulates proliferation of PHA-activated human peripheral blood lymphoblasts and synergizes with low concentrations of IL-2 in the induction of lymphokine-activated killer cells. IL-12 is a 75-kDa heterodimer composed of disulfide-bonded 40-kDa and 35-kDa subunits. mAb prepared against a partially purified preparation of natural IL-12 have been characterized by 1) immunoprecipitation of 125I-labeled IL-12, 2) immunodepletion of IL-12 bioactivity, 3) Western blotting of IL-12, 4) inhibition of [125I]IL-12 binding to its cellular receptor, and 5) neutralization of IL-12 bioactivity. Twenty antibodies immunoprecipitate 125I-labeled IL-12 and immunodeplete IL-12 bioactivity as assessed in the T cell proliferation and lymphokine-activated killer cell induction assays. Western blot analysis demonstrated that each antibody binds to the 75-kDa heterodimer and to the 40-kDa subunit. An IL-12R-binding assay identified 12 individual antibodies that inhibited the binding of [125I]IL-12 to its cellular receptor. Two inhibitory antibodies, 4A1 and 7B2, were tested in the neutralization assay and found to block IL-12 bioactivity whereas one noninhibitory antibody, 8E3, was shown not to neutralize IL-12 bioactivity. Antibodies 4A1 and 8E3 can simultaneously bind to the 75-kDa heterodimer demonstrating that inhibitory and noninhibitory epitopes are spatially distinct on the 40-kDa protein. The ability of antibodies specific for the 40-kDa subunit of IL-12 to block receptor binding of [125I]IL-12 and to neutralize IL-12 bioactivity suggests that localized determinants on the 40-kDa subunit may be necessary for binding to the IL-12 cellular receptor.

Animals↗
PubMed1990

Measurement of biologically active interleukin-1 by a soluble receptor binding assay.

A soluble receptor binding assay has been developed for measuring human interleukin-1 alpha (IL-1 alpha), human IL-1 beta, and mouse IL-1 alpha. The assay is based on a competition between unlabeled IL-1 and 125I-labeled mouse recombinant IL-1 alpha for binding to soluble IL-1 receptor prepared from mouse EL-4 cells. The assay measures only biologically active IL-1 folded in its native conformation. The ratio of human IL-1 alpha to human IL-1 beta can be measured in the same sample by a pretreatment step which removes human IL-1 beta from samples prior to assay. This technique has been used to monitor the purification of recombinant IL-1, and may be utilized to specifically and accurately measure bioactive IL-1 in human serum and cell culture supernatants.

Animals↗
PubMed1990

Does the maximum entropy method improve sensitivity?

Maximum entropy reconstruction has been used in several fields to produce visually striking reconstructions of positive objects (images, densities, spectra) from noisy, indirect measurements. In magnetic resonance spectroscopy, this technique is notable for its apparent noise suppression and its avoidance of the artifacts that affect discrete Fourier transform spectra of short (zero-extended) data records. In the general case where the length of the reconstructed spectrum exceeds that of the data record or where a convolution kernel is incorporated in the reconstruction, no known analytical solution to the reconstruction problem exists. Consequently, knowledge of the properties of maximum entropy reconstruction has been mainly anecdotal, based on a small selection of published reconstructions. However, in the limiting case where the lengths of the reconstructed spectrum and the data record are the same and a convolution kernel is not applied, the problem can be solved analytically. The solution has a simple structure that helps explain several commonly observed features of maximum entropy reconstructions--for example, the biases in the recovered intensities and the fact that noise near the baseline is more successfully suppressed than is noise superimposed on broad features in the spectrum. The solution also shows that the noise suppression offered by maximum entropy reconstruction could (in this special case) be equally well obtained by a "cosmetic" device: simply displaying the conventional Fourier transform reconstruction using a certain nonlinear plotting scale for the vertical (y) coordinate.

Journal Article↗
PubMed1990

Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells.

A cytokine that can synergize with interleukin 2 to activate cytotoxic lymphocytes was purified to homogeneity. The protein, provisionally called cytotoxic lymphocyte maturation factor (CLMF), was isolated from a human B-lymphoblastoid cell line that was induced to secrete lymphokines by culture with phorbol ester and calcium ionophore. The purification method, utilizing classical and high-performance liquid chromatographic techniques, yielded protein with a specific activity of 8.5 x 10(7) units/mg in a T-cell growth factor assay. Analysis of the purified protein by sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated that CLMF is a 75-kDa heterodimer composed of disulfide-bonded 40-kDa and 35-kDa subunits. Determination of the N-terminal amino acid sequences of the two subunits revealed that both subunits are not related to any previously identified cytokine. Purified CLMF stimulated the proliferation of human phytohemagglutinin-activated lymphoblasts by itself and exerted additive effects when used in combination with suboptimal amounts of interleukin 2. Furthermore, the purified protein was shown to synergize with low concentrations of interleukin 2 in causing the induction of lymphokine-activated killer cells.

Amino Acid Sequence↗
PubMed1989

Purification of homogeneity and amino acid sequence analysis of a receptor protein for interleukin 1.

The interleukin 1 (IL-1) receptor from mouse EL-4 thymoma cells was purified to homogeneity by a method which utilized ligand affinity chromatography and classical chromatographic techniques. After solubilization of the receptor from intact cells with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the IL-1 binding activity was purified greater than 23,000-fold. Analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot, and ligand blot demonstrated that a single protein of molecular mass of approximately 80 kDa is the IL-1 binding polypeptide. The purified protein bound IL-1 with a dissociation constant of approximately 1.1 X 10(-10) M, which is indistinguishable from the affinity of the cell-bound receptor. The amino acid composition of this protein is strikingly similar to the composition deduced from the sequence of a cDNA coding for an IL-1 receptor from EL-4 cells. Protein sequence analysis of Staphylococcus aureus V-8 protease-derived peptides yields data consistent with the sequence proposed from cloned cDNA. These studies have demonstrated that the high affinity IL-1 receptor on EL-4 cells is the 80-kDa protein.

Amino Acids↗
PubMed1989

Two high-affinity interleukin 1 receptors represent separate gene products.

Interleukin 1 (IL-1) is a polypeptide hormone that mediates a broad range of biological activities and interacts with surface receptors on numerous cell types. Equilibrium binding studies have identified a class of IL-1 receptors on T cells, fibroblasts, and epithelial cells that have 2- to 5-fold higher affinity than the receptors on bone marrow cells, pre-B cells, and macrophage cell lines. Affinity cross-linking with human 125I-labeled IL-1 alpha (125I-IL-1 alpha) labels an approximately 100-kDa protein on T cells and fibroblasts and an approximately 80-kDa protein on pre-B cells and macrophage cell lines. Monoclonal and polyclonal antibodies specific for the IL-1 receptor on T cells and fibroblasts block human 125I-IL-1 alpha binding to T cells, fibroblasts, and epithelial cells but cannot block IL-1 binding to bone marrow cells, pre-B cells, and macrophages. These antibodies immunoprecipitate the IL-1 receptor-human 125I-IL-1 alpha complex from T cells and fibroblasts but not from pre-B cells and macrophage cell lines. An S1 nuclease protection assay demonstrated that T cells and fibroblasts contain identical IL-1 receptor mRNA but that pre-B cells and macrophages do not contain this receptor mRNA. Taken together, the data demonstrate that mouse T cells, fibroblasts, and epithelial cells express an identical IL-1 receptor, whereas the IL-1 receptor on pre-B cells, macrophages, and bone marrow cells represents a different gene product.

Animals↗
PubMed1988

Expression of porcine cholecystokinin cDNA in a murine neuroendocrine cell line. Proteolytic processing, sulfation, and regulated secretion of cholecystokinin peptides.

The cDNA for porcine preprocholecystokinin (pre-pro-CCK) was engineered for expression in mammalian cells under the control of the Rous sarcoma virus-long terminal repeat promoter. This expression construct was transfected into the murine anterior pituitary cell line, AtT-20. A stable cell line (AtT-20/CCK) was derived that expresses CCK mRNA indistinguishable from the CCK mRNA found in pig brain or gut. The AtT-20/CCK cells carry out proteolytic processing and sulfation reactions to generate authentic sulfated CCK8 from pro-CCK. The cells also store and secrete CCK-immunoreactive peptides. This secretion can be stimulated with corticotropin releasing factor, the natural secretagogue for anterior pituitary cells. In contrast, monkey kidney epithelial cells (COS cells), which are transiently transfected to express CCK, predominantly secrete nonsulfated pro-CCK into the medium. These studies show that a murine neuroendocrine cell line contains the complete processing machinery required to generate authentic porcine CCK8. The processing events include simultaneous proteolytic processing at one and two basic amino acid sites and sulfation of tyrosine residues. The cell line thus duplicates exactly the processing patterns found to occur in pig brain cortex.

Amino Acid Sequence↗
PubMed1987

Structural characterization of human interferon gamma. Heterogeneity of the carboxyl terminus.

Natural human interferon gamma(IFN-gamma) was purified from the conditioned medium of peripheral blood leukocytes using selective silica gel adsorption and antibody-affinity chromatography. SDS-PAGE and Western blot analysis demonstrated three major species with molecular masses of 25 kDa, 20 kDa and 17 kDa. Structural analysis of this natural IFN-gamma preparation demonstrated a pyroglutamate residue at the amino terminus and a heterogeneous carboxyl terminus. The longest and most predominant polypeptide was 138 amino acids in length, which is five residues shorter than the sequence predicted from the cDNA. The presence of multiple-carboxyl-terminal forms indicated possible proteolytic processing during induction or protein purification. Limited proteolytic digestion of full-length recombinant IFN-gamma with endoproteinase Lys-C and trypsin revealed that the carboxyl-terminal 15 residues could be released under conditions in which the core portion of the polypeptide chain remained intact. Thus, the heterogeneity of natural IFN-gamma may be explained by partial proteolytic degradation of the molecule and differences in the degree of glycosylation as previously reported [Rinderknecht, E., O'Conner, B. H. & Rodriguez, H. (1984) J. Biol. Chem. 259, 6790-6797].

Chromatography, Affinity↗
PubMed1987

Detergent solubilization of the interleukin 1 receptor.

Interleukin 1 (IL 1) receptors were solubilized from membranes prepared from murine EL-4 thymoma cells with the zwitterionic detergent 3[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). Binding of IL 1 to the solubilized receptor was detected by a polyethylene glycol (PEG) precipitation procedure. Concentrations of CHAPS from 4 to 8 mM were effective in solubilizing the IL 1 receptor. At 10 mM CHAPS, there was some loss in binding activity, whereas 2 mM CHAPS was completely ineffective in solubilizing the receptor. Detergent concentrations of 4 mM were routinely used. The solubilized receptor retains the ability to bind 125I-IL 1 in a specific and saturable manner. Scatchard analysis reveals a single type of high affinity binding site having an apparent dissociation constant (KD) of approximately 1.2 X 10(-10) M. Nearly identical KD values are observed for membrane fractions. There are approximately 400 to 500 fmol receptor/mg protein in the detergent extract, corresponding to a two- to threefold enrichment in the Bmax observed for membranes. There is no loss in receptor activity as determined by complete recovery of the total number of binding sites from membranes after solubilization. Binding kinetics show that apparent steady state for the solubilized receptor is reached after 60 min at 37 degrees C. The binding of 125I-IL 1 is essentially irreversible because relatively little bound ligand can be dissociated from the receptor on the addition of excess unlabeled IL 1 at 37 degrees C. Both human IL 1 alpha and IL 1 beta compete for binding of 125I-IL 1 to the soluble receptor, confirming that IL 1 alpha and IL 1 beta bind to the same receptor. Other recombinant proteins, including interferon-alpha A, interferon-gamma, and interleukin 2 have no inhibitory effect.

Animals↗