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BACKGROUND: Phosphoenolpyruvate carboxykinase (PEPCK) is a metabolic enzyme in the gluconeogenesis pathway, where it catalyzes the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and CO2. The substrates for Escherichia coli PEPCK are OAA and MgATP, with Mn2+ acting as a cofactor. Analysis of PEPCK structures have revealed amino acid residues involved in substrate/cofactor coordination during catalysis. METHODS: Key residues involved in coordinating the different substrates and cofactor bound to E. coli PEPCK were mutated. Purified mutant enzymes were used for kinetic assays. The structure of some mutant enzymes were determined using X-ray crystallography. RESULTS: Mutation of residues D269 and H232, which comprise part of the coordination sphere of Mn2+, reduced kcat by 14-fold, and significantly increased the Km values for Mn2+ and OAA. Mutation of K254 a key residue in the P-loop motif that interacts with MgATP, significantly elevated the Km value for MgATP and reduced kcat. R65 and R333 are key residues that interacts with OAA. The R65Q and R333Q mutations significantly increased the Km value for OAA and reduced kcat respectively. CONCLUSIONS: Our results show that mutation of residues involved in coordinating OAA, MgATP and Mn2+ significantly reduce PEPCK activity. K254 plays an important role in phosphoryl transfer, while R333 is involved in both OAA decarboxylation and phosphoryl transfer by E. coli PEPCK. GENERAL SIGNIFICANCE: In higher organisms including humans, PEPCK helps to regulate blood glucose levels, hence PEPCK is a potential drug target for patients with non-insulin dependent diabetes mellitus.

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Elevated levels of p56-Lck kinase activity were achieved in an interleukin 2 (IL-2)-dependent cloned cytolytic T cell CTLL-2 through gene transfer approaches. CTLL-2-Lck cells remained dependent on IL-2 for growth and survival in culture but exhibited markedly elevated, IL-2-independent cytolytic activity against a variety of tumor targets. This immune cell effector function was similar to the non-major histocompatibility complex-restricted cytolytic activity previously described for lymphokine activated killer (LAK) cells, and involved a cytolytic mechanism that was independent of protein synthesis in either the T cells or the tumor targets. Characterization of CTLL-2-Lck cells revealed markedly elevated levels of both the alpha (CD11a) and beta (CD18) chains of the cell adhesion molecule lymphocyte function-associated 1 (LFA-1) and increased binding of these T cells to a recombinant protein representing the extracellular domain of the LFA-ligand, intercellular adhesion molecule 1 (ICAM-1). Antibodies to CD11a partially abrogated cytolytic killing of tumor target cells by CTLL-2-Lck cells, suggesting that the upregulation in LFA protein levels potentially accounts at least in part for the phenotype of these T cells. Gene transfer-mediated elevations in p56-Lck kinase activity in an IL-3-dependent myeloid cell clone 32D.3 also resulted in increased LFA-1 expression, demonstrating that the findings are not unique to CTLL-2 cells. In addition to upregulation of LFA-1 expression, CTLL-Lck cells also exhibited more efficient exocytosis of cytotoxic granules upon activation with Ca(2+)-ionophore and phorbol ester, relative to control transfected and untransfected CTLL-2 cells. The findings functionally link the Lck kinase to a T cell effector pathway involved in cell-mediated cytotoxicity.

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We determined the expression of intercellular adhesion molecules (ICAM) on neuro-2a cells in order to evaluate whether they were involved in cytolysis of murine neuroblastoma. Fluorescence-activated cell sorting analysis revealed that the control neomycin-resistance-gene-transduced line (neuro-2a/LN) had poor expression of ICAM-1 (mean channel fluorescence, MCF = 3.7). An ICAM-1-positive transfectant of neuro-2a (neuro-2a/ICAM-1+) (MCF = 64.3) was generated to evaluate directly the role of this adhesion molecule in cytolysis. Neuro-2a/ICAM-1+ was more sensitive to LAK killing (69.7% at an effector-to-target ratio of 100:1) compared to neuro-2a/LN (48.6%) (P < 0.001). Blocking of neuro-2a/LN and neuro-2a/ICAM-1+ lysis with anti-ICAM-1 monoclonal antibodies (mAbs) did not account for all the LFA-1-dependent killing. These data indicate that even in neuro-2a/ICAM-1+ cells, other LFA-1 ligands participated in the effector-target interaction. Therefore, we examined these cell lines for ICAM-2 expression. Both neuro-2a/LN and neuro-2a/ICAM-1+ lines expressed ICAM-2 (MCF = 16.4 and 16.5). ICAM-2 accounted for the majority of the LFA-1-dependent killing in the ICAM-1-negative target, neuro-2a/LN, while ICAM-1 played a primary role in the cytolysis of the ICAM-1+ transfectant. Inhibition of lysis in the presence of anti-ICAM-1 and ICAM-2 mAbs was comparable to that seen with the addition of anti-LFA-1 mAb, indicating that other LFA-1 ligands were not involved in this system. ICAM-1 expression was associated with decreased in vivo tumorigenicity, mice inoculated with neuro-2a/ICAM-1+ cells had a significantly longer survival compared to those receiving neuro-2a/LN cells (median survival time 35.5 versus 24.5 days) (P < 0.001). It is important to note that ICAM-1 transfection of murine neuroblastoma did not alter its metastatic potential. We conclude that transfection of mouse neuroblastoma with ICAM-1 increases its sensitivity to in vitro lysis and reduces its in vivo tumorigenicity. In ICAM-1-negative murine neuroblastoma cells, ICAM-2 plays a primary role in cell-mediated lysis.

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A rapid induction of adhesion to immobilized intercellular adhesion molecule (ICAM)-1 occurs when cytotoxic T lymphocytes (CTL) are stimulated with either soluble anti-T cell receptor (TCR) monoclonal antibodies (mAb) or with immobilized alloantigen, and this binding is blocked by the addition of anti-lymphocyte function-associated (LFA)-1 mAbs. Requirements for activating LFA-1 adhesion to ICAM-1 are similar to those found for induction of binding to immobilized fibronectin (FN), but distinct from those for activating CD8-mediated adhesion to class I major histocompatibility complex. A distinct role for LFA-1 in co-signaling for TCR-dependent degranulation could not be demonstrated. In contrast, both CD8 and the FN-binding integrin provide costimulatory signals for this response. Thus, if co-signaling via LFA-1 occurs, it clearly differs from that provided by CD8 or the FN-binding integrin. On the basis of antibody blocking effects, alloantigen-dependent activation of adhesion to ICAM-1 involves both the TCR and CD8. These results support a view of CTL activation as a cascade of adhesion and signaling events, with different coreceptors making distinct contributions.

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Lymphocytes activated by antigen receptor cross-linking or phorbol esters adhere avidly to surfaces bearing intercellular adhesion molecule 1 (ICAM-1) through the adhesion receptor lymphocyte function-associated antigen 1 (LFA-1). It is not known whether avid adhesion by stimulated lymphocytes is due to higher affinity binding of ICAM-1 or due solely to post-receptor mechanisms. We have used a recombinant, soluble form of the ICAM-1 molecule to measure the affinity of binding to LFA-1 on unstimulated T cells and T cells stimulated with phorbol esters. The affinity was found to be too low for direct measurements, requiring instead the use of competition protocols in which ICAM-1 competes for binding with radiolabeled Fab from a monoclonal antibody specific for LFA-1. By analysis of the equilibrium and kinetics of competitive binding, we found that the affinity on unstimulated T cells is very low, about 100 microM. Activation of the T cells by phorbol esters caused a small increase in average binding affinity. Further analysis suggested that the change in average affinity reflected the conversion of a fraction of LFA-1 molecules to a state with a 200-fold higher affinity.

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Leukocyte traffic in immune-inflammatory responses requires regulated adhesion of leukocyte subsets to vascular endothelium. We show that fibrinogen or normal human plasma enhances by 2- to 5-fold the adhesion of cells of myeloid and lymphoid lineage to endothelium. This mechanism is mediated by fibrinogen binding to complementary membrane receptors on leukocytes and endothelial cells. Using an affinity chromatography purification strategy, genetically engineered transfectants, and direct binding studies to the isolated recombinant protein, we identified a novel hematopoietic fibrinogen receptor participating in this adhesion pathway as intercellular adhesion molecule 1 (ICAM-1). Accordingly, a new model can be proposed, in which fibrinogen binding to a variety of vascular cell receptors mediates a specific pathway of cell to cell adhesion by bridging together leukocytes and endothelial cells.

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To study the signaling role of CD11a/CD18 in the early events of T cell activation we have examined the induction of transcription of two important cytokines, namely TNF alpha and IL-2. Human peripheral blood T cells were stimulated with PMA/ionophore or immobilized anti-CD3 mAb (OKT3) with or without CD11a/CD18 engagement. Induced cytokine production by immobilized OKT3 was enhanced (3- to 10-fold) in cells adhering to OKT3 and ICAM-1 coimmobilized surfaces and anti-CD11a mAb abolished this enhancement effect. Similarly, inhibition of the PMA/ionophore-induced CD11a/CD18-mediated homotypic aggregations of T cells by mAbs specific for either CD11a or ICAM-1 reduced the induced cytokine production by more than 70%. We have also observed that greatly enhanced cytokine production resulted from cellular interactions between activated T cells and monolayers of endothelial cells. This enhancement was inhibited by a combination of CD11a-, CD18-, and ICAM-1-specific mAbs implicating a role of CD11a/CD18 in leukocyte adhesion to endothelium and diapedesis as part of the inflammatory process. By Northern analyses induced TNF alpha mRNA expression was significantly enhanced by the engagement of CD11a/CD18 in all the conditions mentioned above. These results, together with our previous studies on monocytes, lead to the conclusion that engagement of the CD11/CD18 family of receptors results in the transduction of cellular signals that quantitatively enhance the expression of important leukocyte-mediated immune and inflammatory responses.

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The synthesis of two protein reactive (aminostyryl)pyridinium fluorescent dyes, N-ethyl-N-[4-[2-[4-(1-methylpyridinio)]ethyl]phenyl]glycine chloride N-hydroxysuccinimide ester (SuASP, 2) and 4-[4-[2-[4-(N,N- dimethylamino)phenyl]ethenyl]pyridinio]butyrate N-hydroxysuccinimide ester (ASPSu, 3), is reported. Both form amide linkage through the activated succinimidyl ester with primary amines. The two analogues differ by the position of the pyridinium positive change relative to the activated ester. SuASP forms an amide linkage that positions the positive charge distal to the protein surface, while ASPSu places the positive charge proximal. The synthesis of SuASP utilizes a palladium coupling reaction for the arylation of 4-vinylpyridine, while the major connection for ASPSu is accomplished through an aldol condensation between 4-(N,N-dimethylamino)benzaldehyde and picoline. Both reagents are shown to label covalently bovine serum albumin.

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Little is known in quantitative terms about forces between cells generated during adhesion and recognition, or about the contribution of any one set of molecular associations to the development of these forces. To determine the forces involved in adhesion dependent on lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1), we have measured the junctional avidity between single cell pairs consisting of a cloned T cell that expresses LFA-1 and a fibroblast cell that expresses MHC class II molecules and ICAM-1 after transfection. Micromanipulation was used to induce conjugation of cell pairs and to determine the force required to separate the conjugate. T cell adhesion to three related fibroblast cell lines was compared: the parent line that does not express ICAM-1 or other LFA-1 counter-receptors, and two transfectants that have high and moderate levels of surface ICAM-1 expression. The force needed to separate the conjugates varied with the fibroblast ICAM-1 expression levels. The T cell adhesion to ICAM-1-expressing fibroblasts was strong, and the critical separation stresses measured for the three cell lines were 1.4 x 10(3) dyn/cm2 (1 dyn=10(-5) N) for the ICAM-1-negative fibroblast, 4.98 x 10(3) dyn/cm2 for the fibroblast with a moderate level of ICAM-1 expression, and 6.25 x 10(3) dyn/cm2 for the fibroblast line with the highest ICAM-1 expression. The dependence of adhesion strength on the LFA-1/ICAM-1 complex was confirmed by the use of blocking antibodies, which showed the contribution from the interaction of CD4/MHC class II to be negligible.

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Immunoprecipitation of biosynthetically labelled interleukin-4 (IL-4) secreted by activated D10 cells yielded three bands after separation under non-reducing conditions by electrophoresis through gradient SDS polyacrylamide gels. This triplet consists of a closely spaced doublet at 23 and 22 kDa (23/22 kDa), and a third band at 18.5 kDa (19 kDa). The 23/22 kDa doublet was converted to the 19 kDa form by enzymatic removal of the N-linked sugars, indicating that the two glycoforms were derived from the 19-kDa core polypeptide. Under reducing conditions, the 19-kDa polypeptide migrated at 14.5 kDa, consistent with the size predicted from the complementary DNA (cDNA). Under non-reducing conditions, IL-4 retained biological activity after electrophoresis and transfer to nitrocellulose. Applying a biological assay, proliferation of the NK cell line, to fractionated nitrocellulose replicas, we found that IL-4 activity was detected over a relatively broad range of molecular weights, reflecting the multiple bands found by immunoprecipitation. This was true not only for IL-4 produced by D10 cells but also for splenic cells activated in vitro. All of the immunoprecipitated IL-4 species were active in inducing proliferation of the NK cell line. However, when the D10 cell line was used to detect IL-4, the 19-kDa species was significantly more active than the higher molecular weight species. These results suggest that different forms of IL-4 may have different functional properties.

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To examine the role of lymphocyte function-associated antigen 1 (LFA-1) expression on murine B cells as it pertains to their function in T cell activation, we carried out antigen-presentation assays in tissue culture wells coated with a purified, secreted form of the murine intercellular adhesion molecule 1 (ICAM-1). We observed a significant decrease in the concentration of antigen required to activate a T cell hybridoma and primary T cells in wells coated with ICAM-1. This effect was dependent on the amount of ICAM-1 used to coat the wells and was also observed in wells coated with anti-LFA-1-monoclonal antibodies and was blocked by soluble anti-LFA-1 antibodies. The effect on antigen dose was most pronounced in assays carried out with an ICAM-1-deficient mutant B lymphoma cell line, small resting primary B cells, and unfractionated primary B cells at low concentrations. No decrease in the antigen dose was observed if the B cells were chemically fixed or treated with ricin, or when antigen was presented by a HeLa cell line transfected with murine class II major histocompatibility complex (MHC) genes, indicating that the immobilized ICAM-1 was mediating its effect through B cell LFA-1, and that B cell protein synthesis was required. The enhancing effect was also observed if the B cells were prepulsed with antigen, indicating that improved uptake or processing of antigen, or increased class II MHC expression were unlikely mechanisms.

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These studies demonstrate that the murine intercellular adhesion molecule-1 (ICAM-1) performs at least two roles in enhancing T cell activation. These two roles are evident in both of our experimental systems: with ICAM-1 expressed on the surface of transfected fibroblast cells, and with purified ICAM-1 immobilized on plastic. First, as has been documented by many investigators, ICAM-1 mediates adhesion between ICAM-1- and lymphocyte function-associated Ag-1 (LFA-1)-bearing cells. This adhesive interaction occurs even in the absence of T cell stimulation, although it is increased by addition of phorbol ester and calcium ionophore. Although ICAM-1 expression does markedly increase intercellular adhesion, the increase is significantly less than the improvement ICAM-1 expression makes in the Ag-presenting ability of MHC class II-transfected fibroblast cells. We have investigated whether this difference is due to LFA-1-mediated signaling, and we present data that demonstrates that although ICAM-1 does not deliver costimulatory signals required for T cell activation, the interaction of LFA-1 with ICAM-1 does synergize with TCR-transduced signals. This synergy is observed for ICAM-1 on live and on chemically fixed accessory cells, and for purified ICAM-1 molecules, but in all cases occurs only when the ICAM-1 and the TCR ligands are on the same surface. Finally, when the ICAM-1 is present on the surface of accessory cells, it enhances T cell activation by changing the Ag dose-dependence of the T cell, but when ICAM-1 and CD3 mAb are co-immobilized, ICAM-1 increases the peak response of the T cell without affecting the dose dependence of the response.

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Cell adhesion molecules in the immune system are believed to play an important role in lymphocyte-target cell conjugate formation. One such molecule, intercellular adhesion molecule 1 (ICAM-1), is important in the function, aggregation, and adherence of leukocytes. Here, we report the isolation and characterization of the murine ICAM-1 gene. We report that the murine ICAM-1 gene is a member of the Ig gene superfamily, has limited homology to its human counterpart, and is expressed in cells of lymphocytic and myeloid lineages. Transfection of the ICAM-1 cDNA into MHC class II-transfected fibroblasts leads to enhancement of the Ag-specific T cell response when the transfectants are used as APC.

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Activation of B cells to proliferation and antibody secretion is dependent on soluble lymphokines secreted by activated T cells. Activation of T cells results from physical contact between B cells and T cells through binding of the T-cell antigen receptor to a complex of antigen and class II major histocompatibility complex (MHC) molecules. To determine whether this interaction also contributes to B-cell activation by mechanisms other than those mediated by soluble T cell-derived lymphokines, I examined the ability of isolated T-cell plasma membranes to stimulate proliferation in cultures of unfractionated B cells. Membranes prepared from a cloned antigen-specific helper T-cell line induced substantial proliferation provided that the T cells had been mitogen-activated before isolation of membranes. Membranes from splenic Con A-treated blasts also stimulated B-cell proliferation, suggesting that this activity may be a common property of some subsets of activated T cells. Induction of B-cell proliferation was not found to be antigen-dependent or MHC-restricted, indicating no significant contribution by the T-cell receptor for antigen. The presence of interleukins 4 and 5 in membrane fractions was indicated by proliferation of lymphokine-sensitive cell lines, although culture supernatants from mitogen-activated T cells proved to be far more potent sources of these activities. The combined effect of membranes and lymphokine-containing culture supernatants in B-cell cultures was greater than their added effects in separate cultures. This observation suggests that lymphokines or molecules with mitogenic activity for B cells other than those found in abundance in culture supernatants may be present on activated T-cell membranes.

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I-Ad, purified from A20-1.11 cells by affinity chromatography, was incorporated into supported planar membranes by incubation of I-Ad-containing phospholipid vesicles with clean glass coverslips. Such planar membranes present a peptide digest of ovalbumin to the ovalbumin-specific, I-Ad-restricted T-cell hybridoma 3DO-54.8, resulting in the antigen-specific release of interleukin 2. However, when the same material was provided in the form of small unilamellar vesicles, no response was obtained. Antigen presentation by the I-Ad-containing planar membranes was inhibited by the monoclonal antibody MKD6 (anti-I-Ad) but not by the antibody 10-2.16 (anti-I-Ak). The antibody GK1.5, which recognizes the T-cell surface antigen L3T4, was also inhibitory. In contrast to the results with purified I-Ad, crude membrane preparations from A20-1.11 cells were effective in antigen presentation in both planar and vesicular forms.

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Phospholipid vesicles containing the transmembrane protein H-2Kk spontaneously fuse to form planar membranes when incubated on treated glass surfaces. Pattern photobleaching of fluorescent lipid probes indicates that these planar membranes are continuous and that the lipids are as mobile as they are in conventional fluid bilayers or monolayers. H-2Kk molecules in these planar membranes are immobile. These membranes stimulate cytotoxic T lymphocytes when cultured with immune spleen cells. The response to H-2Kk in planar membranes is greatly enhanced by the addition of supernatant from concanavalin A-stimulated spleen cells, indicating that relatively little antigen processing or presentation by accessory cells occurs. Cytotoxic T cells induced by purified alloantigen are found to be as susceptible to antibody blockade as are effectors from conventional mixed lymphocyte culture, where the antibody is directed against a T-cell surface antigen reputed to strengthen target cell adhesion through an interaction independent of major histocompatibility antigens.

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A number of fluorescent peptide-lipid conjugates have been synthesized. Peptides with ten or eleven amino acids are linked through a single lysine residue to the headgroup of phosphatidylethanolamine, fluorescently labelled on one acyl chain, using homobifunctional disuccinimidyl crosslinking reagents. Peptide-lipids can be further derivatized with the hapten dinitrophenyl. Purified peptide-lipids have been incorporated into dimyristoylphosphatidylcholine monolayers at the interface of air and phosphate-buffered saline, at concentrations of up to 11 mol%. For equal average molecular areas, monolayers containing peptide-lipids have higher surface pressures than pure lipid monolayers; for equal surface pressures, peptide-lipid monolayers have higher average molecular areas than pure lipid monolayers. When the peptide-lipid monolayers are transferred to hydrophobic glass slides, the fluorescence appears uniformly distributed. Fluorescence recovery after photobleaching measurements indicate that peptide-lipids diffuse in the monolayer with coefficient 1.5 X 10(-9) cm2/s, which is much smaller than that of typical lipids in fluid membranes. In addition, the diffusion coefficient of peptide-lipids decreases with increasing peptide-lipid concentration. We conclude that the peptide portion of the peptide-lipid associates with the lipid monolayer and/or that peptide-lipids oligomerize.

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DNA molecules, several persistence lengths long in sedimentation equilibrium at speeds high enough to maintain fairly close packing, show a dense, sharply-bounded turbid phase and an isotropic phase (as with shorter fragments) and also an intermediate, somewhat turbid region. The concentration distribution in the isotropic phase is in satisfactory agreement with a simple extension of scaled particle theory in which semiflexible chains are equivalent to straight rods of the same length. The net intermolecular interactions, as inferred from the Zimm cluster integral, are purely repulsive. As in our previous study with short fragments, the results are compatible with a hard-core electrostatic radius, decreasing with increasing salt concentration. However, for the longer fragments it is necessary to infer either a slightly greater mass per unit length or a slightly smaller electrostatic radius for closest agreement with scaled particle theory. The properties of the solution at the boundary with the turbid, presumably strongly ordered phase are consistent with those found for shorter fragments and with theoretical scaling expectation for a hard, asymmetric particle.

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H-2Kk, a transmembrane protein coded for by the mouse major histocompatibility complex, was modified by trypsinization to remove its intracellular segment and was incorporated into lipid monolayers made on an alkylated glass substrate. Cloned cytotoxic T-lymphocytes specific for H-2Kk were incubated on the monolayer. More cytotoxic T-cells bound to monolayers containing the H-2Kk fragment (tH-2Kk) than to control monolayers made without H-2Kk. Preincubation of monolayers with alloantisera against H-2Kk reduced binding of CTL to levels seen with control monolayers. This method offers the possibility of studying specific binding of cytotoxic T-cells to well-defined model membranes.

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