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C R Wagner

Publications and source records attributed to C R Wagner.

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

Molecular basis for nonadditive mutational effects in Escherichia coli dihydrofolate reductase.

Recently, two sets of single, double, and quadruple residue changes within the hydrophobic substrate binding pocket of Escherichia coli dihydrofolate reductase (5,6,7,8-tetrahydrofolate+ oxidoreductase, EC 1.5.1.3) were shown to exhibit nonadditive mutational effects [Huang, Z., Wagner, C. R., & Benkovic, S. J. (1994) Biochemistry 33, 11576--11585]. In particular, the analysis of data for the L28Y, L54F, and L28Y-L54F mutations revealed nonadditive changes in the free energy associated with the substrate and cofactor binding, hydride transfer, and product release steps. Construction of a related set of mutant proteins including L28F and L28F-L54F permits a comparison of similar energy changes and provides a means for assessing differences in the interactions of Phe28 and Tyr28 with both the ligands and the side chains at residue 54. We find a single functional group change, from Phe C4-H to Tyr C4-OH, can influence the additivity of mutational effects and serve as a probe to monitor the appearance of differing enzyme conformations along the reaction pathway through changes in the interaction energy (delta GI). The comparison of additivity/nonadditivity in free energy changes for three interrelated double mutational cycles (WT --> L28F-L54F, WT --> L28Y-L54F, and L28F --> l28Y-L54F) demonstrates that the side chains of positions 28 and 54 interact cooperatively to facilitate hydride transfer by preferentially influencing the enzyme--substrate ground-state complexes. The delta GI data for individual steps also provide evidence for multiple conformations of the enzyme operating during the catalytic cycle. The fact that there are no published examples of the synergistic enhancement of favorable mutational effects is consistent with the expectation that the binding/active site surface of wild-type dihydrofolate reductase has been optimized.

Base Sequence↗
PubMed1995

Cardiac allograft vasculopathy. Association with cell-mediated but not humoral alloimmunity to donor-specific vascular endothelium.

BACKGROUND: Cardiac allograft vasculopathy (CAV) is an accelerated form of coronary artery disease responsible for the majority of late deaths after cardiac transplantation. Although most consider this complication a manifestation of chronic allograft rejection, it has not been established whether this disease is a consequence of humoral or cell-mediated alloreactivity. METHODS AND RESULTS: Human aortic endothelial cells (HAECs) were isolated from donor aortas obtained at the time of organ acquisition for 52 cardiac allograft recipients. Serum and peripheral blood mononuclear cells were obtained from these 52 allograft recipients at several time points during the first year after transplantation. Lymphocyte proliferation (LP) in response to donor-specific HAECs and alloantibody binding to interferon-gamma-treated donor-specific HAECs were performed and correlated with clinical parameters, including HLA matching, acute cellular rejection, and coronary artery disease on surveillance angiography. Ten of the 52 patients studied had angiographic or autopsy evidence of coronary artery disease in the first posttransplantation year (CAV+ group). The CAV+ group had higher LP responses to their donor HAECs at 1 week, 3 months, and 6 months after transplantation compared with the CAV- group (1 week: 1439 +/- 222 versus 824 +/- 141 counts per minute [cpm], P = .026; 3 months: 1282 +/- 388 versus 884 +/- 94 cpm, P = .07; 6 months: 2504 +/- 635 versus 1540 +/- 209 cpm, P = .036; CAV+ versus CAV-, respectively). Only 8 of the 52 patients had donor-specific alloantibodies, and there was no relation between antibody presence and CAV. Other clinical parameters that correlated with CAV included the level of HLA-DR mismatch and the presence of late acute rejection. CONCLUSIONS: CAV is associated with donor-specific cell-mediated alloreactivity to vascular endothelium. Humoral immunity does not appear to have a major role in this disease.

Adult↗
PubMed1995

Hamster monomorphic arylamine N-acetyltransferase: expression in Escherichia coli and purification.

N-Acetylation is a major pathway in the metabolism of hydrazine and arylamine drugs, and has been associated with carcinogen bioactivation. Monomorphic hamster liver N-acetyltransferase (NAT1) cDNA was cloned from hamster liver cells by reverse transcriptase-coupled polymerase chain reaction. The determined nucleotide sequence was identical to that reported for NAT1. The NAT1 coding region was subcloned into the pG1 yeast expression vector, but cell extracts provided only transient acetyltransferase activity. In addition, cDNA was subcloned into the expression vectors pFLAG-ATS and pFLAG-MAC. The latter vectors encoded a tac promoter and appended a low-molecular weight (1 kDa) hydrophilic FLAG marker peptide to the amino terminus of NAT1. Unexpectedly, periplasmic export of FLAGATS-NAT1 by the ompA signal peptide of pFLAG-ATS proved to be detrimental to enzyme activity. High acetyltransferase activity, however, was obtained when the fusion protein was expressed in the cytosol. Enzyme purified to homogeneity by immunoaffinity chromatography exhibited substrate specificity comparable to that of the hamster-derived protein.

Amino Acid Sequence↗
PubMed1994

Phenotypic variations in resting and activated levels of ICAM-1 expression by cultured human aortic endothelial cells.

ICAM-1 is an inducible glycoprotein important in the adhesion, activation, and transmigration of circulating leukocytes across the vascular endothelial monolayer, and it likely plays a key role in the allogeneic response. To determine the reproducibility and significance of variations in resting levels of cell surface ICAM-1, 3 individual measurements of ICAM-1 levels were performed on 26 individual isolates of human aortic endothelial cells (HAECs) both at rest and following activation by allogeneic lymphocytes, using flow cytometry. Resting HAEC ICAM-1 levels varied 10-fold (range 6-60 mean fluorescence channels) depending on the isolate studied. There were strong correlations (r = 0.71 to 0.77, P < 0.0001) between the three measurements (performed no closer than weekly intervals on separate cultures), attesting to the consistency of the phenotypic expression. Constitutive expression of ICAM-1 was not affected by cell age, based upon comparing a subset of these isolates across 3 population doublings. Levels of HAEC ICAM-1 following allogeneic lymphocyte activation varied 15-fold (range 20-300 mean fluorescent channels) and, more important, correlated with resting ICAM-1 levels (r = 0.58, P = 0.002). Finally, constitutive ICAM-1 expression was related to TNF-alpha-induced ICAM-1 levels based upon a subset of the isolates studied. These data suggest that phenotypic, and likely genetic, differences in quiescent endothelial cell adhesion molecule expression can influence inflammatory responses including alloresponsiveness to the vasculature.

Aorta↗
PubMed1994

Nonadditivity of mutational effects at the folate binding site of Escherichia coli dihydrofolate reductase.

The function of the hydrophobic residues Leu28, Phe31, Ile50, and Leu54 at the folate binding site in Escherichia coli dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) has been studied by a combination of site-specific mutagenesis and reaction kinetics. Studies suggest that the overall protein structure and kinetic sequence for the reaction did not change for the mutant proteins compared to the wild-type enzyme. Two sets of mutated reductases have been constructed. The first set, in which the side chains of the targeted amino acids are spatially well separated (approximately 8 A), includes two single mutants (L28Y and L54F) and a double mutant (L28Y-L54F). This set features residues that increased the side chain surface area and the potential for substrate interactions. Unexpectedly, nonadditivity in the free energy changes for the thermodynamics of ligand binding and in the rates of hydride transfer and product release is observed. The progressive increase in dihydrofolate binding is reversed for the sterically more crowded double mutant, with delta delta G ca. 3 kcal mol-1 less favorable than anticipated. On the other hand, the decrease in the rate constant for hydride transfer noted with the single mutants relative to the wild-type enzyme is reversed for the double mutant, so that delta delta G not equal to is ca. 2 kcal mol-1 more favorable. The second set of mutant proteins includes two double mutants (L28A-F31A and I50A-L54G) in which the selected amino acids are separated by three to four intervening amino acids and a quadruple mutant (L28A-F31A-I50A-L54G) in which the two sets L28A-F31A and I50A-L54G are spatially distinct. This set deleted the side chain surface area to lower the opportunity for substrate interactions. Nonadditivity in the free energy changes associated with key kinetic and thermodynamic parameters is again observed. The decrease in dihydrofolate binding found with the two double mutants is not observed with the quadruple mutant, which binds the substrate with delta delta G ca. 6.5 kcal mol-1 more favorable than expected. Similarly, the quadruple mutant has a larger rate constant for hydride transfer (-delta delta G not equal to congruent to 1.7 kcal mol-1) than predicted. One interpretation for the nonadditivity is that these residues interact through binding of the folate substrate, which serves to link molecularly remote side chain moieties within the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1994

An H2-T MHC class Ib molecule presents Listeria monocytogenes-derived antigen to immune CD8+ cytotoxic T cells.

Mouse spleen T cells can adoptively transfer immunity to Listeria monocytogenes; this activity was markedly enhanced by stimulation with Con A in vitro before transfer. The enhanced and prolonged protection against L. monocytogenes in vivo was correlated with enhanced lysis in vitro of target cells infected with strains of L. monocytogenes that produce listeriolysin O (LLO). One of the targets of such cytotoxic cells from BALB/c (H2d) mice was a peptide that corresponded to amino acids 91 to 99 (p91-99) of the LLO molecule, which satisfies the binding motif of H2-Kd. Listeria-immune CD3+CD8+, but not CD3+CD8-, cells could also lyse H-2-incompatible, infected target cells. Immune cells from C57BL/6 (H2b) mice lysed allogeneic H-2d target cells infected with L. monocytogenes or a Bacillus subtilis transformant that secretes LLO, but did not lyse targets pulsed with p91-99. This H2-unrestricted cytolysis was therefore directed at a fragment of the LLO molecule other than p91-99. Listeria-infected bone marrow macrophages from congenic and recombinant strains of mice were lysed only when they shared the H2-T region or were Qa1-compatible with the immune cytotoxic cells; sharing of the H2-D, Q, or M region was insufficient. Thus, the immune response to L. monocytogenes included cytolytic CD8+ cells that recognized endogenously processed Listeria-derived Ags in the context of the class Ia H2-K molecule, as well as a class Ib H2-T molecule.

Amino Acid Sequence↗
PubMed1993

The pattern of cytokine messenger RNA expression in human aortic endothelial cells is different from that of human umbilical vein endothelial cells.

Endothelial cells most readily available and most frequently used in investigations of alloimmunity and cytokine expression and function are derived from human umbilical veins. It is unclear whether cells derived from fetal venous tissue are relevant to phenomena related to the adult allograft, especially in areas such as cardiac allograft vasculopathy, a chronic rejection process directed against the coronary arteries. Human aortic endothelial cells (HAECs) were compared to human umbilical vein endothelial cells (HUVECs) for their constitutive expression of poly (A)+ RNA coding for a group of cytokines known to stimulate smooth muscle cell proliferation, including acidic fibroblast growth factor, basic fibroblast growth factor, transforming growth factor alpha, transforming growth factor beta, platelet-derived growth factor A-chain, platelet-derived growth factor B-chain and amphiregulin. Poly (A)+ RNA coding for basic fibroblast growth factor and transforming factor-beta was consistently expressed by all nine isolates of HAECs, but platelet-derived growth factor A- and B-chain were expressed in only six of the nine isolates. In most cases this was related to the presence of transforming growth factor alpha expression. In contrast, HUVECs consistently expressed basic fibroblast growth factor, transforming growth factor-beta, and both platelet-derived growth factor chains. Transforming growth factor alpha expression was never seen in the HUVEC isolates. No endothelial cell isolate expressed mRNA coding for acidic fibroblast growth factor or amphiregulin. There appear to be differences between cytokine gene expression patterns by endothelial cells from different vascular beds.

Amphiregulin↗
PubMed1993

Regulation of human aortic endothelial cell-derived mesenchymal growth factors by allogeneic lymphocytes in vitro. A potential mechanism for cardiac allograft vasculopathy.

Cardiac allograft vasculopathy is thought to be triggered by an alloreactive response to the donor coronary vasculature, resulting in smooth muscle cell proliferation and ultimate occlusion of the donor coronary arteries. To determine whether allogeneic lymphocytes are capable of regulating endothelial-derived smooth muscle cell (SMC) growth factors, human aortic endothelial cells (HAECs) were exposed to allogeneic lymphocytes. The HAEC-lymphocyte co-cultures were assessed for (a) lymphocyte proliferation in response to the allogeneic HAECs; (b) release of soluble factors that stimulate human aortic SMC proliferation; and (c) alteration of HAEC mRNA levels for a panel of known SMC growth factors. Co-culture conditioned medium increased SMC proliferation, compared to medium conditioned by HAECs alone. HAECs exposed to allogeneic lymphocytes increased their expression of mRNA for basic fibroblast growth factor, transforming growth factors alpha and beta, and platelet derived growth factor A and B chains. These results demonstrate that allogeneic lymphocytes are capable of inducing HAECs to increase mRNA levels for several mesenchymal growth factors and to release bioactive products capable of stimulating SMC cell proliferation in vitro. Additionally, the data support the hypothesis that alloreactive lymphocytes can stimulate allogeneic donor endothelial cells to produce growth factors that may contribute to the intimal thickening seen in cardiac allograft vasculopathy.

Base Sequence↗
PubMed1993

The modulation of human aortic endothelial cell ICAM-1 (CD-54) expression by serum containing high titers of anti-HLA antibodies.

Allograft recipients who have preformed antibodies to MHC determinants or develop these antibodies post-transplantation have a higher incidence of cellular rejection and graft loss. It is unclear whether this association is an etiologic one or whether the presence of these antibodies solely identifies individuals with a more pronounced alloimmunologic response. To determine whether antibodies to MHC determinants have a direct role in enhancing cell-mediated immunity, specifically in altering effector-target cell adhesion, the expression of endothelial cell surface intercellular adhesion molecule-1 (ICAM-1) in response to serum with high-titer anti-HLA antibodies was investigated. The target cells used were a pool of blood group O human aortic endothelial cells (HAECs) representing a wide range of HLA-A, B, C, and DR phenotypes. The test serum was serum pooled from 30 highly sensitized individuals (panel-reactive antibody 80%). Antibody binding to HAECs, and HAEC expression of class I and class II major histocompatibility (MHC) antigens and ICAM-1 were assessed by flow cytometry. General HAEC metabolic changes were assessed by 3H-uridine incorporation as a measure of RNA synthesis. Test serum resulted in almost a 14-fold increase in HAEC surface ICAM-1 expression compared with control serum, and titrations of test serum yielded a strong correlation between IgG bound to HAECs and HAEC ICAM-1 expression (r = 0.92). Test serum induced no change in expression of HAEC class I or class II MHC antigens, or 3H-uridine incorporation. The HAEC ICAM-1-inducing ability of the test serum was retained by concentrating the high molecular weight (> 100 kilodaltons) fraction of the test serum, isolation and purification of IgG from the test serum, and lost by absorbing this fraction with pooled platelets, suggesting that the activity was mediated by antibodies directed against MHC class I determinants. These data suggest that the presence of anti-HLA antibodies is more than a marker for individuals with greater alloreactive responsiveness. Anti-HLA antibodies may directly and specifically alter adhesion of effector cells to the allograft.

Aorta↗
PubMed1992

Complementary perturbation of the kinetic mechanism and catalytic effectiveness of dihydrofolate reductase by side-chain interchange.

The variable residue Leu-28 of Escherichia coli dihydrofolate reductase (DHFR) and the corresponding residue Phe-31 in murine DHFR were interchanged, and the impact on catalysis was evaluated by steady-state and pre-steady-state analysis. The E. coli L28F mutant increased the pH-independent kcat from 11 to 50 s-1 but had little effect on Km(H2F). An increase in the rate constant for dissociation of H4F from E.H4F.NH (from 12 to 80 s-1) was found to be largely responsible for the increase in kcat. Unexpectedly, the rate constant for hydride transfer increased from 950 to 4000 s-1 with little perturbation of NADPH and NADP+ binding to E. Consequently, the flux efficiency of the E. coli L28F mutant rose from 15% to 48% and suggests a role in genetic selection for this variable side chain. The murine F31L mutant decreased the pH-independent kcat from 28 to 4.8 s-1 but had little effect on Km(H2F). A decrease in the rate constant for dissociation of H4F from E.H4F.NH (from 40 to 22 s-1) and E.H4F (from 15 to 0.4 s-1) was found to be mainly responsible for the decrease in kcat. The rate constant for hydride transfer decreased from 9000 to 5000 s-1 with minor perturbation of NADPH binding. Thus, the free energy differences along the kinetic pathway were generally similar in magnitude but opposite in direction to those incurred by the E. coli L28F mutant. This conclusion implies that DHFR hydrophobic active-site side chains impart their characteristics individually and not collectively.

Animals↗
PubMed1992

A high-mobility-group protein and its cDNAs from Drosophila melanogaster.

We have identified, purified, and characterized a high-mobility-group (HMG) protein and its cDNAs from Drosophila melanogaster. This protein, HMG D, shares most of the characteristics of vertebrate HMG proteins; it is extractable from nuclei with 0.35 M NaCl, is soluble in 5% perchloric acid, is relatively small (molecular weight of 12,000), has both a high basic (24%) and high acidic (24%) amino acid content, and is a DNA-binding protein. HMG D exhibits characteristics of both the vertebrate HMG 1 and 2 class and the HMG 14 and 17 class of proteins. Its amino acid sequence is similar (36% amino acid identity) to that of HMG1, while its size and selective extraction with ethidium bromide are similar to properties of the HMG 14 and 17 class of proteins. HMG D is encoded by a single-copy gene that maps to 57F8-11 on the right arm of chromosome 2. Two transcripts are observed during embryogenesis; the protein is relatively stable throughout development. By the biochemical criteria of size, solubility, and amino acid content, HMG D appears to be the major HMG protein of D. melanogaster.

Amino Acid Sequence↗
PubMed1991

Cytomegalovirus-induced regulation of major histocompatibility complex class I antigen expression in human aortic smooth muscle cells.

Cardiac allograft vasculopathy (accelerated transplant atherosclerosis) is considered by most to involve a chronic allogeneic immune response to one or more constituents in the coronary vascular wall. Recent evidence suggests that there is an association between cytomegalovirus infection and the development of cardiac allograft vasculopathy (CAV). To determine whether CMV directly infects and/or potentially influences immunogenicity of vascular tissue, human umbilical vein (HU-VECs) or human aortic (HAECs) endothelial cells and human aortic smooth muscle cells (HASMCs) were isolated, cultured, and infected with CMV strain AD 169. Infection was detected using an immunoperoxidase-labeled monoclonal antibody to CMV immediate-early antigen (L-14). The presence and relative quantity of MHC class I and II antigens were determined flow cytometrically using monoclonal antibodies to monomorphic class I and class II HLA determinants. Gamma interferon was used as a positive control stimulant for the upregulation of MHC determinants. Both pooled HUVECs as well as 2 cell lines of HAECs served as targets for CMV infection though less than 10% of the cells were infected despite inocula of 10 pfu/cell. Infection of the pooled HUVECs resulted in no significant changes in the cell surface density of either MHC class I or II determinants. In contrast, HASMCs were excellent targets for CMV infection with virtually 100% of cells infected. CMV infection of 2 distinct HASMC cultures resulted in an increase of 254 +/- 158 relative fluorescence units (RFUs) in MHC class I antigen expression, as assessed by fluorescence intensity, in a variable portion of the HASMCs. A second population of cells exhibited a decrease of 73 +/- 16 RFUs in MHC class I antigen expression. No significant change in MHC class II antigen expression was noted. These results demonstrate that while HUVECs and HAECs are targets of CMV infection, human aortic smooth muscle cells can more readily be infected by CMV. Furthermore, CMV can regulate smooth muscle cell MHC class I expression, hence potentially altering immunogenicity. A pathophysiologic link between cardiac allograft vasculopathy and CMV disease can therefore be hypothesized.

Aorta↗
PubMed1990

Site directed mutagenesis: a tool for enzyme mechanism dissection.

Protein engineering has become the principle means of examining the active site of an enzyme to identify and quantify the roles of specific residues in ligand binding, specificity and catalysis. Site-specific mutagenesis has extended our knowledge gained from X-ray crystallography, and has provided striking proof that the intricate active-site geometry is supported by the remainder of the protein infrastructure for maximum catalytic efficiency.

Animals↗
PubMed1990

Collaborative treatment of juvenile firesetters: assessment and outreach.

Assessment, preventive intervention, and interdisciplinary collaboration between the fire department and mental health services are highlighted as key factors in the identification and treatment of juvenile firesetters and their families. An interagency pilot program in New York City is described and the importance of "aggressive" outreach is emphasized.

Acting Out↗
PubMed1989

The molecular weight of the endothelial cell IL-1 receptor is 78,000.

Human endothelial cells have been shown to be distinctive from other IL-1 responsive cells (e.g. murine thymocytes), in the equal units of murine and human IL-1 do not have the same endothelial cell stimulatory effect, as measured by increased lymphocyte adherence. In this paper, the cell surface IL-1 receptors of human endothelial cells, fibroblasts and T-lymphocytes have been compared, to investigate whether endothelial cells have a unique receptor for IL-1. IL-1 receptors were isolated by both immunoprecipitation and chemical cross-linking to the ligand. Both techniques demonstrated that human endothelial cells are similar to human T-lymphocytes and fibroblasts, in that they all have a 78,000 mol. wt IL-1 receptor.

Cells, Cultured↗