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C Daniel

Publications and source records attributed to C Daniel.

At least 19 recordsLinked to original sources
PubMed1998

Mutational analysis of the STAT6 SH2 domain.

The SH2 domain of the STAT family of transcription factors is essential for STAT binding to phosphorylated cytoplasmic domains of activated cytokine receptors. Furthermore, the same domain mediates dimerization of activated STAT monomers, a prerequisite for DNA binding by this family of proteins. To identify amino acid residues within the STAT protein that mediate these various interactions, we have carried out an extensive mutational analysis of the Stat6 SH2 domain. Recombinant proteins carrying C-terminal deletions or double alanine substitutions were expressed in mammalian and insect cells and assayed for DNA binding, transcription activation, tyrosine phosphorylation, and the ability to interact with a tyrosine-phosphorylated peptide derived from the interleukin-4 receptor signaling chain. From these studies, we have identified amino acids that are required for both DNA binding and interleukin-4 receptor interaction, as well as residues that when mutated impair only one of the two functions. Our results suggest that the structural homology between the SH2 domain of Stat6 and that of the distantly related Src protein may be higher than predicted on the basis of primary amino acid sequence comparisons. However, the two types of SH2 domains may differ at their C-terminal ends.

Alanine↗
PubMed1998

Inhibition of an in vitro CD4+ T cell alloresponse using altered peptide ligands.

In this study, we explore the potential of altered peptide ligands (APLs) to modulate the alloresponse of CD4+ T cells using elements of the murine hemoglobin (Hb) Ag model. We first demonstrated that the T cell 2.102, specific for the Hb(64-76)/I-Ek complex, was alloreactive against splenocytes of the H-2p haplotype. Using Ab-blocking and transfection experiments, we further showed that this alloreactivity was restricted to the class II molecule I-Ep. We tested a panel of APLs previously shown to antagonize the Hb response of 2.102 and found that these peptides could also effectively inhibit the alloresponse to I-Ep. Importantly, these peptides were able to antagonize the alloresponse of naive T cells derived from mice transgenic for the 2.102 TCR, as well as Th1 and Th2 cell lines. The antagonism required the presence of both I-Ep and I-Ek on the same APC. Our study demonstrates the effectiveness of APLs to antagonize the primary alloresponse of specific T cells and provides a basis for the development of immunotherapeutics for use in transplantation and immune-mediated diseases.

Amino Acid Substitution↗
PubMed1998

Effects of subchronic exposure to cadmium chloride on endocrine and metabolic functions in rainbow trout Oncorhynchus mykiss.

Adult and juvenile rainbow trout, Oncorhynchus mykiss, were exposed for 30 days to CdCl2 in water (10 and 25 micrograms Cd/L for adults, and 1 and 5 micrograms Cd/L for juveniles) to investigate effects of subchronic exposures to Cd on the hypothalamo-pituitary-interrenal and the hypothalamo-pituitary-thyroid axis, and on metabolic status. liver size and glycogen content and body mass gain were significantly reduced in the exposed adult fish and similar changes were detected in the juveniles. Plasma cortisol levels increased after exposure to the lower dose but decreased at the higher dose. Plasma T3 and T4 tended to decrease in the exposed adult fish. No significant differences in plasma glucose or cholesterol levels were detected between the controls and the exposed fish, plasma calcium levels were lower in the exposed fish. The results indicate that long-term exposures to Cd at sublethal doses have adverse effects on the physiological status of the fish and that these effects are mediated partly through alterations of endocrine function.

Animals↗
PubMed1998

A basis for alloreactivity: MHC helical residues broaden peptide recognition by the TCR.

The high frequency of alloreactive T cells is a major hindrance for transplantation; however, the molecular basis for alloreactivity remains elusive. We examined the I-Ep alloreactivity of a well-characterized Hb(64-76)/I-Ek-specific murine T cell. Using a combinatorial peptide library approach, we identified a highly stimulatory alloepitope mimic and observed that the recognition of the central TCR contact residues (P3 and P5) was much more flexible than that seen with Hb(64-76)/I-Ek, but still specific. Therefore, alloreactive T cells can recognize a self-peptide/MHC surface; however, the allogeneic MHC molecule changes the recognition requirements for the central region of the peptide, allowing a more diverse repertoire of ligands to be recognized.

Animals↗
PubMed1998

Genetic analysis of brahma: the Drosophila homolog of the yeast chromatin remodeling factor SWI2/SNF2.

The Drosophila brahma (brm) gene encodes an activator of homeotic genes related to the yeast chromatin remodeling factor SWI2/SNF2. Here, we report the phenotype of null and dominant-negative brm mutations. Using mosaic analysis, we found that the complete loss of brm function decreases cell viability and causes defects in the peripheral nervous system of the adult. A dominant-negative brm mutation was generated by replacing a conserved lysine in the ATP-binding site of the BRM protein with an arginine. This mutation eliminates brm function in vivo but does not affect assembly of the 2-MD BRM complex. Expression of the dominant-negative BRM protein caused peripheral nervous system defects, homeotic transformations, and decreased viability. Consistent with these findings, the BRM protein is expressed at relatively high levels in nuclei throughout the developing organism. Site-directed mutagenesis was used to investigate the functions of conserved regions of the BRM protein. Domain II is essential for brm function and is required for the assembly or stability of the BRM complex. In spite of its conservation in numerous eukaryotic regulatory proteins, the deletion of the bromodomain of the BRM protein has no discernible phenotype.

Amino Acid Sequence↗
PubMed1998

Stat proteins control lymphocyte proliferation by regulating p27Kip1 expression.

The proliferation of lymphocytes in response to cytokine stimulation is essential for a variety of immune responses. Recent studies with signal transducer and activator of transcription 6 (Stat6)-deficient mice have demonstrated that this protein is required for the normal proliferation of lymphocytes in response to interleukin-4 (IL-4). In this report, we show that the impaired IL-4-induced proliferative response of Stat6-deficient lymphocytes is not due to an inability to activate alternate signaling pathways, such as those involving insulin receptor substrates, or to a failure to upregulate IL-4 receptor levels. Cell cycle analysis showed that the percentage of Stat6-deficient lymphocytes that transit from the G1 to the S phase of the cell cycle following IL-4 stimulation is lower than that of control lymphocytes. Although the regulation of many genes involved in the control of cytokine-induced proliferation is normal in Stat6-deficient lymphocytes, protein levels of the cdk inhibitor p27Kip1 were found to be markedly dysregulated. p27Kip1 is expressed at significantly higher levels in Stat6-deficient lymphocytes than in control cells following IL-4 stimulation. The higher level of p27Kip1 expression seen in IL-4-stimulated Stat6-deficient lymphocytes correlates with decreased cdk2-associated kinase activity and is the result of the increased accumulation of protein rather than altered mRNA expression. Similarly, higher levels of p27Kip1 protein expression are also seen following IL-12 stimulation of Stat4-deficient lymphocytes than are seen following stimulation of control cells. These data suggest that Stat proteins may control the cytokine-induced proliferative response of activated T cells by regulating the expression of cell cycle inhibitors so that cyclin-cdk complexes may function to promote transition from the G1 to the S phase of the cell cycle.

Animals↗
PubMed1998

Occupational asthma due to chromium.

We describe a 28-year-old subject employed as a roofer in a construction company since the age of 19, who developed work-related symptoms of a cough, shortness of breath, wheezing, rhinitis and headaches. A description of a usual day at work suggested that the symptoms worsened while he was sawing corrugated fiber cement. Baseline spirometry was normal, and there was a mild bronchial hyperresponsiveness to carbachol. A skin patch test to chromium was negative. A specific inhalation challenge showed a boderline fall in forced expiratory volume in 1 s (FEV1) after exposure to fiber cement dust. Exposure to nebulization of potassium dichromate (K2Cr2O7), at 0.1 mg.ml-1 for 30 min, was followed by an immediate fall by 20% FEV1. Simultaneously, a significant increase in bronchial hyperresponsiveness was demonstrated.

Adult↗
PubMed1998

Mycophenolic acid: a new approach to the therapy of experimental mesangial proliferative glomerulonephritis.

Mycophenolate mofetil (MMF) represents a powerful immunosuppressant in organ transplantation. The aim of this study was to determine the anti-inflammatory effects of MMF on mesangial cells. Cultured rat mesangial cells were exposed to mycophenolic acid (MPA) in concentrations of 0.1 to 10 microM. MPA inhibited the proliferation of these cells in a dose-dependent manner. A maximum of 98% inhibition was obtained by a 2-d exposure of mesangial cells to > or =5 microM MPA. As expected, the addition of > or =75 microM guanosine prevented the antiproliferative effect of MPA completely. Subsequently, in vivo studies were performed in the anti-Thy1.1 nephritis model. Sixty-six male Wistar rats were investigated: healthy rats (n = 15), treated healthy rats (n = 6), nephritic rats (n = 15), and treated nephritic rats (n = 30). MMF therapy (40 mg/kg body wt per d) of nephritic animals was initiated 2 d before (n = 3) and 6 h (n = 15) or 2 d (n = 12) after induction of nephritis. Renal histology was analyzed at days +6 and +9 after initiation of disease. Therapy of nephritic rats by MMF resulted in a significant amelioration of glomerular histology, assessed by glomerular cellularity, synthesis of alpha-smooth muscle actin, extracellular matrix deposition, and glomerular hypertrophy. Proteinuria, expressed as areas under the curve of protein/creatinine ratios versus time, showed a clear tendency toward a reduction by MMF therapy. Healthy control rats were not negatively affected by exposure to MMF. In summary, this study shows that mesangial cell proliferation can be significantly inhibited by MPA in vitro and in vivo. MMF represents a new approach to the therapy of experimental mesangial cell-mediated forms of glomerulonephritis.

Animals↗
PubMed1998

[Chemotherapy of small cell bronchogenic carcinoma in a patient on hemodialysis for chronic renal failure. Apropos of a case].

Optimal dosage for chemotherapy administered to patients on chronic hemodialysis remains a difficult question. We report the case of a patient with chronic renal failure on hemodialysis for 9 years who was treated for small-cell lung cancer. The chemotherapy protocol combined carboplatin and etoposide at doses adapted to creatinine clearance. Tumor regression was observed after 6 cycles without major intolerance.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1997

Antigenic mapping of human thyroglobulin--topographic relationship between antigenic regions and functional domains.

We characterized 26 mAb to human thyroglobulin to obtain a topographic map of the thyroglobulin antigenic surface. Among these mAb, three bind thyroglobulin peptides that are located in the primary sequence of thyroglobulin at either the N terminus or in the middle part of the molecule, three bind thyroglobulin via epitopes comprising the thyroid-hormone moiety, and three bind thyroglobulin through epitopes involved in the recognition of the molecule by its receptor. The 18 remaining mAb bind thyroglobulin through undetermined epitopes; most of these epitopes are resistant to trypsinization. We used two methods to map the antigenic regions of thyroglobulin: all 26 mAb were grouped, by means of cross-inhibition experiments, in 11 clusters corresponding to 11 antigenic regions of the thyroglobulin surface; by means of thyroglobulin peptides of decreasing size, obtained by time-controlled tryptic digestion, we analyzed the relative distance between pairs of epitopes in sandwich immunoassays. By combining these two methods, we organized most of the 11 antigenic regions on a topographic representation of the thyroglobulin surface. This new topographic map of thyroglobulin led us to some unexpected features of the thyroglobulin structure. First, antigenic region 8 located far from the N-terminal region is in close contact with two remote N-terminal antigenic regions (1 and 4), both involved in hormone formation. This antigenic region is likely to play a role in the correct positioning of hormonogenic tyrosines so as to optimize iodination-coupling reactions. Secondly, the domain involved in the binding of thyroglobulin to its receptor, probed by three mAb, is shared by two distinct mid-molecule antigenic regions, one being the main autoantigenic region of thyroglobulin.

Alkylation↗
PubMed1995

ISWI, a member of the SWI2/SNF2 ATPase family, encodes the 140 kDa subunit of the nucleosome remodeling factor.

The generation of an accessible heat shock promoter in chromatin in vitro requires the concerted action of the GAGA transcription factor and NURF, an ATP-dependent nucleosome remodeling factor. NURF is composed of four subunits and is biochemically distinct from the SWI2/SNF2 multiprotein complex, a transcriptional activator that also appears to alter nucleosome structure. We have obtained protein microsequence and immunological evidence identifying the 140 kDa subunit of NURF as ISWI, previously of unknown function but highly related to SWI2/SNF2 only in the ATPase domain. The ISWI protein is localized to the cell nucleus and is expressed throughout Drosophila development at levels as high as 100,000 molecules/cell. The convergence of biochemical and genetic studies on ISWI and SWI2/SNF2 underscores these ATPases and their close relatives as key components of independent systems for chromatin remodeling.

Adenosine Triphosphatases↗
PubMed1995

Human pancreatic lipase. Importance of the hinge region between the two domains, as revealed by monoclonal antibodies.

Several monoclonal antibodies (mAbs) were prepared against human pancreatic lipase (HPL). Two enzyme-linked immunosorbent assay (ELISA) procedures were set up for screening hybridomas producing specific antibodies. Four mAbs (81-23, 146-40, 315-25, and 320-24) of the IgG1 isotype were found to react with HPL in both simple sandwich and double sandwich ELISAs, while mAb 248-31, of the IgG2b isotype, reacted only with HPL in a double sandwich ELISA. The results of Western blot analysis carried out with native and SDS-denatured HPLs indicated that mAb 248-31 recognized only native HPL, while all the other mAbs recognized both forms of HPL. Since mAb 248-31 did not recognize SDS-denatured HPL, it was not possible to localize its epitope. To carry out epitope mapping along the primary sequence of HPL, four fragments (14, 26, 30, and 36 kDa) resulting from a limited chymotryptic cleavage of HPL were characterized by Western blotting as well as N-terminal amino acid sequence analysis. Of the above five anti-HPL mAbs, four (81-23, 248-31, 315-25, and 320-24) were found to inhibit the lipolytic activity of HPL (in both the presence and absence of bile salts and colipase), while mAb 146-40 had no inhibitory effects. The epitope recognized by mAb 146-40 was found to be located in the N-terminal domain (Lys1-Phe335). Combined immunoinactivation and epitope mapping studies showed that three inhibitory mAbs (81-23, 315-25, and 320-24) recognize overlapping epitopes from the hinge region between the N- and C-terminal domains of HPL, belonging to the 26-kDa fragment. In the presence of lipids, a significant decrease has been observed in the bending angle between the N- and C-terminal domains of the HPL tertiary structure (van Tilbeurgh, H., Egloff, M. P., Martinez, C., Rugani, N., Verger, R. and Cambillau, C. (1993) Nature 362, 814-820). From the present immunochemical data, we further propose that locking the hinge movement with mAbs may induce lipase immunoinactivation.

Animals↗
PubMed1995

Decreased interleukin-2 receptor and cell cycle changes in murine lymphocytes exposed in vitro to low doses of cadmium chloride.

Relationships between in vitro cadmium-related cell cytotoxicity, ultrastructural changes and altered cell cycle were determined at 21-72 h after mitogenic stimulation of C57BL/6 mouse spleen lymphocytes with concanavalin A (Con A). Relatively low doses, 0.6-10 microM cadmium (Cd), added at 4 h after the mitogen activation, induced a significant cell cytotoxicity and reduced the lymphoblastic activity of the cells. Cytometric analysis of the lymphoid cell cycle at 72 h revealed that at concentrations > or = 0.6 microM Cd, the number of cells arrested in G0 + G1 phase increased, whereas the proportions of cells of the S and G2 + M phases were substantially reduced. Staining of cells with fluorescent anti-CD25 monoclonal antibody showed a cadmium-related decreased number and relative mean fluorescence of CD25+ cells, demonstrating a decreased level of interleukin-2 receptor (IL-2R). Furthermore, immunogold ultramicroscopic assay was developed for determination of intracellular interleukin-2 (IL-2) in cadmium-treated lymphocytes. The level of cytoplasmic and nuclear IL-2, localized in situ by colloidal gold ultraimmunocytochemical technique, has been estimated as markedly decreased in cells treated with > or = 1.2 microM Cd, as compared with the untreated controls. Disorganization/fragmentation of mitochondrion cristae and dilatation of cisternae of the Golgi apparatus appeared as the major ultrastructural change in 1.2 microM Cd-treated lymphocytes. Interestingly, addition of cadmium in the incubation medium, up to 4 h after mitogen activation, also interacted with lymphoproliferative mechanisms of cells in G0 + G1, S and G2 + M phase. Overall, multiple ultrastructural changes of Cd-treated lymphoid cells were clearly related with the reduced cell viability and reduced number of activated lymphoblasts.

Animals↗
PubMed1995

Immunolocalization of freezing-tolerance-associated proteins in the cytoplasm and nucleoplasm of wheat crown tissues.

A protein family associated with the development of freezing tolerance in wheat has been identified. This protein family is Gramineae-specific and coordinately regulated by low temperature. Antibodies directed against the 50 kDa (WCS120) protein recognize at least 5 members of this family. Using these antibodies, the cellular content and location of this protein family was determined in cold-acclimated wheat seedlings. Western analyses of subcellular fractions indicated the presence of all members of the family in the cytosolic and purified nuclear fractions. These proteins accumulated to 0.9% of soluble proteins after 21 days of cold acclimation in winter wheat. This represents a cellular concentration of 1.34 microM. Immunohistochemical localization showed that these proteins are highly expressed in the vascular transition zone. No detectable expression was found in mature xylem, in the shoot apical meristem or lateral root primordia. This differential tissue expression suggests that the sensitive cells near the regions where water tends to freeze first require a higher amount of these proteins. This observation is consistent with the fact that regrowth after freezing stress is highly dependent on the viability of this region of the crown. Electron microscopy analysis using immunogold labelling showed that these proteins are present in the cytoplasm and in the nucleoplasm. They are not found in cell walls or other organelles. In vitro cryoprotective assays indicated that the WCS120 protein (PD50 of 10 micrograms ml-1 or 0.2 microM) are as effective as BSA and sucrose (at 250 mM) against freezing denaturation of lactate dehydrogenase. These results suggest that this protein family may be involved in a general mechanism of protection in the soluble fraction of the cell. Their presence in the nucleoplasm may also suggest a possible protective function of the transcriptional machinery. The high hydrophilicity, the abundance and stability of these proteins to boiling suggest that they may provide a particular micro-environment needed for cell survival in the sensitive vascular transition zone during freezing stress.

Antibodies↗
PubMed1994

Mapping of linear antigenic sites on the S glycoprotein of a neurotropic murine coronavirus with synthetic peptides: a combination of nine prediction algorithms fails to identify relevant epitopes and peptide immunogenicity is drastically influenced by the nature of the protein carrier.

The elucidation of the antigenic structure of the S glycoprotein of murine coronaviruses will provide further understanding of the complex pathogenicity of these viruses. In order to identify linear antigenic determinants, the primary structure of the S glycoprotein of murine hepatitis virus strain A59 was analyzed with a combination of nine epitope prediction algorithms. Fifteen potential epitopes were synthesized chemically and injected into BALB/c mice to study their biological relevance. This approach failed to identify novel important epitopes. Furthermore, the algorithms were unable to identify as antigenic the previously mapped immunodominant epitope A [C. Daniel, R. Anderson, M. J. Buchmeier, J. O. Fleming, W. J. M. Spaan, H. Wege, and Talbot, P. J. (1993). J. Virol. 67, 1185-1194]. Interestingly, peptide A coupled to KLH induced an immune response that simulated the immune response induced by the corresponding region of the protein much more accurately than when the same peptide was coupled to BSA. This included drastically enhanced competition with monoclonal antibodies and protection from virus challenge. These findings emphasize the shortcomings of amino acid sequence-based epitope prediction algorithms and demonstrate the critical importance of the carrier when synthetic peptides are considered as potential vaccines.

Amino Acid Sequence↗
PubMed1994

Increased viral titers and enhanced reactivity of antibodies to the spike glycoprotein of murine coronavirus produced by infection at pH 6.

Infection of cell monolayers by murine coronavirus A59 at pH 6 rather than 7 yielded a ten-fold increase in the infectious titer and a remarkable enhancement of the reactivities of monoclonal and polyclonal antibodies against the spike glycoprotein in immunoblotting, immunoprecipitation and enzyme-linked immunosorbent assays. These observations are very useful for detecting antibodies against the S glycoprotein of coronaviruses and enhancing infectious titers.

Animals↗
PubMed1994

Monoclonal antibodies to human pancreatic procolipase: production and characterization by competitive binding studies.

Hybridomas secreting monoclonal antibodies (MAbs) specific for human pancreatic colipase were established and 11 clones were selected by using a dot immunobinding assay. Characterization of the MAbs was carried out by using direct and competitive epitope mapping methods, including ELISA and inactivation of colipase-dependent pancreatic lipase. Monoclonal antibodies showed four distinct patterns of reactivity. Monoclonal antibody 5.30 (group I) inhibited colipase-dependent lipase activity. The dissociation constant of the inactive antibody-antigen complex was 10(-9) M. Monoclonal antibodies 48.30, 66.24, and 153.23 (group II) had no effect on activity although they bound competitively with MAb 5.30 to antigen as shown by their capacity to displace MAb 5.30 from the antibody-antigen complex and by ELISA additivity test. Dissociation constants calculated from the displacement curves were 0.9 10(-9) M, 0.6 10(-9) M, and 2 10(-9) M, respectively. Noninhibitory MAbs 13.29, 16.25, and 33.30 bound competitively with MAbs of group II but not with MAb 5.30 (group I). Monoclonal antibodies of group IV (MAbs 17.6, 18.1, 37.39, and 169.29) had no effect on activity and did not react with immobilized antigen. None of the MAbs reacted in ELISA with reduced and carboxymethylated human procolipase, indicating that epitopes involved conformationally dependent determinants on protein antigen. Anti-human colipase MAbs showed no cross-reactivity with porcine or equine procolipases. Monoclonal antibodies described here appear to be useful tools for studying surface hydrophobic domain of colipase and/or interaction between colipase and lipase in its active conformation (open lid).

Animals↗