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Biomedical subjects

C Wagner

Publications and source records attributed to C Wagner.

At least 91 records · Page 5Linked to original sources
PubMed2000

Intracutaneous genetic immunization with autologous melanoma-associated antigen Pmel17/gp100 induces T cell-mediated tumor protection in vivo.

Using the differentiation antigen Pmel17/gp100 to genetically immunize C57BL/6 mice (H-2(b)), we and colleagues noticed that only mice that had received the human homolog but not animals injected with the murine counterpart were protected against the growth of syngeneic B16 melanoma cells. The goal of this study was to determine whether the state of nonresponsiveness to the autoantigen Pmel17/gp100 can be broken by immunization with a plasmid DNA construct encoding the autologous form of the molecule. A construct containing the murine form of Pmel17 was administered intradermally to DBA/2 mice (H-2(d)), which were then investigated for the presence of Pmel17/gp100-specific immunity. We show that administration of plasmid DNA coding for the autologous melanoma-associated antigen Pmel17/gp100 protects DBA/2 mice against the growth of Pmel17-positive M3 melanoma cells but not against Pmel17-negative M3 melanoma cells or unrelated P815 mastocytoma cells. Cell depletion experiments demonstrated that this protective effect is mediated by T lymphocytes. The notion that Pmel17/gp100 represents the biologically relevant target in this system was supported by the observations (i) that recipients of Pmel17/gp100 DNA mount an antigen-specific cytotoxic T lymphocyte response and (ii) that M3 tumors growing in mice immunized with autologous Pmel17/gp100 had lost expression of this melanoma-associated antigen whereas M3 melanomas appearing in control-vector-treated animals were still Pmel17/gp100-positive. These results indicate that intracutaneous genetic immunization with autologous melanoma-associated antigen Pmel17/gp100 encoding plasmid DNA can lead to protection against melanoma cells as a result of the induction of a melanoma-associated antigen-specific and protective T-cell-mediated immune response. J Invest Dermatol 115:1082-1087 2000

Animals↗
PubMed2000

Isolated hypermethioninemia: measurements of S-adenosylmethionine and choline.

The concentrations of methionine and S-adenosylmethionine (AdoMet) in plasma and free choline and phospholipid-bound choline in both plasma and red blood cells from individuals with isolated hypermethioninemia have been measured. The only genetic abnormalities identified in these individuals have been inactivating mutations in MAT1A, the gene that encodes the subunit of the isozymes of methionine adenosyltransferase (MAT), MAT I, and MAT III, expressed only in adult liver. These measurements were performed to learn more about AdoMet metabolism and to test the working hypotheses that inadequate delivery of AdoMet, or of choline or a choline derivative, from liver to brain might be a cause of the neurologic disease often found in humans with the most severe losses of MAT I/III activity. In striking contrast to the elevations of plasma AdoMet reported in control humans with hypermethioninemia resulting from methionine loading, plasma AdoMet levels were generally below the mean reference value in the MAT I/II-deficient hypermethioninemic patients. This is interpreted as a result of subnormal formation of AdoMet in liver due to the deficient activity of MAT I/III and resultant lower-than-normal delivery of AdoMet from liver to plasma. A low plasma AdoMet concentration in the presence of an elevated methionine provides a useful diagnostic tool that pinpoints the cause of a case of hypermethioninemia as defective MAT I/III activity. Plasma-free choline concentrations were also generally somewhat below normal in the hypermethioninemic patients. However, neither plasma AdoMet nor plasma choline concentrations were strikingly lower in MAT I/III-deficient individuals with neurologic abnormalities than in those without. These results thus fail to provide support for the working hypotheses in question.

Choline↗
PubMed2000

Screening for abnormalities of the protein C anticoagulant pathway using the ProC Global assay. Results of a European multicenter evaluation.

ProC Global is a new global clotting assay designed to evaluate the functionality of the protein C anticoagulant pathway. It is based on the ability of endogenous activated protein C, generated by activation of protein C by Protac, to prolong an activated partial thromboplastin time, and the results are expressed in protein C activation time normalized ratio (PCAT-NR), after normalization. This multicenter trial involving five European laboratories was designed in order to determine the ability of the ProC Global assay to distinguish patients with and without abnormalities of the protein C pathway. The PCAT-NR was significantly lower in the patients with a thrombotic history not on oral anticoagulant treatment (n = 627) than in the healthy controls (n = 148), even after exclusion from both groups of the patients with abnormality of the protein C pathway. Using receiver operator characteristics analysis, the cut-off level of PCAT-NR = 0.80 was found to provide the best sensitivity-specificity ratio. All the carriers of the factor V Leiden mutation (n = 73), as well as all the patients with activated protein C resistance (n = 42), had a PCAT-NR below 0.80. The ProC Global assay performed well in patients with combined defects (97.0%, n = 33) or protein C deficiency (91.3%, n = 46), but it failed to detect all of them, and one patient with combined defects as well as four patients with a low protein C level had a PCAT-NR above the cut-off level. The sensitivity of the assay for protein S deficiency (n = 58) was weak (only 69.0%) and, surprisingly, more than 40% of the 375 patients without any of these abnormalities of the protein C pathway had a PCAT-NR below the cut-off level.

Activated Protein C Resistance↗
PubMed2000

Crossover from a square to a hexagonal pattern in faraday surface waves

We report on surface wave pattern formation in a Faraday experiment operated at a very shallow filling level, where modes with a subharmonic and harmonic time dependence interact. Associated with this distinct temporal behavior are different pattern selection mechanisms, favoring squares or hexagons, respectively. In a series of bifurcations running through a pair of superlattices the surface wave pattern transforms between the two incompatible symmetries. The close analogy to two- and three-dimensional crystallography is pointed out.

Journal Article↗
PubMed2000

Microarray-based identification of a novel Streptococcus pneumoniae regulon controlled by an autoinduced peptide.

We have identified in the Streptococcus pneumoniae genome sequence a two-component system (TCS13, Blp [bacteriocin-like peptide]) which is closely related to quorum-sensing systems regulating cell density-dependent phenotypes such as the development of genetic competence or the production of antimicrobial peptides in lactic acid bacteria. In this study we present evidence that TCS13 is a peptide-sensing system that controls a regulon including genes encoding Blps. Downstream of the Blp TCS (BlpH R) we identified open reading frames (blpAB) that have the potential to encode an ABC transporter that is homologous to the ComA/B export system for the competence-stimulating peptide ComC. The putative translation product of blpC, a small gene located downstream of blpAB, has a leader peptide with a Gly-Gly motif. This leader peptide is typical of precursors processed by this family of transporters. Microarray-based expression profiling showed that a synthetic oligopeptide corresponding to the processed form of BlpC (BlpC*) induces a distinct set of 16 genes. The changes in the expression profile elicited by synthetic BlpC* depend on BlpH since insertional inactivation of its corresponding gene abolishes differential gene induction. Comparison of the promoter regions of the blp genes disclosed a conserved sequence element formed by two imperfect direct repeats upstream of extended -10 promoter elements. We propose that BlpH is the sensor for BlpC* and the conserved sequence element is a recognition sequence for the BlpR response regulator.

Alleles↗
PubMed2000

High sensitivity C-reactive protein: biological variations and reference limits.

Serum C-reactive protein (CRP) concentration was determined for 3605 subjects using an immunonephelometric assay improved to provide greater sensitivity. Subjects were from 5 to 75 years old and belonging to 1003 nuclear families recruited from the Stanislas Cohort Study between January 1994 and August 1995. Sample values for CRP ranged from 0.17 mg/l to 100 mg/l. Geometric means (mean - SD; mean +/- SD) were in the 5-14 years old group 0.37 (0.17-1.07) mg/l, in the 15-28 years old group 0.47 (0.17-1.38) mg/l and in the 29-75 years old group 0.98 (0.34-2.85) mg/l. For women, the geometric means were 0.38 (0.17-1.10) mg/l, 0.62 (0.20-1.90) mg/l and 0.98 mg/l (0.31-3.13) mg/l respectively. The interindividual variability ranged from 138% to 759% among different age classes. Biological factors associated with CRP concentration variations were examined and accounted for 25% of the CRP variability in men and 40% in women. The main biological factors statistically associated with CRP concentration variations in men were: drugs, leukocyte count, body mass index, tobacco consumption, age, and in women: drugs, leukocyte count, age, body mass index and hemoglobin concentration. These factors were used to define the exclusion and partition criteria when obtaining the reference samples. Medians for reference values ranged from 0.20 to 0.68 mg/l in males and from 0.20 to 0.78 mg/l in women.

Adolescent↗
PubMed2000

Psychosocial impact of the lipodystrophy syndrome in HIV infection.

Lipodystrophy is a poorly understood condition associated with antiretroviral therapy in HIV infection. The symptoms may include some combination of central fat accumulation, peripheral fat depletion, and metabolic disturbance. A qualitative survey of 33 HIV-infected heterosexual women and gay men with lipodystrophy assessed psychosocial impact and effect on quality of life. Dominant themes included erosion of self-image and self-esteem, problems in social and sexual relations, threats to locus of control, forced HIV disclosure, and demoralization and depression. Another theme was clinicians' minimization of the importance of lipodystrophy. Further research is required to fully understand the psychosocial impact of lipodystrophy and to develop strategies that help individuals cope.

Adult↗
PubMed1999

Inhibition of glycine N-methyltransferase by 5-methyltetrahydrofolate pentaglutamate.

Glycine N-methyltransferase (EC 2.1.1.20) catalyzes the methylation of glycine by S-adenosylmethionine to form sarcosine and S-adenosylhomocysteine. The enzyme was previously shown to be abundant in both the liver and pancreas of the rat, to consist of four identical monomers, and to contain tightly bound folate polyglutamates in vivo. We now report that the inhibition of glycine N-methyltransferase by (6S)-5-CH(3)-H(4)PteGlu(5) is noncompetitive with regard to both S-adenosylmethionine and glycine. The enzyme exhibits strong positive cooperativity with respect to S-adenosylmethionine. Cooperativity increases with increasing concentrations of 5-CH(3)-H(4)PteGlu(5) and is greater at physiological pH than at pH 9.0, the pH optimum. Under the same conditions, cooperativity is much greater for the pancreatic form of the enzyme. The V(max) for the liver form of the enzyme is approximately twice that of the pancreatic enzyme, while K(m) values for each substrate are similar in the liver and pancreatic enzymes. For the liver enzyme, at pH 7.0 half-maximal inhibition is seen at a concentration of about 0.2 microM (6S)-5-CH(3)-H(4)PteGlu(5), while at pH 9.0 this value is increased to about 1 microM. For the liver form of the enzyme, 50% inhibition with respect to S-adenosylmethionine at pH 7.4 occurs at about 0.27 microM. The dissociation constant, K(s), obtained from binding data at pH 7.4 is 0.095. About 1 mol of (6S)-5-CH(3)-H(4)PteGlu(5) was bound per tetramer at pH 7.0, and 1.6 mol were bound at pH 9.0. The degree of binding and inhibition were closely parallel at each pH. At equal concentrations of (6R,6S)- and (6S)-5-CH(3)-H(4)PteGlu(5), the natural (6S) form was about twice as inhibitory. These studies indicate that glycine N-methyltransferase is a highly allosteric enzyme, which is consistent with its role as a regulator of methyl group metabolism in both the liver and the pancreas.

Animals↗
PubMed1999

Aspartate 142 is involved in both hydrolase and dehydrogenase catalytic centers of 10-formyltetrahydrofolate dehydrogenase.

The enzyme 10-formyltetrahydrofolate dehydrogenase (FDH) catalyzes conversion of 10-formyltetrahydrofolate to tetrahydrofolate in either a dehydrogenase or hydrolase reaction. The hydrolase reaction occurs in a 310-residue amino-terminal domain of FDH (N(t)-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. N(t)-FDH shares some sequence identity with several 10-formyltetrahydrofolate-utilizing enzymes. All these enzymes have a strictly conserved aspartate, which is Asp(142) in the case of N(t)-FDH. Replacement of the aspartate with alanine, asparagine, glutamate, or glutamine in N(t)-FDH resulted in complete loss of hydrolase activity. All the mutants, however, were able to bind folate, although with lower affinity than wild-type N(t)-FDH. Six other aspartate residues located near the conserved Asp(142) were substituted with an alanine, and these substitutions did not result in any significant changes in the hydrolase activity. The expressed D142A mutant of the full-length enzyme completely lost both hydrolase and dehydrogenase activities. This study shows that Asp(142) is an essential residue in the enzyme mechanism for both the hydrolase and dehydrogenase reactions of FDH, suggesting that either the two catalytic centers of FDH are overlapped or the dehydrogenase reaction occurs within the hydrolase catalytic center.

Amidohydrolases↗
PubMed1999

A salt-induced kinetic intermediate is on a new parallel pathway of lysozyme folding.

Lysozyme folds through two competing pathways. A fast pathway leads directly from a collapsed state to the native protein, whereas folding on a slow pathway proceeds through a partially folded intermediate (I(1)). At NaCl concentrations above 100 mM, a second transient intermediate (I(2)) is induced as judged by the appearance of an additional apparent rate constant in the refolding kinetics. Monitoring the time course of native molecules and of both intermediates shows that the NaCl-induced state (I(2)) is located on neither of the two folding pathways observed at low-salt concentrations. These results suggest that I(2) is a metastable high-energy intermediate at low-ionic strength and is located on a third folding pathway. The folding landscape of lysozyme seems to be complex with several high-energy intermediates located on parallel folding routes. However, the experiments show no evidence for partially folded states on the fast direct pathway.

Animals↗
PubMed1999

Domain organization and molecular characterization of 13 two-component systems identified by genome sequencing of Streptococcus pneumoniae.

In bacteria, adaptive responses to environmental stimuli are often initiated by two-component signal transduction systems (TCS). The prototypical TCS comprises two proteins: a histidine kinase (HK, hk) and a response regulator (RR rr). Recent research has suggested that compounds that inhibit two-component systems might have good antibacterial activity. In order to identify TCS that are crucial for growth or virulence of Streptococcus pneumoniae, we have examined the genomic sequence of a virulent S. pneumoniae strain for genes that are related to known histidine kinases or response regulators. Altogether 13 histidine kinases and 13 response regulators have been identified. The protein sequences encoded by these genes were compared with sequences deposited in public databases. This analysis revealed that two of the 13 pneumococcal TCSs have been described before (ciaRH and comDE) and two are homologous to the yycFG and the phoRP genes of Bacillus subtilis. All the pneumococcal response regulators contain putative DNA binding motifs within the C-terminal output domain, implying that they are involved in transcriptional control. Two of these response regulators are obviously the first representatives of a new subfamily containing an AraC-type DNA-binding effector domain. To assess the regulatory role of these transcription factors, we disrupted each of the 13 response regulator genes by insertional mutagenesis. All the viable mutant strains with disrupted response regulator genes were further characterized with regard to growth in vitro, competence, and experimental virulence. Two response regulator genes could not be inactivated, indicating that they may regulate essential cellular functions. The possibility of using these systems as targets for the development of novel antibacterials will be discussed.

Amino Acid Motifs↗
PubMed1999

Intermediates can accelerate protein folding.

The effect of intermediates on the rate of protein folding is explored by applying Kramers' theory of diffusive barrier crossing in the high friction limit. Intermediates are represented as local minima in the transition barrier. We observe that very large or very small additional barriers created by the intermediates slow down the folding process. The rate of folding markedly increases, however, when the additional barriers become >1 kBT but leave the overall barrier height unchanged. This rate-enhancing effect is caused by a favorable entropic contribution to the free energy of activation, and it increases with the number of intermediates up to a limiting value. From these calculations, we conclude that optimized transition barriers should contain partially folded high energy intermediates.

Animals↗
PubMed1999

Labeling of the internal pool of GP IIb-IIIa in platelets by c7E3 Fab fragments (abciximab): flow and endocytic mechanisms contribute to the transport.

Abciximab is a new antiplatelet therapeutic in ischemic cardiovascular disease. The drug, chimeric Fab fragments of a murine monoclonal antibody (MoAb) (c7E3), blocks GP IIb-IIIa function. However, its capacity to reach all receptor pools in platelets is unknown. Electron microscopy and immunogold labeling were used to localize abciximab in platelets of patients receiving the drug for up to 24 hours. Studies on frozen-thin sections showed that c7E3 Fab, in addition to the surface pool, also labeled the surface-connected canalicular system (SCCS) and alpha-granules. Analysis of gold particle distribution showed that intraplatelet labeling was not accumulative and in equilibrium with the surface pool. After short-term incubations of platelets with c7E3 Fab in vitro, gold particles were often seen in lines within thin elements of the SCCS, some of which appeared in contact with alpha-granules. Little labeling was associated with Glanzmann's thrombasthenia platelets, confirming that the channels contained bound and not free c7E3 Fab. Endocytosis of abciximab in clathrin-containing vesicles was visualized by double staining and constitutes an alternative mechanism of transport. The remaining free pool of GP IIb-IIIa was evaluated with the MoAb AP-2; flow cytometry showed it to be about 9% on the surface of nonstimulated platelets but 33% on thrombin-activated platelets. The ability of drugs to block all pools of GP IIb-IIIa and then to be associated with secretion-dependent residual aggregation must be considered when evaluating their efficiency in a clinical context.

Abciximab↗
PubMed1999

Overproduction of the Opi1 repressor inhibits transcriptional activation of structural genes required for phospholipid biosynthesis in the yeast Saccharomyces cerevisiae.

Transcription of structural genes required for phospholipid biosynthesis in the yeast Saccharomyces cerevisiae is repressed by high concentrations of inositol and choline. The ICRE (inositol/choline-responsive element), which is necessary and sufficient for regulation by phospholipid precursors, functions as a binding site for the heterodimeric Ino2/Ino4 activator. ICRE-dependent transcription becomes constitutive in the absence of the Opi1 repressor. Opi1 contains a leucine zipper motif and two glutamine-rich stretches. In this work we describe a molecular analysis of OPI1 function and expression. Opi1 mutant variants altered at the leucine zipper and a glutamine-rich region, respectively, were no longer functional repressors. In contrast, an Opi1 deletion variant lacking the N-terminal 106 amino acids still mediated negative regulation. Although the leucine zipper suggests that Opi1 may act as a DNA-binding protein, our data do not support a direct interaction with the ICRE. Despite its function as an antagonist of INO2 and INO4, expression of OPI1 is stimulated by an upstream ICRE. Overexpression of OPI1 under control of the GAL1 promoter severely inhibited activation of ICRE-dependent genes, leading to inositol-requiring cells. Growth inhibition of GAL1-OPI1 was observed with INO2 and INO4 alleles activated by either the natural promoter or a heterologous control region. Although induction of GAL1-OPI1 strongly repressed ICRE-dependent gene expression, the concentration of the Ino2/Ino4 activator remained unchanged. This finding suggests that differential expression of phospholipid biosynthetic genes may occur even in the presence of a constant amount of the specific activator.

Basic Helix-Loop-Helix Proteins↗
PubMed1999

Sensory nerve endings in the anterior cruciate ligament (Lig. cruciatum anterius) of sheep.

This study examines the structure of sensory nerve endings in the sheep anterior cruciate ligament (ACL). Three types of nerve endings are found: free nerve endings (FNE), Ruffini corpuscles, and lamellated corpuscles. The FNE (more than 100) are found subsynovially. The afferent nerve fibres are either thin myelinated axons (Adelta) or C fibres with diameters of 1-2 microm. FNE have been reported to function as thermoreceptors and polymodal nociceptors. In addition, FNE are also seen between fascicles of collagen fibres, often close to blood vessels. Part of this group may be efferent autonomic fibres controlling local blood flow. The corpuscles are seen subsynovially and between fascicles of connective tissue close to the attachment points of the ACL. A ligament contains about 20 Ruffini corpuscles, which are mainly located in the subsynovial connective tissue. They consist of cylinders formed from perineural cells surrounding the afferent myelinated axons (diameters 4-5 microm) with enlarged nerve terminals anchored between collagen fibres. These enter in bundles from the surrounding connective tissue at one open pole, pass through the length of the cylinder, and leave at the other pole. Functionally, Ruffini corpuscles have been described as slowly adapting stretch receptors. Lamellated corpuscles (usually between 5 and 15) are found in the subsynovial connective tissue. The afferent myelinated axon has a diameter of 4-6 microm, and the nerve terminal is located in the centre of numerous layers formed by lamellated terminal glial cells and by a perineural capsule. They are known to function as rapidly adapting pressure receptors. The most important function of the ACL is its mechanical function, but additional sensory functions must be considered triggering reflex mechanisms in case of extreme positioning or overload.

Animals↗
PubMed1999

Up-regulation of intracellular calcium, cyclic adenosine monophosphate and fibronectin synthesis in tubuar epithelial cells by complement.

The terminal complement complex C5b-9 is known to participate in inflammatory processes including glomerular or tubulointerstitial injury. Injury appears to be a direct consequence of C5b-9-mediated cell stimulation. In that context we studied activation of tubular epithelial cells by C5b-9 particularly with regard to fibronectin synthesis and the transmembrane signals involved. C5b-9 in sublytic concentrations caused a rise of intracellular calcium and of cAMP, followed by an increase in abundance of fibronectin-specific mRNA and accumulation of protein. Stabilized cAMP or increasing the cAMP level by forskolin enhanced fibronectin synthesis with similar kinetics. The effect of cAMP could be enhanced by adding a calcium ionophore. Since the fibronectin gene is known to have a cAMP-responsive element, the data suggest that C5b-9 increases fibronectin synthesis via generation of cAMP.

Bucladesine↗