Biomedical subjects

C Ebel

Publications and source records attributed to C Ebel.

At least 19 recordsLinked to original sources

PubMed2004

Patient satisfaction with care for genital herpes: insights from a global survey.

OBJECTIVE: To describe patient experiences and views regarding genital herpes management. METHODS: Between February 2002 and January 2003, subjects with genital herpes were recruited via the International Herpes Alliance website and through banners on additional sites. Surveys were available in English, French, Spanish, Italian, and German and assessed views on access to care, diagnosis, related emotional experiences, educational resources, counselling, pharmacotherapy, and satisfaction with care. RESULTS: 2075 patient responses from 78 countries were analysed. 49% reported their diagnosis was by culture (or other direct detection) and 9% by antibody test, while 34% reported they had been diagnosed by examination alone. 65% used a prescription antiviral therapy, 18% a topical antiviral therapy, and 17% an alternative therapy. Of 901 subjects who reported on frequency of antiviral use, only 30% reported a frequency consistent with a suppressive regimen while 59% of respondents said they would be likely to take daily therapy if it reduced the frequency of outbreaks. Patient satisfaction with management of physical symptoms was independently associated with duration of initial visit >or=15 minutes (adjusted odds ratio (OR) = 4.52), receiving a prescription (adj OR = 2.34) and receipt of a brochure/fact sheet (adj OR = 2.14). Satisfaction with attention to emotional issues also correlated with the first two of these factors. CONCLUSIONS: Genital herpes management may be improved by including the use of confirmatory laboratory testing, employing a full range of antiviral therapy options, providing educational materials, and committing more time to counselling at the initial visit.

Adult↗

PubMed2003

Amphipols: polymeric surfactants for membrane biology research.

Membrane proteins classically are handled in aqueous solutions as complexes with detergents. The dissociating character of detergents, combined with the need to maintain an excess of them, frequently results in more or less rapid inactivation of the protein under study. Over the past few years, we have endeavored to develop a novel family of surfactants, dubbed amphipols (APs). APs are amphiphilic polymers that bind to the transmembrane surface of the protein in a noncovalent but, in the absence of a competing surfactant, quasi-irreversible manner. Membrane proteins complexed by APs are in their native state, stable, and they remain water-soluble in the absence of detergent or free APs. An update is presented of the current knowledge about these compounds and their demonstrated or putative uses in membrane biology.

Animals↗

PubMed2001

Induction of H(2)O(2) synthesis by beta-glucan elicitors in soybean is independent of cytosolic calcium transients.

Soybean cell suspension cultures have been used to investigate the role of the elevation of the cytosolic Ca(2+) concentration in beta-glucan elicitors-induced defence responses, such as H(2)O(2) and phytoalexin production. The intracellular Ca(2+) concentration was monitored in transgenic cells expressing the Ca(2+)-sensing aequorin. Two lines of evidence showed that a transient increase of the cytosolic Ca(2+) concentration is not necessarily involved in the induction of H(2)O(2) generation: (i) a Bradyrhizobium japonicum cyclic beta-glucan induced the H(2)O(2) burst without increasing the cytosolic Ca(2+) concentration; (ii) two ion channel blockers (anthracene-9-carboxylate, A9C; 5-nitro-2-(3-phenylpropylamino)-benzoate, NPPB) could not prevent a Phytophthora soja beta-glucan elicitor-induced H(2)O(2) synthesis but did prevent a cytosolic Ca(2+) concentration increase. Moreover, A9C and NPPB inhibited P. sojae beta-glucan-elicited defence-related gene inductions as well as the inducible accumulation of phytoalexins, suggesting that the P. sojae beta-glucan-induced transient cytosolic Ca(2+) increase is not necessary for the elicitation of H(2)O(2) production but is very likely required for phytoalexin synthesis.

Aequorin↗

PubMed2001

Biophysical characterization of the C-propeptide trimer from human procollagen III reveals a tri-lobed structure.

Procollagen C-propeptide domains direct chain association during intracellular assembly of procollagen molecules. In addition, they control collagen solubility during extracellular proteolytic processing and fibril formation and interact with cell surface receptors and extracellular matrix components involved in feedback inhibition, mineralization, cell growth arrest, and chemotaxis. At present, three-dimensional structural information for the C-propeptides, which would help to understand the underlying molecular mechanisms, is lacking. Here we have carried out a biophysical study of the recombinant C-propeptide trimer from human procollagen III using laser light scattering, analytical ultracentrifugation, and small angle x-ray scattering. The results show that the trimer is an elongated molecule, which by modeling of the x-ray scattering data appears to be cruciform in shape with three large lobes and one minor lobe. We speculate that each of the major lobes corresponds to one of the three component polypeptide chains, which come together in a junction region to connect to the rest of the procollagen molecule.

Cell Line↗

PubMed2001

Differences in the oligomeric states of the LDH-like L-MalDH from the hyperthermophilic archaea Methanococcus jannaschii and Archaeoglobus fulgidus.

L-Malate (MalDH) and L-lactate (LDH) dehydrogenases belong to the same family of NAD-dependent enzymes. To gain insight into molecular relationships within this family, we studied two hyperthermophilic (LDH-like) L-MalDH (proteins with LDH-like structure and MalDH enzymatic activity) from the archaea Archaeoglobus fulgidus (Af) and Methanococcus jannaschii (Mj). The structural parameters of these enzymes determined by neutron scattering and analytical centrifugation showed that the Af (LDH-like) L-MalDH is a dimer whereas the Mj (LDH-like) L-MalDH is a tetramer. The effects of high temperature, cofactor binding, and high phosphate concentration were studied. They did not modify the oligomeric state of either enzyme. The enzymatic activity of the dimeric Af (LDH-like) L-MalDH is controlled by a pH-dependent transition at pH 7 without dissociation of the subunits. The data were analyzed in the light of the crystallographic structure of the LDH-like L-MalDH from Haloarcula marismortui. This showed that a specific loop at the dimer-dimer contact regions in these enzymes controls the tetramer formation.

Amino Acid Sequence↗

PubMed2001

Functional determinants of the Epstein-Barr virus protease.

Herpesvirus proteases are essential for the production of progeny virus. They cleave the assembly protein that fills the immature capsid in order to make place for the viral DNA. The recombinant protease of the human gamma-herpesvirus Epstein-Barr virus (EBV) was expressed in Escherichia coli and purified. Circular dichroism indicated that the protein was properly folded with a secondary structure content similar to that of other herpesvirus proteases. Gel filtration and sedimentation analysis indicated a fast monomer-dimer equilibrium of the protease with a K(d) of about 60 microM. This value was not influenced by glycerol but was lowered to 1.7 microM in the presence of 0.5 M sodium citrate. We also developed an HPLC-based enzymatic assay using a 20 amino acid residue synthetic peptide substrate derived from one of the viral target sequences for the protease. We found that conditions that stabilised the dimer also led to a higher enzymatic activity. Through sequential deletion of amino acid residues from either side of the cleavage site, the minimal peptide substrate for the protease was determined as P5-P2'. This minimal sequence is shorter than that for other herpesvirus proteases. The implications of our findings are discussed with reference to the viral life-cycle. These results are the first ever published on the EBV protease and represent a first step towards the development of protease inhibitors.

Amino Acid Sequence↗

PubMed2001

Assembly and enzymatic properties of the catalytic domain of human complement protease C1r.

The catalytic properties of C1r, the protease that mediates activation of the C1 complex of complement, are mediated by its C-terminal region, comprising two complement control protein (CCP) modules followed by a serine protease (SP) domain. Baculovirus-mediated expression was used to produce fragments containing the SP domain and either 2 CCP modules (CCP1/2-SP) or only the second CCP module (CCP2-SP). In each case, the wild-type species and two mutants stabilized in the proenzyme form by mutations at the cleavage site (R446Q) or at the active site serine residue (S637A), were produced. Both wild-type fragments were recovered as two-chain, activated proteases, whereas all mutants retained a single-chain, proenzyme structure, providing the first experimental evidence that C1r activation is an autolytic process. As shown by sedimentation velocity analysis, all CCP1/2-SP fragments were dimers (5.5-5.6 S), and all CCP2-SP fragments were monomers (3.2-3.4 S). Thus, CCP1 is essential to the assembly of the dimer, but formation of a stable dimer is not a prerequisite for self-activation. Activation of the R446Q mutants could be achieved by extrinsic cleavage by thermolysin, which cleaved the CCP2-SP species more efficiently than the CCP1/2-SP species and yielded enzymes with C1s-cleaving activities similar to their active wild-type counterparts. C1r and its activated fragments all cleaved C1s, with relative efficiencies in the order C1r < CCP1/2-SP < CCP2-SP, indicating that CCP1 is not involved in C1s recognition.

Binding Sites↗

PubMed2001

Characterization of a novel complex from halophilic archaebacteria, which displays chaperone-like activities in vitro.

We isolated a protein, P45, from the extreme halophilic archaeon Haloarcula marismortui, which displays molecular chaperone activities in vitro. P45 is a weak ATPase that assembles into a large ring-shaped oligomeric complex comprising about 10 subunits. The protein shows no significant homology to any known protein. P45 forms complexes with halophilic malate dehydrogenase during its salt-dependent denaturation/renaturation and decreases the rate of deactivation of the enzyme in an ATP-dependent manner. Compared with other halophilic proteins, the P45 complex appears to be much less dependent on salt for its various activities or stability. In vivo experiments showed that P45 accumulates when cells are exposed to a low salt environment. We suggest, therefore, that P45 could protect halophilic proteins against denaturation under conditions of cellular hyposaline stress.

Adenosine Triphosphatases↗

PubMed2001

Non-ideality by sedimentation velocity of halophilic malate dehydrogenase in complex solvents.

We have investigated the potential of sedimentation velocity analytical ultracentrifugation for the measurement of the second virial coefficients of proteins, with the goal of developing a method that allows efficient screening of different solvent conditions. This may be useful for the study of protein crystallization. Macromolecular concentration distributions were modeled using the Lamm equation with the approximation of linear concentration dependencies of the diffusion constant, D = D(o) (1 + k(D)c), and the reciprocal sedimentation coefficient s = s(o)/(1 + k(s)c). We have studied model distributions for their information content with respect to the particle and its non-ideal behavior, developed a strategy for their analysis by direct boundary modeling, and applied it to data from sedimentation velocity experiments on halophilic malate dehydrogenase in complex aqueous solvents containing sodium chloride and 2-methyl-2,4-pentanediol, including conditions near phase separation. Using global modeling for three sets of data obtained at three different protein concentrations, very good estimates for k(s) and s degrees and also for D degrees and the buoyant molar mass were obtained. It was also possible to obtain good estimates for k(D) and the second virial coefficients. Modeling of sedimentation velocity profiles with the non-ideal Lamm equation appears as a good technique to investigate weak inter-particle interactions in complex solvents and also to extrapolate the ideal behavior of the particle.

Diffusion↗

PubMed2001

Signal transduction via both human low-affinity IgG Fc receptors, Fc gamma RIIa and Fc gamma RIIIb, depends on the activity of different families of intracellular kinases.

In contrast to IgG Fc receptor II (Fc gamma RIIa), the function of Src-family kinases, Syk and phosphoinositide 3-kinase (PI3K) in signal transduction of glycosylphosphatidylinositol-anchored Fc gamma RIIIb has not been analyzed in detail. Therefore pharmacological inhibitors were used to define the role of specific kinases in signalling of Fc gamma RIIa and Fc gamma RIIIb in myeloid cells. We demonstrate that the broad tyrosine kinase inhibitor genistein, the Src-family kinase inhibitor PP2, and the Syk kinase inhibitor piceatannol inhibit [Ca2+]i rise induced by both low-affinity Fc gamma R in neutrophils. Genistein and PP2 additionally prevent Fc gamma R-triggered hydrogen peroxide generation. In contrast, wortmannin, a PI3K inhibitor, which blocks Fc gamma RIIIb activation completely, abolishes Fc gamma RIIa-mediated [Ca2+]i flux only in the beginning. In addition, herbimycin A, a further specific inhibitor of Src-family kinases leads to a delayed Fc gamma RIIa-induced [Ca2+]i rise in THP-1 cells. In summary, our data demonstrate differences between both low-affinity IgG Fc receptors, and provide evidence for an essential role of Src-family kinases, Syk and PI3K in Fc gamma RIIIb-mediated signalling.

Androstadienes↗

PubMed2001

Differential mRNA degradation of two beta-tubulin isoforms correlates with cytosolic Ca2+ changes in glucan-elicited soybean cells.

Transgenic soybean (Glycine max) culture cells expressing apoaequorin, a Ca2+ indicator, were exposed to glucan fragments derived from Phytophthora sojae or to chitin oligomers. The effects of these elicitors on cytosolic Ca2+ concentrations and on mRNA levels of two beta-tubulin isoforms, tubB1 and tubB2, were investigated. The glucan elicitors, to which the cells are known to react with a biphasic cytosolic Ca2+ increase, induced a down-regulation of the tubB1 mRNA levels while the tubB2 mRNA level remained constant. The decrease of tubB1 mRNA level was observed after 1 hour of glucan treatment. In contrast, chitin oligomers, known to provoke a monophasic Ca2+ increase of short duration, did not affect the tubB1 mRNA level. Pre-incubation with 10 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, an extracellular Ca2+ chelator, blocked the cytosolic Ca2+ increase as well as the decrease of tubB1 mRNA levels induced by glucan elicitors. Likewise, pre-incubation with 1 mM neomycin, which reduced only the second glucan-induced Ca2+ peak, blocked the decrease of tubB1 mRNA level. Experiments with cordycepin, a transcription inhibitor, indicated that glucan fragments induced the degradation of tubB1 mRNA. In conclusion, the glucan-induced cytosolic Ca2+ changes are correlated with a strong increase in tubB1 mRNA degradation.

Base Sequence↗

PubMed2000

Self-assembly of the vascular endothelial cadherin ectodomain in a Ca2+-dependent hexameric structure.

Vascular endothelial cadherin (VE-cadherin) is a transmembrane protein essential for endothelial cell monolayer integrity (Gulino, D., Delachanal, E., Concord, E., Genoux, Y., Morand, B., Valiron, M. O., Sulpice, E., Scaife, R., Alemany, M., and Vernet, T. (1998) J. Biol. Chem. 273, 29786-29793). This molecule belongs to the cadherin family of cell-cell adhesion receptors, for which molecular details of homotypic interactions are still lacking. In this study, a recombinant fragment encompassing the four N-terminal modules of VE-cadherin (VE-EC1-4) was shown to associate, in solution, as a stable Ca(2+)-dependent oligomeric structure. Cross-linking experiments combined with mass spectrometry demonstrated that this oligomer is a hexamer. Gel filtration chromatography experiments and analytical ultracentrifugation analyses revealed the existence of an equilibrium between the hexameric and monomeric species of VE-EC1-4. The concentration at which 50% of VE-EC1-4 is in its hexameric form was estimated as 1 microm. The dimensions of the hexamer, measured by cryoelectron microscopy to be 233 +/- 10 x 77 +/- 7 A, are comparable to the thickness of adherens endothelial cell-cell junctions. Altogether, the results allow us to propose a novel homotypic interaction model for the class II VE-cadherin, in which six molecules of cadherin form a hexamer.

Animals↗

PubMed2000

Insights into the molecular relationships between malate and lactate dehydrogenases: structural and biochemical properties of monomeric and dimeric intermediates of a mutant of tetrameric L-[LDH-like] malate dehydrogenase from the halophilic archaeon Haloarcula marismortui.

L-Malate (MalDH) and L-lactate (LDH) dehydrogenases belong to the same family of NAD-dependent enzymes. LDHs are tetramers, whereas MalDHs can be either dimeric or tetrameric. To gain insight into molecular relationships between LDHs and MalDHs, we studied folding intermediates of a mutant of the LDH-like MalDH (a protein with LDH-like structure and MalDH enzymatic activity) from the halophilic archaeon Haloarcula marismortui (Hm MalDH). Crystallographic analysis of Hm MalDH had shown a tetramer made up of two dimers interacting mainly via complex salt bridge clusters. In the R207S/R292S Hm MalDH mutant, these salt bridges are disrupted. Its structural parameters, determined by neutron scattering and analytical centrifugation under different conditions, showed the protein to be a tetramer in 4 M NaCl. At lower salt concentrations, stable oligomeric intermediates could be trapped at a given pH, temperature, or NaCl solvent concentration. The spectroscopic properties and enzymatic behavior of monomeric, dimeric, and tetrameric species were thus characterized. The properties of the dimeric intermediate were compared to those of dimeric intermediates of LDH and dimeric MalDHs. A detailed analysis of the putative dimer-dimer contact regions in these enzymes provided an explanation of why some can form tetramers and others cannot. The study presented here makes Hm MalDH the best characterized example so far of an LDH-like MalDH.

Anilino Naphthalenesulfonates↗

PubMed2000

On the domain structure and the polymerization state of the sendai virus P protein.

The phosphoproteins (P) of paramyxoviruses and rhabdoviruses are cofactors of the viral polymerase (L) and chaperones of soluble nucleoprotein preventing its polymerization and nonspecific binding to cellular RNA. The primary sequences of six paramyxovirus P proteins were compared, and although there was virtually no sequence similarity, there were two regions with similar secondary structure predictions in the C-terminal part of P: the predicted multimerization domain and the X-protein, the sequence that binds to N in the N:RNA template. The C-terminal part of the Sendai virus P protein, the multimerization domain including the binding site for the polymerase, and the X-protein were expressed in Escherichia coli. All three polypeptides folded with secondary structures similar to those predicted. The C-terminal part of P is a very elongated molecule with most of its length encompassing the multimerization domain. Both the multimerization domain and the C-terminal part of P were found to form tetramers, whereas the X-protein was monomeric.

Amino Acid Sequence↗

PubMed2000

Characterization of trans-splicing in Euglenoids.

We have looked for trans-splicing of nuclear mRNAs in several Euglenoid species. In Cyclidiopsis acus, Phacus curvicauda, Rhabdomonas costata and Menoidium pellucidum we showed that several premRNAs chosen at random are matured by a transsplicing process: we identified SL-RNA genes whose 5' ends (SLs for spliced leader-sequences) were transferred to the 5' extremities of mRNAs. The SL-RNA genes are located on repeated DNA fragments which also encode 5S rRNA in P. curvicauda and C. acus. The potential secondary structures of SL-RNAs are compared to those previously characterized in two other Euglenoids: Euglena gracilis and Entosiphon sulcatum. In another Euglenoid species, Distigma proteus, since none of the mRNAs examined were trans-spliced, it is possible that trans-splicing does not occur. Phylogeny based on 5S rRNA sequences suggests that the species which have, or have had, chloroplasts (E. gracilis, P. curvicauda, C. acus) diverged early from the others.

Animals↗

PubMed2000

Halophilic adaptation of enzymes.

It is now clear that the understanding of halophilic adaptation at a molecular level requires a strategy of complementary experiments, combining molecular biology, biochemistry, and cellular approaches with physical chemistry and thermodynamics. In this review, after a discussion of the definition and composition of halophilic enzymes, the effects of salt on their activity, solubility, and stability are reviewed. We then describe how thermodynamic observations, such as parameters pertaining to solvent-protein interactions or enzyme-unfolding kinetics, depend strongly on solvent composition and reveal the important role played by water and ion binding to halophilic proteins. The three high-resolution crystal structures now available for halophilic proteins are analyzed in terms of haloadaptation, and finally cellular response to salt stress is discussed briefly.

Adaptation, Physiological↗

PubMed2000

Probing protein-sugar interactions.

We have investigated the partial specific volumes (2) (ml/g), hydration, and cosolvent interactions of rabbit muscle aldolase by equilibrium sedimentation in the analytical ultracentrifuge and by direct density increment (partial differential/partial differentialc(2))(mu) measurements over a range of sugar concentrations and temperature. In a series of sugars increasing in size, glucose, sucrose, raffinose, and alpha-cyclodextrin, (partial differential/ partial differentialc(2))(mu) decreases linearly with the solvent density rho(0). These sugar cosolvents do not interact with the protein; however, the interaction parameter B(1) (g water/g protein) mildly increases with increasing sugar size. The experimental B(1) values are smaller than values calculated by excluded volume (rolling ball) considerations. B(1) relates to hydration in this and in other instances studied. It decreases with increasing temperature, leading to an increase in (2) due to reduced water of hydration electrostriction. The density increments (partial differential/ partial differentialc(2))(mu), however, decrease in concave up form in the case of glycerol and in concave down form for trehalose, leading to more complex behavior in the case of carbohydrates playing a biological role as osmolytes and antifreeze agents. A critical discussion, based on the thermodynamics of multicomponent solutions, is presented.

Animals↗

PubMed1999

Relative role of anions and cations in the stabilization of halophilic malate dehydrogenase.

Halophilic malate dehydrogenase unfolds at low salt, and increasing the salt concentration stabilizes, first, the folded form and then, in some cases, destabilizes it. From inactivation and fluorescence measurements performed on the protein after its incubation in the presence of various salts in a large range of concentrations, the apparent effects of anions and cations were found to superimpose. A large range of ions was examined, including conditions that are in general not of physiological relevance, to explore the physical chemistry driving adaptation to extreme environments. The order of efficiency of cations and anions to maintain the folded form is, for the low-salt transition, Ca(2+) approximately Mg(2+) > Li(+) approximately NH(4)(+) approximately Na(+) > K(+) > Rb(+) > Cs(+), and SO(4)(2)(-) approximately OAc(-) approximately F(-) > Cl(-), and for the high-salt transition, NH(4)(+) approximately Na(+) approximately K(+) approximately Cs(+) > Li(+) > Mg(2+) > Ca(2+), and SO(4)(2)(-) approximately OAc(-) approximately F(-) > Cl(-) > Br(-) > I(-). If a cation or anion is very stabilizing, the effect of the salt ion of opposite charge is limited. Anions of high charge density are always the most efficient to stabilize the folded form, in accordance with the order found in the Hofmeister series, while cations of high charge density are the most efficient only at the lower salt concentrations and tend to denature the protein at higher salt concentrations. The stabilizing efficiency of cations and anions can be related in a minor way to their effect on the surface tension of the solution, but the interaction of ions with sites only present in the folded protein has also to be taken into account. Unfolding at high salt concentrations corresponds to interactions of anions of low charge density and cations of high charge density with the peptide bond, as found for nonhalophilic proteins.

Acetates↗