Biomedical subjects

E Leclerc

Publications and source records attributed to E Leclerc.

17 recordsLinked to original sources

PubMed2006

Copper induces conformational changes in the N-terminal part of cell-surface PrPC.

Prion diseases are caused by misfolding of the cellular prion protein, PrPC. In vitro studies have shown that PrP binds copper via the octarepeat region lying within the unstructured N-terminal segment of the protein, but the significance of copper in PrP metabolism remains unclear. Here, six specific antibodies recognizing different epitope regions of PrP were used to measure the effect of copper on the conformation of the molecule at the cell surface. Binding of an antibody, E149, to an epitope within the octarepeat domain of PrP is halved in the presence of copper, whereas binding of antibodies recognizing epitope motifs C-terminal to residue 90 of PrP remain relatively unaltered under equivalent conditions. These experiments strongly suggest that copper induces localized conformational change within the N-terminal portion of cell-surface PrPC.

Amino Acid Sequence↗

PubMed2005

Study of osteoblastic cells in a microfluidic environment.

Bone tissue engineering consists of culturing osteoblastic cells onto synthetic three-dimensional (3D) porous scaffolds. The organization of bone cells into 3D scaffolds is crucial for ex vivo tissue formation. Diffusional rates of nutrients could be greatly improved by perfusing media through the 3D microporous scaffolds. However, bone cells cultured in vitro are responsive to a variety of different mechanical signals including fluid flow and shear stresses. In this work, we attempt to study osteoblastic cells behaviour cultured within microdevices allowing continuous and homogenous feeding of cells. We have fabricated polydimethylsiloxane PDMS microdevices with a 3D microstructured channel network. Mouse calvarial osteoblastic cells MC3T3-E1 were seeded at 2x10(6)cells/ml and cultured into the microdevices under flow rates of 0, 5, 35 microl/min. Cells attached and proliferated well in the designed microdevices. Cell viability was found around 85% up to 1 to 2 weeks for shear stress value under 5 mPa. The alkaline phosphatase (ALP) activity was enhanced 3- and 7.5-fold inside the microdevices under static and dynamic flow of 5 microl/min as compared to flat static cultures in PDMS coated Petri dishes. Therefore, osteoblastic cells could be successfully cultured inside the microdevices under dynamic conditions and their ALP activity was enhanced. These results are promising for bone cell growth and differentiation as well as future tissue regeneration using larger 3D microfluidic microdevices.

3T3 Cells↗

PubMed2004

Fabrication of microstructures in photosensitive biodegradable polymers for tissue engineering applications.

Combining the MEMS technology and biology requirements for tissue engineering, the fabrication processes of microstructured chambers and microchannels made in biodegradable photosensitive polymers are presented. The fabrication processes, based on softlithography are very fast and flexible. Various single and multistepwise microstructures could be achieved using the biodegradable polymers. Microstructures down to 50microm, which are suitable for liver reconstructs, could be fabricated. As the pCLLA acrylate photosensitive polymer has interesting property for implantable bioreactors, that is, its softness, we examined the ability of various mammalian cells to grow and spread on it. With Hep G2 cells, human umbilical blood vessel endothelial cells (HUVEC), 3T3-L1 mouse fibroblasts, static cultures could be successfully performed on single stepwise microstructures. Then, by using this photosensitive biodegradable polymer, a microstructure with simple fluidic channels is fabricated and a perfusion experiment could be carried out. Both cell cultures and perfusion experiments suggested the possibility to use the present photosensitive polymer as microfluidic supports for biodegradable bioreactors for implantation applications.

3T3-L1 Cells↗

PubMed2002

Unusual property of prion protein unfolding in neutral salt solution.

The unfolding of cellular prion protein and its refolding to the scrapie isoform are related to prion diseases. Studies in the literature have shown that structures of proteins, either acidic or basic, are stabilized against denaturation by certain neutral salts, for example, sulfate and fluoride. Contrary to these observations, the full-length recombinant prion protein (amino acid residues 23-231) is denatured by these protein structure stabilizing salts. Under identical concentrations of salts, the structure of the sheep prion protein, which contains a greater number of glycine groups in the N-terminal unstructured segment than the mouse protein, becomes more destabilized. In contrast to the full-length protein, the C-terminal 121-231 prion protein fragment, consisting of all the structural elements of the protein, viz., three alpha-helices and two short beta-strands, is stabilized against denaturation by these salts. We suggest that an increase in the concentration of the anions on the surface of the prion protein molecule due to their preferential interaction with the glycine residues in the N-terminal segment destabilizes the structure of the prion protein by perturbing the prion helix 1 which is the most soluble of all the protein alpha-helices reported so far in the literature. The present results could be relevant to explain the observed structural conversion of the prion protein by anionic nucleic acids and sulfated glycosaminoglycans.

Amino Acid Sequence↗

PubMed2002

DNA-induced partial unfolding of prion protein leads to its polymerisation to amyloid.

The full-length mouse recombinant prion protein (23-231 amino acid residues) contains all of its structural elements viz. three alpha-helices and a short two-stranded antiparallel beta-sheet in its C-terminal fragment comprising 121-231 amino acid residues. The incubated mixture of this prion protein fragment and nucleic acid results in the formation of amyloid fibres evidenced from electron microscopy, birefringence and fluorescence of the fibre bound Congo Red and Thioflavin T dyes, respectively. The secondary structure of the amyloid formed in nucleic acid solution is similar to the in vivo isolated prion protein 27-30 amyloid but unlike in it, a hydrophobic milieu is absent in the 121-231 amyloid. Thermal denaturation study demonstrates a partial unfolding of the protein fragment in nucleic acid solution. We propose that nucleic acid catalyses unfolding of prion protein helix 1 followed by a nucleation-dependent polymerisation of the protein to amyloid.

Amyloid↗

PubMed2001

Antibodies inhibit prion propagation and clear cell cultures of prion infectivity.

Prions are the transmissible pathogenic agents responsible for diseases such as scrapie and bovine spongiform encephalopathy. In the favoured model of prion replication, direct interaction between the pathogenic prion protein (PrPSc) template and endogenous cellular prion protein (PrPC) is proposed to drive the formation of nascent infectious prions. Reagents specifically binding either prion-protein conformer may interrupt prion production by inhibiting this interaction. We examined the ability of several recombinant antibody antigen-binding fragments (Fabs) to inhibit prion propagation in cultured mouse neuroblastoma cells (ScN2a) infected with PrPSc. Here we show that antibodies binding cell-surface PrPC inhibit PrPSc formation in a dose-dependent manner. In cells treated with the most potent antibody, Fab D18, prion replication is abolished and pre-existing PrPSc is rapidly cleared, suggesting that this antibody may cure established infection. The potent activity of Fab D18 is associated with its ability to better recognize the total population of PrPC molecules on the cell surface, and with the location of its epitope on PrPC. Our observations support the use of antibodies in the prevention and treatment of prion diseases and identify a region of PrPC for drug targeting.

Animals↗

PubMed2001

Immobilized prion protein undergoes spontaneous rearrangement to a conformation having features in common with the infectious form.

It is hypothesized that infectious prions are generated as the cellular form of the prion protein (PrP(C)) undergoes pronounced conformational change under the direction of an infectious PrP(Sc) template. Conversion to the infectious conformer is particularly associated with major structural rearrangement in the central portion of the protein (residues 90-120), which has an extended flexible structure in the PrP(C) isoform. Using a panel of recombinant antibodies reactive with different parts of PrP, we show that equivalent major structural rearrangements occur spontaneously in this region of PrP immobilized on a surface. In contrast, regions more towards the termini of the protein remain relatively unaltered. The rearrangements occur even under conditions where individual PrP molecules should not contact one another. The propensity of specific unstructured regions of PrP to spontaneously undergo large and potentially deleterious conformational changes may have important implications for prion biology.

Animals↗

PubMed2001

Anti-DNA antibodies are a major component of the intrathecal B cell response in multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of unknown cause that afflicts the central nervous system. MS is typified by a highly clonally restricted antigen-driven antibody response that is confined largely to the central nervous system. The major antigenic targets of this response and the role of antibody in disease pathogenesis remain unclear. To help resolve these issues, we cloned the IgG repertoire directly from active plaque and periplaque regions in MS brain and from B cells recovered from the cerebrospinal fluid of a patient with MS with subacute disease. We found that high-affinity anti-DNA antibodies are a major component of the intrathecal IgG response in the patients with MS that we studied. Furthermore, we show DNA-specific monoclonal antibodies rescued from two subjects with MS as well as a DNA-specific antibody rescued from an individual suffering from systemic lupus erythematosus bound efficiently to the surface of neuronal cells and oligodendrocytes. For two of these antibodies, cell-surface recognition was DNA dependent. Our findings indicate that anti-DNA antibodies may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as MS and systemic lupus erythematosus.

Adult↗

PubMed2001

Phosphorylation of serine residues affects the conformation of the calmodulin binding domain of human protein 4.1.

We have previously characterized the calcium-dependent calmodulin (CaM)-binding domain (Ser76-Ser92) of the 135-kDa human protein 4.1 isoform using fluorescence spectroscopy and chemically synthesized nonphosphorylated or serine phosphorylated peptides [Leclerc, E. & Vetter, S. (1998) Eur. J. Biochem. 258, 567-671]. Here we demonstrate that phosphorylation of two serine residues within the 17-residue peptide alters their ability to adopt alpha helical conformation in a position-dependent manner. The helical content of the peptides was determined by CD-spectroscopy and found to increase from 36 to 45% for the Ser80 phosphorylated peptide and reduce to 28% for the Ser84 phosphorylated peptide; the di-phosphorylated peptide showed 32% helical content. Based on secondary structure prediction methods we propose that initial helix formation involves the central residues Leu82-Phe86. The ability of the peptides to adopt alpha helical conformations did not correlate with the observed binding affinities to CaM. We suggest that the reduced CaM-binding affinities observed for the phosphorylated peptides are more likely to be the result of unfavorable sterical and electrostatic interactions introduced into the CaM peptide-binding interface by the phosphate groups, rather than being due to the effect of phosphorylation on the secondary structure of the peptides.

Arginine↗

PubMed2000

Selection and characterization of single chain Fv fragments against murine recombinant prion protein from a synthetic human antibody phage display library.

We describe the selection of single chain Fv fragments (scFv) against recombinant murine prion protein (mPrP) from a synthetic human antibody phage display library. Six different antibodies were isolated after three rounds of panning against full-length mPrP. All antibodies recognized a truncated form of mPrP containing residues (121-231). The amino acid sequence of the CDR3 of the scFv fragments has been determined. Five of the antibodies have been over-expressed, purified and their affinity for full-length mPrP determined by ELISA. The observed binding affinities vary from 30 nM to 2.7 microM.

Amino Acid Sequence↗

PubMed1999

Serine/threonine phosphorylation of calmodulin modulates its interaction with the binding domains of target enzymes.

The interaction of serine/threonine-phosphorylated calmodulin with synthetic peptides corresponding to the calmodulin-binding domains of six enzymes has been studied by fluorescence spectroscopy. For five peptides, the dissociation constant of the calmodulin-peptide complex (K(d)) increased when calmodulin was phosphorylated. An increase of more than one order of magnitude was observed with peptides derived from smooth-muscle myosin light-chain kinase and cAMP phosphodiesterase. In contrast, only a slight increase in K(d) was noted with two peptides derived from the plasma membrane Ca(2+)-ATPase and for the peptide derived from nitric oxide synthase. No significant change in affinity was detected with the peptide derived from calcineurin. In contrast, a decrease in the dissociation constant was observed with the peptide derived from the Ca(2+)-calmodulin dependent kinase II. Phosphorylation also affected the peptide-calmodulin binding stoichiometry: a decrease from two to one binding sites was observed with the peptides derived from myosin light-chain kinase and phosphodiesterase.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1999

Asymmetric Multiblock Copolymers at the Gas-Liquid Interface: Phase Diagram and Surface Pressure.

A theoretical model of copolymers made of N blocks is studied at the air-water interface. Each block is made of a sequence A of ZA hydrophobic and of a sequence B of ZB hydrophilic monomers. The A and B sequences cannot cross the interface. The conformation of an adsorbed polymer is determined as a random walk of N elements whose size is the Flory radius of a single sequence. The structure of the interfacial layer is determined as a function of alpha = ZA/ZB and of the surface concentration using scaling law arguments. Only three different regions are found in the phase diagram to describe the change of surface regime as a function of the total surface concentration. The energy of flower-like micelles of polymers is calculated and compared with the energy of adsorbed macromolecules in order to determine the surface concentration at saturation. The surface pressure is also calculated as a function of the surface concentration in the three different regions of the phase diagram. It is found that these surface pressure isotherms are not affected by the solvent quality except when the properties of the interfacial layer are dominated by a purely two-dimensional behavior (semidiluted regime of the whole polymer or of the A sequences on the air side of the interface). Finally the properties of this model are compared with experimental data obtained with protein adsorbed layers and encouraging agreement is found although proteins are much more complicated polymers than this crude model. Copyright 1999 Academic Press.

Journal Article↗

PubMed1999

Polymerization of murine recombinant prion protein in nucleic acid solution.

Recombinant prion protein has been used earlier to understand the structural properties of cellular prion protein PrP(C) and to understand conformational change of PrP(C) to its isoform, PrP(Sc) which is believed to be responsible for the prion disease. Here we report that murine recombinant prion protein, MoPrP(C) polymerizes in the presence of nucleic acid. The aggregation process and the properties of the aggregates have been monitored by physical, biochemical and ultrastructural studies. An increase in the turbidity at 0,90 degrees light scattering is observed when the protein is added to nucleic acid. An increase in the fluorescence of anilino naphthalene sulfonic acid dye (ANS) accompanying a blue shift in its emission maxima is observed when the aggregate obtained from prion protein and DNA reaction is added to it. The kinetics of the increase of the ANS fluorescence during aggregation process show lag periods which depend linearly on the nucleic acid concentration but show a biphasic dependence on the protein concentration. The change in the fluorescence properties of the dye in the presence of the aggregates obtained in the present study and in the presence of the protein PrP 27-30 amyloid isolated in vivo reported in literature are similar. The dye Congo Red binds to the aggregates resulting from the aggregation reaction.The ultrastructural analysis revealed polymeric structures with amyloid like morphologies and smaller oligomeric structures. In addition, condensed nucleic acid structures are also observed which are morphologically different from histone induced condensed nucleic acid structures but are similar to Human Immunodeficiency Virus-1 nucleocapsid protein, NCp7, induced nucleic acid structures. The aggregates show resistance to degradation by proteinase K treatment. Charge neutralization resulting from the MoPrP(C)-DNA interaction and accompanying structural changes in the molecules may explain the observed effects.

Anilino Naphthalenesulfonates↗

PubMed1998

Characterization of a calcium-dependent calmodulin-binding domain in the 135-kD human protein 4.1 isoform.

The putative calmodulin binding domain of non-erythroid protein 4.1, previously suggested by Kelly et al. [Kelly, G. M., Zelus, B. D. & Moon, R. T. (1991) J. Biol. Chem. 266, 12469-12473] has been synthesized, and its binding to calmodulin has been studied by fluorescence spectroscopy. For this purpose, the peptide has been N-terminally dansylated. The 4.1 peptide Dns-Abu-S76RGLSRLFSSFLKRPKS92, binds calmodulin in a calcium-dependent way with high affinity (Kd = 23 +/- 6 nM). The peptide inhibits bovine-heart phosphodiesterase with an IC50 of 50 nM. Since the sequence of the peptide shows two putative consensus sites of phosphorylation by cAMP-dependent protein kinase or Ca2+-calmodulin protein-kinase II, the interaction of the two mono-phosphorylated peptides (P4.1 Ser(80-P) and P4.1 Ser(84-P)) and the di-phosphorylated peptide (P4.1 Ser(80-P)/Ser(84-P)) with calmodulin has been investigated. A decrease of affinity by a factor 1.5-8 has been observed for the phosphorylated peptides. CD measurements have shown an increase of the content of alpha helices in the peptides when bound to calmodulin.

Amino Acid Sequence↗

PubMed1995

[A computerized nursing care and services management tool].

The Quebec government has initiated a project to review the funding of health care establishments by determining the average cost of a hospital stay. A committee of nursing directors studied existing nursing care measurement tools and decided to develop a new tool better suited to computerized care plans. The new tool is called MESSII, after the French acronym "Mesure des soins et des services infirmiers informatisée," meaning computerized measurement of nursing care and services. This article presents the development and validation stages of MESSII.

Humans↗

PubMed1993

Interaction of heme with amphiphilic peptides: use of hemin-CN to probe the interaction of calmodulin with its target peptides.

UNLABELLED: The interaction of heme with several amphiphilic peptides has been studied by absorption and fluorescence spectroscopy. The binding can be followed by the changes in the absorption spectrum of the heme group or by the decrease in the peptide tryptophan fluorescence due to energy transfer to the heme. Despite their small size, ranging from 26 residues for melittin to 14 for mastoporan, a high affinity for heme-CO and hemin-CN (Kd < 100 nM) may be observed. Spectral shifts in the absorption peaks and appreciable geminate recombination after photodissociation of CO from the complex peptide-heme-CO suggest the formation of a heme pocket, as for the natural heme proteins. APPLICATION: hemin-CN can be used as a probe for the interaction of calmodulin with these target peptides. Amphiphilic peptides such as melittin bind to calmodulin with a high (nM) affinity. While both the peptide and calcium-bound calmodulin bind heme-CO, only the peptide binds hemin-CN. These interactions permit studies of the competition between hemin-CN and calmodulin for binding to the peptide: while hemin-CN quenches the melittin tryptophan fluorescence, addition of calmodulin to the [melittin*hemin-CN] complex displaces the hemin-CN and the melittin tryptophan fluorescence is recovered.

Amino Acid Sequence↗

PubMed1993

Heme binding to calmodulin, troponin C, and parvalbumin, as a probe of calcium-dependent conformational changes.

Heme-CO binds to the active (calcium-bound) form of calmodulin (CaM), but not to the inactive form. Despite a similarity in structure of another calcium-binding protein, skeletal muscle troponin C, both the affinity and the spectral red-shift of the absorption of the heme group are greatly decreased for troponin C relative to calmodulin. Parvalbumin, another calcium-binding protein, shows a twofold greater affinity for heme-CO relative to CaM. Unlike calmodulin and troponin C, the affinity of parvalbumin for heme-CO is even greater in the absence of calcium. The affinity of the tryptic and thrombic fragments of CaM for heme-CO are decreased relative to the entire calmodulin. The binding of heme-CO is specific as demonstrated by the discrimination of the calmodulin, troponin C, and parvalbumin pockets. The interaction of heme-CO with active (calcium-bound) CaM is rapid (ms) as determined by stopped flow measurements. No difference in kinetics was observed for mixing inactive (calcium free) CaM with a solution of [heme-CO plus calcium], indicating that the calcium-binding step and subsequent change in protein conformation are rapid.

Animals↗