Laurent Bédouet

Soluble proteins of the nacre of the giant oyster Pinctada maxima and of the abalone Haliotis tuberculata:: extraction and partial analysis of nacre proteins

Several proteins from nacre of the oyster Pinctada maxima and the abalone Haliotis tuberculata were extracted and partly characterized. Proteins dispersed in aragonite were solubilized during demineralization with acetic acid whereas proteins adsorbed on conchiolin were extracted with sodium dodecyl sulfate and beta-mercaptoethanol. The matrix of Pinctada maxima nacre is composed of one main protein with an apparent molecular weight of 20 kDa (p20). This protein was found in the acetic acid soluble fraction of nacre, as well as in the Laemmli-solubilized extract of conchiolin. In addition, the p20 solubilized with acetic acid can form oligomers made of 6 monomers linked together by disulfide bridges. The first N-terminal 21 amino acids of p20 were determined and no homology with known proteins was found. In Haliotis tuberculata nacre, 5 main proteins were solubilized during demineralization and 3 glycoproteins were detected. Stains-all and Alcian blue staining revealed polyanionic proteins in the extracts isolated from Pinctada maxima and Haliotis tuberculata nacre.

Evolution of Nacre: Biochemistry and Proteomics of the Shell Organic Matrix of the Cephalopod Nautilus macromphalus

Matrix evolutions: We have biochemically characterized the nacre matrix of the cephalopod Nautilus macromphalus, in part by a proteomic approach applied to the acetic acid‐soluble and ‐insoluble shell matrices, as well as to spots obtained after 2D gel electrophoresis. Strikingly, most of the obtained partial sequences are entirely new, whereas a few correspond only partly with bivalvian nacre proteins. Our findings shed new light on the macroevolution of nacre matrix proteins.

Nacre Calcification in the Freshwater Mussel Unio pictorum: Carbonic Anhydrase Activity and Purification of a 95 kDa Calcium-Binding Glycoprotein

The formation of the molluscan shell is finely tuned by macromolecules of the shell organic matrix. Previous results have shown that the acid‐soluble fraction of the nacre matrix of the freshwater paleoheterodont bivalve Unio pictorum shell displays a number of remarkable properties, such as calcium‐binding activity, the presence of extensive glycosylations and the capacity to interfere at low concentration with in vitro calcium carbonate precipitation. Here we have found that the nacre‐soluble matrix exhibits a carbonic anhydrase activity, an important function in calcification processes. This matrix is composed of three main proteinaceous discrete fractions. The one with the highest apparent molecular weight is a 95 kDa glycoprotein that is specific to the nacreous layer. P95, as it is provisionally named, is enriched in Gly, Glx and Asx and exhibits an apparent pI value of ∼4, or ∼7 when chemically deglycosylated. Furthermore, its glycosyl moiety, consisting of sulfated polysaccharides, is involved in calcium binding. Purified fractions of the three main proteins were digested with trypsin, and the resulting peptides were analysed by mass spectrometry. Our results suggest that identical peptides are constitutive domains of the different proteins. Partial primary structures were obtained by de novo sequencing and compared with known sequences from other mollusc shell proteins. Our results are discussed from an evolutionary viewpoint.

Characterization and quantification of chitosan extracted from nacre of the abalone Haliotis tuberculata and the oyster Pinctada maxima.

Abstract: This study was performed to characterize and quantify chitosan by simple physicochemical methods (infrared spectroscopy and potentiometric measurements). These procedures were validated with well-characterized chitosan before being used to investigate chitosan in nacre of the abalone Haliotis tuberculata and of the giant oyster Pinctada maxima. Potentiometric study revealed a chitosan extract from the nacre of H. tuberculata with a degree of deacetylation of around 88% and an intrinsic pK of 6.5. According to infrared and potentiometric data, a low yield (η) of extraction was calculated (η= 0.064%). For experiments performed on the nacre of P. maxima, and in spite of more stringent deacetylation conditions, results suggested that a chitin-protein complex (η= 0.053%) was isolated rather than chitosan.

Proteomics Analysis of the Nacre Soluble and Insoluble Proteins from the Oyster Pinctada margaritifera

Shell nacre is laid upon an organic cell-free matrix, part of which, paradoxically, is water soluble and displays biological activities. Proteins in the native shell also constitute an insoluble network and offer a model for studying supramolecular organization as a means of self-ordering. Consequently, difficulties are encountered in extraction and purification strategies for protein characterization. In this work, water-soluble proteins and the insoluble conhiolin residue of the nacre of Pinctada margaritifera matrix were analyzed via a proteomics approach. Two sequences homologous to nacre matrix proteins of other Pinctada species were identified in the water-soluble extract. One of them is known as a fundamental component of the insoluble organic matrix of nacre. In the conchiolin, the insoluble residue, four homologs of Pinctada nacre matrix proteins were found. Two of them were the same as the molecules characterized in the water-soluble extract. Results established that soluble and insoluble proteins of the nacre organic matrix share constitutive material. Surprisingly, a peptide in the conchiolin residue was found homologous to a prismatic matrix protein of Pinctada fucata, suggesting that prismatic and nacre matrices may share common proteins. The insoluble properties of shell matrix proteins appear to arise from structural organization via multimerization. The oxidative activity, found in the water-soluble fraction of the nacre matrix, is proposed as a leading process in the transformation of transient soluble proteins into the insoluble network of conchiolin during nacre growth.

Coupling Proteomics and Transcriptomics for the Identification of Novel and Variant Forms of Mollusk Shell Proteins: A Study with P. margaritifera

Shell matrix proteins from Pinctada margaritifera were characterized by combining proteomics analysis of shell organic extracts and transcript sequences, both obtained from the shell‐forming cell by using the suppression subtractive hybridization method (SSH) and from an expressed sequence tag (EST) database available from Pinctada maxima mantle tissue. Some of the identified proteins were homologues to proteins reported in other mollusk shells, namely lysine‐rich matrix proteins (KRMPs), shematrins and molluscan prismatic and nacreous layer 88 kDa (MPN88). Sequence comparison within and among Pinctada species pointed to intra‐ and interspecies variations relevant to polymorphism and to evolutionary distance, respectively. In addition, a novel shell matrix protein, linkine was identified. BLAST analysis of the peptide sequences obtained from the shell of P. margaritifera against the EST database revealed the presence of additional proteins: two proteins similar to the Pif97 protein that was identified in the shell of P. fucata, a chitinase‐like protein previously identified in Crassostrea gigas, two chitin‐binding proteins, and two incomplete sequences of proteins unknown so far in mollusk shells. Combining proteomics and transcriptomics analysis we demonstrate that all these proteins, including linkine, are addressed to the shell. Retrieval of motif‐forming sequences, such as chitin‐binding, with functional annotation from several peptides nested in the shell could indicate protein involvement in shell patterning.

Poly(ethylene glycol) methacrylate hydrolyzable microspheres for transient vascular embolization.

Poly(ethylene glycol) methacrylate (PEGMA) hydrolyzable microspheres intended for biomedical applications were readily prepared from poly(lactide-co-glycolide) (PLGA)-poly(ethylene glycol) (PEG)-PLGA crosslinker and PEGMA as a monomer using a suspension polymerization process. Additional co-monomers, methacrylic acid and 2-methylene-1,3-dioxepane (MDO), were incorporated into the initial formulation to improve the properties of the microspheres. All synthesized microspheres were spherical in shape, calibrated in the 300-500 μm range, swelled in phosphate-buffered saline (PBS) and easily injectable through a microcatheter. Hydrolytic degradation experiments performed in PBS at 37 °C showed that all of the formulations tested were totally degraded in less than 2 days. The resulting degradation products were a mixture of low-molecular-weight compounds (PEG, lactic and glycolic acids) and water-soluble polymethacrylate chains having molecular weights below the threshold for renal filtration of 50 kg mol(-1) for the microspheres containing MDO. Both the microspheres and the degradation products were determined to exhibit minimal cytotoxicity against L929 fibroblasts. Additionally, in vivo implantation in a subcutaneous rabbit model supported the in vitro results of a rapid degradation rate of microspheres and provided only a mild and transient inflammatory reaction comparable to that of the control group.

Intra-articular fate of degradable poly(ethyleneglycol)-hydrogel microspheres as carriers for sustained drug delivery

A novel degradable microsphere (MS) for intra-articular drug delivery, composed of a polyethylene glycol (PEG) core containing degradable regions made of short poly-(lactic-co-glycolic acid) (PLGA) sequences - named PEG-hydrogel MS - was injected into the cavity of sheep shoulder joint, and compared to non-degradable MS devoid of hydrolysable crosslinker in terms of location, degradation and inflammation. One week after intra-articular injection both groups of MS were localized beneath the synovial lining of the synovial fringes located at bottom of the shoulder joint, while a fraction of particles remained in synovial fluid. Histological analyses made one and 4 weeks after intra-articular injection showed cell proliferation around the non-degradable MS entrapped within the synovium. By contrast, degradable PEG-hydrogel MS were surrounded by few cells. The degradation of degradable PEG-hydrogel MS within the synovium was slow and was not fully complete after four weeks. Our findings indicate that the tissue entrapment of MS below the synovial lining was independent of the material degradability, while degradable PEG-hydrogel MS are less inflammatory than the non-degradable one. Degradable PEG-hydrogel MS offer several advantages over the non-degradable MS as carriers for a sustained drug delivery in synovial tissue according to the low intensity of inflammatory reaction triggered in synovium.

Synthesis of hydrophilic intra-articular microspheres conjugated to ibuprofen and evaluation of anti-inflammatory activity on articular explants.

The main limitation of current microspheres for intra-articular delivery of non-steroidal anti-inflammatory drugs (NSAIDs) is a significant initial burst release, which prevents a long-term drug delivery. In order to get a sustained delivery of NSAIDs without burst, hydrogel degradable microspheres were prepared by co-polymerization of a methacrylic derivative of ibuprofen with oligo(ethylene-glycol) methacrylate and poly(PLGA-PEG) dimethacrylate as degradable crosslinker. Microspheres (40-100 μm) gave a low yield of ibuprofen release in saline buffer (≈2% after 3 months). Mass spectrometry analysis confirmed that intact ibuprofen was regenerated indicating that ester hydrolysis occurred at the carboxylic acid position of ibuprofen. Dialysis of release medium followed by alkaline hydrolysis show that in saline buffer ester hydrolysis occurred at other positions in the polymer matrix leading to the release of water-soluble polymers (>6-8000 Da) conjugated with ibuprofen showing that degradation and drug release are simultaneous. By considering the free and conjugated ibuprofen, 13% of the drug is released in 3 months. In vitro, ibuprofen-loaded MS inhibited the synthesis of prostaglandin E2 in articular cartilage and capsule explants challenged with lipopolysaccharides. Covalent attachment of ibuprofen to PEG-hydrogel MS suppresses the burst release and allows a slow drug delivery for months and the cyclooxygenase-inhibition property of regenerated ibuprofen is preserved.

A Novel Resorbable Embolization Microsphere for Transient Uterine Artery Occlusion: A Comparative Study with Trisacryl-Gelatin Microspheres in the Sheep Model

PURPOSE To evaluate angiographic recanalization, inflammatory reaction, and uterine damage after sheep uterine artery embolization (UAE) with a novel calibrated resorbable embolization microsphere (REM) and compare the results with control nonresorbable microspheres. MATERIALS AND METHODS Six hormonally artificially cycled sheep underwent bilateral UAE until stasis with either REM or trisacryl-gelatin microspheres (TGMS). At 7 days, control angiograms were obtained to assess the residual vascularization at arterial and parenchymal phases. The animals were then sacrificed for analysis of the presence of microspheres, inflammatory foreign body reaction, and surface areas of uterine damage. RESULTS Mean volume of microspheres injected per uterine artery (UA) or per animal did not differ between groups. At day 7, the flow was normal for six of six UAs that received embolization with REM versus only three of six UAs with TGMS (P = .0455, χ(2) test). Uterine parenchymography showed no defects in six UAs in the REM group versus five defects in six UAs in the TGMS group (P = .0060, χ(2) test). No REM or residual fragments of microspheres were observed on histologic analysis. TGMS were observed in tissues and accompanied by a mild inflammatory response. Necrosis rates were not significantly different between the two products, either in endometrium (REM 23.5% ± 28.8% [median 8.1%] vs TGMS 21.8% ± 23.7% [median 14.6%]) or in myometrium (REM 8.2% ± 22.7% [median 0.0%] vs TGMS 8.8% ± 20.8% [median 0.9%]). Endometrium alteration rate was lower with REM than with TGMS (39.7% ± 25.7% [median 34%] vs 60.6% ± 27.1% [median 71%]; P = .0060, Mann-Whitney test). Myometrium alteration rates were not significantly different between REM (45.7% ± 37.1% [median 63.0%]) and TGMS (37.8% ± 34.0% [median 19.1%]). CONCLUSIONS At 1 week after sheep UAE with REM, the recanalization was complete, the microspheres were completely degraded, and there was no remnant inflammatory response.