Protein selective adsorption properties of a polyethylene terephtalate artificial ligament grafted with poly(sodium styrene sulfonate) (polyNaSS): correlation with physicochemical parameters of proteins.

Immediately after surgical placement of biomaterials, a first step consists in the adsorption of proteins from the biological environment on the artificial surfaces. Because the composition of the adsorbed protein layer modulates the cell response to the implanted material, researchers in the biomaterials field have focused on coating proteins or peptides onto surfaces to improve cell response and therefore the long-term compatibility of the implant. However, some materials used in tissue engineering, mainly synthetic polymers, are too hydrophobic to allow the optimal adsorption of proteins and have to be first submitted to physical or chemical treatments. In our laboratory, we have demonstrated that grafting of poly(sodium styrene sulfonate) (polyNaSS) onto biomaterials can strongly modulate the protein adsorption and the cellular response compared to unmodified surfaces. In this study, we used a liquid chromatography strategy coupled to proteomics to evaluate the adsorptive properties of a polyethylene terephtalate (PET) artificial ligament grafted with polyNaSS, and to identify and analyse proteins adsorbed on PET fibers. Results obtained with platelet rich plasma (PRP) proteins demonstrated that grafting significantly increases the protein adsorption of the PET and also selectively modulates the adsorption of proteins on PET fibers. Finally, regarding physicochemical parameters calculated from the amino acid sequence of identified proteins, we found that the aliphatic index is highly correlated with the selective adsorption of proteins onto the polyNaSS/PET surface. Therefore, the proteomic approach complemented with physicochemical property evaluation could provide a powerful tool for the elaboration of new biomaterials based on protein layer deposition.

New beam monitoring tool for radiobiology experiments at the cyclotron ARRONAX.

The ARRONAX cyclotron is able to deliver alpha particles at 68 MeV. In the frame of radiological research, a new method is studied to infer in situ the deposited dose: it is based on the online measurement of the bremsstrahlung (>1 keV) produced by the interaction of the incident particle with the medium. Experiments are made using bombarded poly(methyl methacrylate) (PMMA)-equivalent water targets in order to characterise this continuous X-ray spectrum. The intensity of the bremsstrahlung spectrum allows for the beam monitoring. A simulation code of the bremsstrahlung has been built, and a good agreement is found with the experimental spectra. With this simulation, it is possible to predict the sensibility of this method: it varies with the target thickness, showing a good sensibility for thin target (<1000 µm) and saturation for thicker ones. Bremsstrahlung spectrum also shows a sensibility on the target's chemical composition.

Biotribocorrosion (tribo-electrochemical) characterization of anodized titanium biomaterial containing calcium and phosphorus before and after osteoblastic cell culture.

The purpose of this study was to investigate the relationship between the osteoblastic cells behavior and biotribocorrosion phenomena on bioactive titanium (Ti). Ti substrates submitted to bioactive anodic oxidation and etching treatments were cultured up to 28 days with MG63 osteoblast-like cells. Important parameters of in vitro bone-like tissue formation were assessed. Although no major differences were observed between the surfaces topography (both rough) and wettability (both hydrophobic), a significant increase in cell attachment and differentiation was detected on the anodized substrates as product of favorable surface morphology and chemical composition. Alkaline phosphatase production has increased (≈20 nmol/min/mg of protein) on the anodized materials, while phosphate concentration has reached the double of the etched material and calcium production increased (over 20 µg/mL). The mechanical and biological stability of the anodic surfaces were also put to test through biotribocorrosion sliding solicitations, putting in evidence the resistance of the anodic layer and the cells capacity of regeneration after implant degradation. The Ti osteointegration abilities were also confirmed by the development of strong cell-biomaterial bonds at the interface, on both substrates. By combining the biological and mechanical results, the anodized Ti can be considered a viable option for dentistry.

EBT2 films response to alpha radiation at 48.3 MeV.

To advance the development of a radiobiological experimental set-up for alpha particle irradiations at the Arronax cyclotron, experiments were performed to get the dose response of Gafchomic EBT2 films for alpha particles at 48.3 MeV. A system has been developed using a thin monitor copper foil and an X-ray spectrometer to measure the beam intensity and to calculate the delivered dose. On the other hand, the authors have irradiated EBT2 films, with 6-MV X rays, to get the dose response of EBT2 films for photons. The dose response curve for alpha particles shows an effect of polymerisation saturation compared with the dose response curve for photons.

Mast cells rescue implantation defects caused by c-kit deficiency.

Various physiologically relevant processes are regulated by the interaction of the receptor tyrosine kinase (c-Kit) and its ligand stem cell factor (SCF), with SCF known to be the most important growth factor for mast cells (MCs). In spite of their traditional role in allergic disorders and innate immunity, MCs have lately emerged as versatile modulators of a variety of physiologic and pathologic processes. Here we show that MCs are critical for pregnancy success. Uterine MCs presented a unique phenotype, accumulated during receptivity and expanded upon pregnancy establishment. Kit(W-sh/W-sh) mice, whose MC deficiency is based on restricted c-Kit gene expression, exhibited severely impaired implantation, which could be completely rescued by systemic or local transfer of wild-type bone marrow-derived MCs. Transferred wild-type MCs favored normal implantation, induced optimal spiral artery remodeling and promoted the expression of MC proteases, transforming growth factor-ß and connective tissue growth factor. MCs contributed to trophoblast survival, placentation and fetal growth through secretion of the glycan-binding protein galectin-1. Our data unveil unrecognized roles for MCs at the fetomaternal interface with critical implications in reproductive medicine.

The osteogenic differentiation improvement of human mesenchymal stem cells on titanium grafted with polyNaSS bioactive polymer.

Osseointegration of metallic implants used in orthopedic surgery requires that osteoprogenitor cells attach and adhere to the surface, then proliferate, differentiate into osteoblasts, and finally produce mineralized matrix. Because the ability of progenitor cells to attach to a scaffold surface during early stages is important in the development of new tissue structures, we developed in our laboratory, a strategy involving grafting of implants with a polymer of sodium styrene sulfonate (polyNaSS) used as a scaffold which enables human mesenchymal stem cells (hMSCs) interactions. In the present study, we investigated the cellular response of hMSCs to polyNaSS surfaces of titanium (Ti). In particular, cell proliferation, cell viability, cell differentiation, and cell spreading were evaluated. Results showed that cell proliferation and cell viability did not differ with any statistical significance between modified and unmodified Ti surfaces. Interestingly, culture of MSCs on polyNaSS surfaces resulted in a significant increase of cell spreading and cell differentiation compared with the other tested surfaces. These results suggest that titanium surface grafted with polyNaSS is a suitable scaffold for bone tissue engineering.

Development of proteomic tools to study protein adsorption on a biomaterial, titanium grafted with poly(sodium styrene sulfonate).

It is known that protein adsorption is the initial interaction between implanted biomaterials and biological environment. Generally, a complex protein layer will be formed on material surfaces within a few minutes and the composition of this layer at the interface determines the biological response to the implanted material, and therefore the long-term compatibility of the biomaterial. Despite different techniques exist to observe protein adsorption on biomaterials, none of them led to the identification of adsorbed proteins. In this paper, we report a chromatographic technique coupled to proteomics to analyse and identify proteins from complex biological samples adsorbed on biomaterial surfaces. This approach is based on (1) elaboration of the chromatographic support containing the biomaterial (2) a chromatography step involving adsorption of proteins on the biomaterial (3) the high-resolution separation of eluted proteins by 2-DE gel and (4) the identification of proteins by mass spectrometry. Experiments were performed with proteins from platelets rich plasma (PRP) adsorbed on a biomaterial which consist in titanium bioactivated with PolyNaSS. Our results show that chromatographic approach combined to 2-DE gels and mass spectrometry provides a powerful tool for the analysis and identification of proteins adsorbed on various surfaces.

Galectin-1 promotes basal and kainate-induced proliferation of neural progenitors in the dentate gyrus of adult mouse hippocampus.

We examined the expression of galectin-1, an endogenous lectin with one carbohydrate-binding domain, in the adult mouse hippocampus after systemic kainate administration. We found that the expression of galectin-1 was remarkably increased in activated astrocytes of the CA3 subregion and dentate gyrus of the hippocampus, and in nestin-positive neural progenitors in the dentate gyrus. Quantitative reverse transcription PCR (RT-PCR) analysis revealed that the galectin-1 mRNA level in hippocampus began to increase 1 day after kainate administration and that a 13-fold increase was attained within 3 days. Western blotting analysis confirmed that the level of galectin-1 protein increased to more than three-fold a week after the exposure. We showed that isolated astrocytes express and secrete galectin-1. To clarify the significance of the increased expression of galectin-1 in hippocampus, we compared the levels of hippocampal cell proliferation in galectin-1 knockout and wild-type mice after saline or kainate administration. The number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells detected in the subgranular zone (SGZ) of galectin-1 knockout mice decreased to 62% with saline, and to 52% with kainate, as compared with the number seen in the wild-type mice. Most of the BrdU-positive cells in SGZ expressed doublecortin and neuron-specific nuclear protein, indicating that they are immature neurons. We therefore concluded that galectin-1 promotes basal and kainate-induced proliferation of neural progenitors in the hippocampus.

Intracellular localisation of galectin-3 has a protective role in chondrocyte survival.

OBJECTIVE: Although galectin-3 (gal-3) is expressed during arthritic disorders, the part it plays has never been described. The aim of the study was to determine the intracellular roles of gal-3 in chondrocytes and cartilage. METHODS: Following treatment with sodium nitroprusside, a cell death inducer, intracellular levels of total and phosphorylated gal-3 were measured by immunoblots in human osteoarthritic (OA) chondrocytes. Cell viability was also assessed by the lactate dehydrogenase activity in conditioned media from OA chondrocytes or from ATDC5 cells transfected with a gal-3-expressing vector. After generating an OA model by intra-articular injection of 0.5% mono-iodoacetate (MIA), histological evaluation of articular cartilage and subchondral bone was performed in wild-type (WT) and gal-3 knockout (KO) mice aged 6 weeks and 4 months. RESULTS: In vitro experiments demonstrated that intracellular gal-3 had a protective role in chondrocyte survival, which involved its phosphorylation. In contrast to 6-week-old mice, 4-month-old gal-3 KO mice, compared with WT mice, presented OA-like cartilage modifications. OA induction via MIA injection in WT mice generated cartilage lesions similar to those found in gal-3 KO animals. Moreover, OA induction showed a significant decrease in subchondral bone surface in the gal-3 KO mice in contrast to the WT group. CONCLUSIONS: Altogether these findings indicate that intracellular gal-3 has a beneficial effect in articular cells, as its absence in KO mice led to cartilage lesions.

Mice deficient in galectin-1 exhibit attenuated physiological responses to chronic hypoxia-induced pulmonary hypertension.

Pulmonary hypertension (PH) is characterized by sustained vasoconstriction, with subsequent extracellular matrix (ECM) production and smooth muscle cell (SMC) proliferation. Changes in the ECM can modulate vasoreactivity and SMC contraction. Galectin-1 (Gal-1) is a hypoxia-inducible beta-galactoside-binding lectin produced by vascular, interstitial, epithelial, and immune cells. Gal-1 regulates SMC differentiation, proliferation, and apoptosis via interactions with the ECM, as well as immune system function, and, therefore, likely plays a role in the pathogenesis of PH. We investigated the effects of Gal-1 during hypoxic PH by quantifying 1) Gal-1 expression in response to hypoxia in vitro and in vivo and 2) the effect of Gal-1 gene deletion on the magnitude of the PH response to chronic hypoxia in vivo. By constructing and screening a subtractive library, we found that acute hypoxia increases expression of Gal-1 mRNA in isolated pulmonary mesenchymal cells. In wild-type (WT) mice, Gal-1 immunoreactivity increased after 6 wk of hypoxia. Increased expression of Gal-1 protein was confirmed by quantitative Western analysis. Gal-1 knockout (Gal-1(-/-)) mice showed a decreased PH response, as measured by right ventricular pressure and the ratio of right ventricular to left ventricular + septum wet weight compared with their WT counterparts. However, the number and degree of muscularized vessels increased similarly in WT and Gal-1(-/-) mice. In response to chronic hypoxia, the decrease in factor 8-positive microvessel density was similar in both groups. Vasoreactivity of WT and Gal-1(-/-) mice was tested in vivo and with use of isolated perfused lungs exposed to acute hypoxia. Acute hypoxia caused a significant increase in RV pressure in wild-type and Gal-1(-/-) mice; however, the response of the Gal-1(-/-) mice was greater. These results suggest that Gal-1 influences the contractile response to hypoxia and subsequent remodeling during hypoxia-induced PH, which influences disease progression.

[Surgical treatment of temporomandibular joint: apropos of 94 cases]. / Prise en charge chirurgicale de l'articulation temporomandibulaire: à propos de 94 cas.

INTRODUCTION: The temporomandibular joint (TMJ) is a complex entity subjected to repeated stress with several symptoms. About one-third of people have at least one of those symptoms but only few (3 to 7%) need treatment. The aim of this retrospective study was to evaluate the results of temporomandibular joint surgery in 94 patients. PATIENTS AND METHODS: Several data were used for decision-making and the surgical technique was adapted to the etiology. The type of postoperative physiotherapy performed depended on the type of pathology. RESULTS: Most patients who underwent surgery between 1989 and 2004 were women (83%). The mean age was 30 years. We performed 179 surgical procedures and among them 151 Dautrey procedures. In 28 cases miniplates were used to avoid recurrences. In 57 cases postoperative physiotherapy was performed. The mean postoperative mouth opening increase was 4.7 mm (+ 23%). There was no infection or lost of plate. The mean of follow-up was about 14 months. DISCUSSION: With a long follow-up and an acceptable number of patients and operations, this retrospective study demonstrated the effectiveness of the Dautrey procedure in TMJ subluxations.

Altered primary afferent anatomy and reduced thermal sensitivity in mice lacking galectin-1.

The transmission of nociceptive information occurs along non-myelinated, or thinly myelinated, primary afferent axons. These axons are generally classified as peptidergic (CGRP-expressing) or non-peptidergic (IB4-binding), although there is a sub-population that is both CGRP-positive and IB4-binding. During neuronal development and following injury, trophic factors and their respective receptors regulate their survival and repair. Recent reports also show that the carbohydrate-binding protein galectin-1 (Gal1), which is expressed by nociceptive primary afferent neurons during development and into adulthood, is involved in axonal pathfinding and regeneration. Here we characterize anatomical differences in dorsal root ganglia (DRG) of Gal1 homozygous null mutant mice (Gal1(-/-)), as well as behavioural differences in tests of nociception. Gal1(-/-) mice have a significantly reduced proportion of IB4-binding DRG neurons, an increased proportion of NF200-immunoreactive DRG neurons, increased depth of central terminals of IB4-binding and CGRP-immunoreactive axons in the dorsal horn, and a reduced number of Fos-positive second order neurons following thermal (cold or hot) stimulation. While there is no difference in the total number of axons in the dorsal root of Gal1(-/-) mice, there are an increased number of myelinated axons, suggesting that in the absence of Gal1, neurons that are normally destined to become IB4-binding instead become NF200-expressing. In addition, mice lacking Gal1 have a decreased sensitivity to noxious thermal stimuli. We conclude that Gal1 is involved in nociceptive neuronal development and that the lack of this protein results in anatomical and functional deficits in adulthood.

Galectin-1 in regenerating motoneurons.

The exogenous application of recombinant galectin-1 has recently been shown to promote the rate of peripheral nerve regeneration. Endogenous neuronal galectin-1 expression has recently been demonstrated to increase after axotomy. Here we demonstrate a significant increase in the endogenous neuronal expression of galectin-1 mRNA in facial motoneurons after either a nerve resection or crush injury in mice. This increase in galectin-1 expression was due in part to the loss of target-derived factor(s) as indicated by both the return of galectin-1 expression to control levels following target re-innervation and the increase in galectin-1 expression after blockade of axonal transport by an interneuronal colchicine injection. Furthermore, interneuronal injections of glial-derived neurotrophic factor into the uninjured nerve also increased galectin-1 mRNA expression within facial motoneurons suggesting that positive signals may also be involved in the regulation of galectin-1 expression. Galectin-1 null mutant mice showed an attenuated rate of functional recovery of whisking movement after a facial nerve crush.

Consanguinity and spousal concordance in Kuwait.

Consanguineous marriage is favored in Kuwait. This research focuses on the relationship of physical and cultural traits to marriage types in Kuwait and examines concordance as a function of consanguinity and marriage duration. In a nonrandom opportunistic sample of 242 couples anthropometric and blood pressure data have been collected as well as data on acculturation, religiosity, Farsi proficiency, level of education, occupation, and attitudes regarding fertility. Significant concordances occur in cultural characteristics among couples in all three types of marriages with respect to the degree of religiosity, acculturation, language similarity, education, and occupation. Non-consanguineous spouses have the highest concordance in educational level, occupation, and degree of acculturation, but the lowest for religiosity and Farsi proficiency. Nonkin marriages seem to be based on personal preferences. In the wider potential nonkin marriage pool spouses show more concordance in stature and education indicating the positive assortative mating for those traits. Non-consanguineous spouses show a significant association for triceps and subscapular skinfold thicknesses hip and waist circumferences, and body fat distribution. Unrelated spouses exhibit more concordance for physical traits than do related spouses. There is a significant correlation between spouses in first and double cousin marriages as well as in spouses in second and less than second cousin unions for systolic and diastolic blood pressure, while non-consanguineous spouses show a significant association in diastolic blood pressure only.

Diabetic patients on peritoneal dialysis need less erythropoietin to maintain adequate hemoglobin.

Peritoneal dialysis (PD) patients have been shown to require less erythropoietin as compared with hemodialysis (HD) patients to maintain similar hemoglobin values. In our unit, we observed that diabetic PD patients required less erythropoietin treatment than did other PD patients. We therefore compared the amount of erythropoietin needed in diabetic and non diabetic patients on PD to maintain a similar hemoglobin value. All polycystic patients were excluded from the study because they rarely require erythropoietin. We also excluded patients with bone marrow disease, active gastrointestinal bleeding, or patients very resistant (requiring more than 25,000 U per week) to Eprex (recombinant human erythropoietin: Janssen-Cilag, North York, Ontario, Canada). Patients not requiring Eprex were also excluded from the study. We calculated the weekly erythropoietin dose in the two groups. We also compared hemoglobin level, iron transferrin saturation, vitamin?12 level, and serum folate. Diabetic patients required a lower weekly erythropoietin dose. Diabetic PD patients in our unti receive an average 4497 U per week compared with 7593 U per week for non diabetic PD patients. The difference (approximately 3000 U per week) is statistically significant.

Two-dimensional database of a Burkitt lymphoma cell line (DG 75) proteins: protein pattern changes following treatment with 5'-azycytidine.

Hypermethylation is an important mechanism for repression of tumor gene suppressor in cancer. The drug 5'-azacytidine (AZC) has been used as demethylating agent to induce the expression of previously silencing genes. In the present work, we attempted to determine, using proteomics, the changes in protein expression profiles following a treatment of an Epstein Barr virus (EBV)-negative Burkitt lymphoma (BL) cell line DG 75. The effects of the treatment in terms of cell viability and growth were first examined. The following observations were made: AZC treatment led to (i) a decrease in cell growth with an arrest of the cell at G0/G1 phase of the cell cycle, (ii) the expression of p16, a tumor-suppressor gene whose expression was dependent on its promoter demethylation. Proteomic study evidenced that AZC treatment affected protein expression in two different ways. Twenty-one polypeptides were down-expressed, while 14 showed an increased expression. Some of the upregulated proteins appeared related to the energy metabolism, to organization of cytoskeletal structures, and to cell viability and protein synthesis. We also established a reference map for proteins in DG 75 cell line, comprising 74 different polypeptides corresponding to 67 proteins. This map will be accessible via Internet as a resource for proteome analyses of B-cells. Taken together, the results presented here highlight new insights into lymphoma cell gene regulations following a treatment of lymphoma cells with AZC and illustrate a use of proteomics to evidence the direct and indirect effects of a drug and the pathways it possibly regulates.

The BPP (protein biochemistry and proteomics) two-dimensional electrophoresis database.

The BPP (protein biochemistry and proteomics) two-dimensional electrophoresis (2-DE) database (http://www-smbh.univ-paris13.fr/lbtp/Biochemistry/Biochimie/bque.htm) was established in 1998. The current release contains 11 reference maps from human hematopoietic and lymphoid cell line samples. These reference maps have now 255 identified spots, corresponding to 84 protein entries. The World Wide Web (WWW) presentation is designed to allow public access to the available 2-DE data together with logical connections to databases providing complementary information.

Uncoupling of chondrocyte death and vascular invasion in mouse galectin 3 null mutant bones.

Galectin 3 is a beta-galactoside binding protein which localizes to the cytoplasm of proliferative, mature, and hypertrophic chondrocytes in the growth plate cartilage of developing long bones. To elucidate the function of galectin 3 during bone development, we examined the epiphyseal femurs and tibias of fetal mice carrying a null mutation for the galectin 3 gene. Detailed histological and ultrastructural studies identified abnormalities in the cells of the proliferative, mature, and hypertrophic zones and in the extracellular matrix of the hypertrophic zone, as well as a reduction in the total number of hypertrophic chondrocytes. The expression patterns of several chondrocyte and bone cell markers were analyzed and revealed a subtle modification of Ihh expression in the galectin 3 mutant growth plate. A striking difference was observed at the chondrovascular junction where many empty lacunae are present. In addition, large numbers of condensed chondrocytes exhibiting characteristic signs of cell death were found in the late hypertrophic zone, indicating that the rate of chondrocyte death is increased in the mutants. These results suggest a role for galectin 3 as a regulator of chondrocyte survival. In addition, this unique phenotype shows that the elimination of chondrocytes and vascular invasion can be uncoupled and indicates that galectin 3 may play a role in the coordination between chondrocyte death and metaphyseal vascularization.

Protein analysis by mass spectrometry and sequence database searching: a proteomic approach to identify human lymphoblastoid cell line proteins.

Lymphoblastoid cell lines correspond to in vitro EBV-immortalized lymphocyte B-cells. These cells display a suitable model for experiments dealing with changes in protein expression occurring upon B-cell differentiation, after drug treatment, or after inhibition of some transcription factors. For all these reasons we have undertaken an effort aimed at developing a hematopoietic cell line protein two-dimensional electrophoresis (2-DE) database, containing B-lymphoblastoid 2-DE maps. In this work, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting analysis was adopted for protein identification. The peptide mass fingerprinting identification and the sequence coverage obtained on colloidal Coomassie blue (CBB) stained gel was close to that obtained using zinc-imidazole staining. Everything considered, CBB being more comfortable for subsequent spot manipulations, CBB staining was chosen for identification of a larger number of polypeptides. The results suggest that reticulation of the gel can interfere preventing the uptake of the enzyme during the in-gel digestion step. Consequently, low molecular mass proteins appear more difficult to identify by mass fingerprinting. Finally, the information provided in this study allows the construction of a new annoted reference map of human lymphoblastoid cell proteins. Among the identified proteins 60% were not yet positioned on 2-DE maps in three of the most important well-documented databases. The annoted map will be accessible via Internet on the LBPP server at URL:http:// www-smbh.univ-paris13.fr/lbtp/index.htm.

Regulation of CD45-induced signaling by galectin-1 in Burkitt lymphoma B cells.

It has been well established that Galectin-1 (GAL1), a beta-galactoside-binding protein, regulates the viability of lymphoid cells. However, the signaling pathway governed by the binding of GAL1 to the cell membrane is not understood. As a first step towards the elucidation of GAL1-initiated signaling events leading to a reduced viability of Burkitt lymphoma B cells, we tried to characterize the initial events induced by the binding of GAL1 to its receptor. This characterization was performed in BL36 cells, a Burkitt lymphoma cell line sensitive to GAL1. The results were as follows: (1) when solubilized cell membrane lysates were affinity bound to immobilized GAL1 and eluted by competition, the tyrosine phosphatase glyco-protein CD45 was found in the eluate, highlighting the role of CD45 as a receptor of GAL1; (2) the phosphatase activity of cell membranes diminished after incubation with GAL1; (3) immunoprecipitation experiments demonstrated that the phosphotyrosine kinase Lyn was dysregulated in cells that have been cultured in medium containing 700 nM GAL1, and (4) that the ratio between two isoforms of Lyn was modified during the treatment with GAL1. The regulation of Lyn therefore seems to be a key event in the action of GAL1.