ABSTRACT
Head injury-induced heterotopic ossification (HO) develops at vicinity of joints and in severe cases requires surgical intervention. Our previous study demonstrated high mRNA levels of osteocalcin (OC), type 1 collagen (COL1), osteonectin and RUNX2/CBFA1 in osteocytes and lining osteoblasts from non-evolutive HO compared to equivalent healthy cells from the proximal femoral shaft of patients receiving prosthesis. This allowed a first molecular characterisation of this pathological bone. The aims of this study is to extend the analysis to 10 more genes and determine those involved in the high OC mRNA level observed in pathological bone samples. RNAs were prepared from normotopic and heterotopic human bone samples digested by collagenase. After cDNA synthesis, mRNA levels were determined by real-time PCR and normalised using beta actin and glyceraldehyde-3-phosphate dehydrogenase. OSTERIX/SP7 expression was observed for the first time in human adult bone biopsies. In HO samples higher levels of SP7 (four- to sevenfold increase) and 1alpha,25-dihydroxy vitamin D(3) receptor (VDR) (two- to threefold increase) were observed compared to control samples. Moreover, SP7 level was correlated to OC and RUNX2 levels. In control samples, OC and SP7 levels were correlated. Our study further confirms that the involvement of SP7 in bone physiology is not only limited to the developmental step. Moreover, our results support the hypothesis that in HO the high level of OC expression could be due not only to an increase in RUNX2, but also in SP7 or VDR or to an imbalance in their respective activities.
Subject(s)
Bone and Bones/pathology , Gene Expression Regulation , Transcription Factors/genetics , Adult , Bone and Bones/metabolism , Choristoma , Female , Gene Expression Profiling , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Male , Middle Aged , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Sp7 Transcription Factor , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , Transcription Factors/metabolismABSTRACT
The influence of dexamethasone on rabbit bone marrow stromal cells differentiation was studied by screening the action of dexamethasone on gene expression. Using differential display, we observed some differential amplifications. The use of five of thirteen different primers combination allowed to identify one or more differential bands. One of them was identified as moesin gene. Real-time PCR confirmed a significant reduction of moesin gene expression following dexamethasone treatment. The decrease of expression for this protein, involved in cytoskeletal organization, could explain the effects of dexamethasone treatment on bone marrow stromal cells differentiation.
Subject(s)
Bone Marrow Cells/cytology , Dexamethasone/pharmacology , Gene Expression Regulation , Microfilament Proteins/biosynthesis , Stromal Cells/cytology , Animals , Base Sequence , Cytoskeleton/metabolism , DNA Primers/chemistry , DNA, Complementary/metabolism , Female , Gene Expression Profiling , Glucocorticoids/pharmacology , Microfilament Proteins/metabolism , Molecular Sequence Data , Osteoblasts/metabolism , RNA, Messenger/metabolism , Rabbits , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The role of micrometer and submicrometer surface roughness on the interaction of cells with titanium model surfaces of well-defined topography was investigated using human bone-derived cells (MG63 cells). The early phase of interactions was studied using a kinetic morphological analysis of adhesion, spreading and proliferation of the cells. By SEM and double immunofluorescent labeling of vinculin and actin, it was found that the cells responded to nanoscale roughness by a higher cell thickness and a delayed apparition of the focal contacts. A singular behavior was observed on nanoporous oxide surfaces, where the cells were more spread and displayed longer and more numerous filopods. On electrochemically microstructured surfaces with hemispherical cavities, arranged in a hexagonal pattern, the MG63 cells were able to go inside, adhere and proliferate in cavities of 30 or 100 microm in diameter, whereas they did not recognize the 10 microm diameter cavities. Cells adopted a 3D shape when attaching inside the 30 microm diameter cavities. Condensation of actin cytoskeleton correlated with vinculin-positive focal contacts on cavity edges were observed on all microstructured surfaces. Nanotopography on surfaces with 30 microm diameter cavities had little effect on cell morphology compared to flat surfaces with same nanostructure, but cell proliferation exhibited a marked synergistic effect of microscale and nanoscale topography.
Subject(s)
Biocompatible Materials/chemistry , Electrochemistry/methods , Osteoblasts/cytology , Osteoblasts/physiology , Titanium/chemistry , Adaptation, Physiological , Cell Adhesion , Cell Count , Cell Culture Techniques/methods , Cell Division , Cell Line , Cell Size , Humans , Materials Testing , Surface Properties , Time FactorsABSTRACT
The influence of temperature on cellular fatty acid composition and on heat stress tolerance was studied in the two species of Pectinatus, an anaerobic gram-negative bacterium. Cellular fatty acid (FA) patterns were determined for Pectinatus species cultivated in MRS medium at various defined conditions of temperature and pH. Our study shows that fluctuations of growth temperature and pH induced important changes in the ratio of unsaturated FAs (UFAs) to saturated FAs (SFAs). The major differences in the FA composition as a function of growth temperature concerned C15:0 and C17:0 for the SFAs and C15:1 and C17:1 for the UFAs. The most significant adaptation of lipid composition to lower growth temperatures was the strong increase of UFAs, particularly for C15:1 and C17:1 concomitantly with a decrease of SFAs (C15:0 and C17:0). When the pH of the culture medium was lowered from 6.2 to 4.0, a notable drop in the synthesis of the UFAs C15:1 and C17:1 was observed together with an important increase of C18-cyclopropane (C18-cyc) and high carbon number SFAs. Thermal modifications also provoked changes in Pectinatus behaviour. We observed that P. cerevisiiphilus was more heat sensitive than P. frisingensis. Mild exponential phase cells were treated for 1 h, at 40 degrees C for P. cerevisiiphilus or at 41 degrees C for P. frisingensis. This thermal adaptation induced tolerance against heat challenge (49 and 50 degrees C for P. cerevisiiphilus and P. frisingensis, respectively). Survival of P. cerevisiiphilus and P. frisingensis adapted cells was, respectively, 3400- and 790-fold higher than control. Interestingly, adapted cells of P. cerevisiiphilus were more thermotolerant than P. frisingensis pretreated cells.
Subject(s)
Gram-Negative Anaerobic Bacteria/physiology , Temperature , Fatty Acids/analysis , Gram-Negative Anaerobic Bacteria/chemistry , Hydrogen-Ion ConcentrationABSTRACT
The O(2) evolution activity of immobilized chloroplast membranes in different environments (albumin-glutaraldehyde matrix, urethane polymer and alginate beads) is presented. As previously shown, the stability of photosystem II (PS II) of lettuce thylakoids appears to be increased by the immobilization process. For understanding such stability, some spectral investigations have been made about the energy distribution between the immobilized photosystems. The low-temperature (77 K) fluorescence emission and photoacoustic spectroscopy are well adapted to solid particle studies. Especially, it has been shown that the fluorescence ratio (F(735)/F(695)) and photoacoustic ratio (PA(676)/PA(440)) are good indicators of the functional level of native and immobilized thylakoids. Such ratios are also given after storage and after continuous illumination conditions. Some results about the role played by glutaraldehyde (in the case of albumin-glutar-aldehyde matrix) in the stabilization process are also reported.
ABSTRACT
Immobilization of lettuce (Lactuca sativa) thylakoids has been performed by using glutaraldehyde and bovine serum albumin. Confirming previous reports, a stabilization of the O(2) evolution activity of the photosystem II (PSII) under storage and functional conditions has been observed. The present work is devoted to the role played by mono-and divalent cations, during the immobilization process itself, on the O(2) production. Four types of measurements have been employed: kinetic measurements, low temperature (77 K) fluorescence emission, photoacoustic (PA) spectroscopy, and electron microscopy observations. We show that the effect of glutaraldehyde is complex because it acts as an inhibitor, a stabilizing agent, and a cross-linking reactive. In the present studies, the thylakoids are immobilized within a polymeric insoluble albumin matrix. The highest activity yield and the best storage conditions are obtained when 0.15 mm Na(+) (or K(+)), 1 mm Mg(2+), and 0.1 mm Mn(2+) are present in the resuspending media before the immobilization. Due to modifications of the ionic content during such a process, structural differences are observed on the stacking degree of thylakoids. No modification of the fluorescence and PA spectra after the immobilization are found. Furthermore, a correlation between activities and spectral changes have been shown: when the activities increase, the F(735) to F(695) ratio increases and the PA(676) to PA(440) ratio decreases.
