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Biomedical subjects

E Mathieu

Publications and source records attributed to E Mathieu.

At least 55 records · Page 3Linked to original sources
PubMed1994

Characterization and purification of osteogenic cells from murine bone marrow by two-color cell sorting using anti-Sca-1 monoclonal antibody and wheat germ agglutinin.

Osteogenic cells were sorted from bone marrow of 5-fluorouracil (5-FU)-treated mice based on light scatter characteristics, Sca-1 expression, and their binding to wheat germ agglutinin (WGA). Four sort gates were established using forward (FSC) and perpendicular (SSC) light scatter and were denominated as FSChigh SSClow, FSClow SSChigh, FSClow SSClow, and FSChigh SSChigh cell. Cells from the FSChigh SSChigh gate, but not from the other gates, synthesized alkaline phosphatase, collagen, and osteocalcin and formed a mineralized matrix in culture. The number of osteoprogenitor cells was significantly enriched after depleting the 5-FU bone marrow from cells of the lymphoid and myeloid lineage, eg, T cells, B cells, natural killer cells, granulocytes, macrophages, and erythrocytes. Approximately 95% of the FSChigh SSChigh cell population of this "lineage-negative" (Lin-) marrow expressed the Sca-1 antigen (Sca-1+) and bound WGA. Three additional sort windows were established based on WGA binding intensity and were denominated as Sca-1+ WGAdull, Sca-1+ WGAmedium, and Sca-1+ WGAbright. Cells from the Sca-1+ WGAbright gate, but not from the other gates, synthesized bone proteins and formed a mineralized matrix. However, they lost this capacity upon subcultivation. Further immunophenotypic characterization showed that FSChigh SSChigh Lin- Sca-1+ WGAbright cells expressed stromal (KM16) and endothelial (Sab-1 and Sab-2) markers, but not hematopoietic surface markers such as c-kit and Thy1.2. Sorted FSChigh SSChigh Lin- Sca-1+ WGAbright cells form three-dimensional nodules that stain with the von Kossa technique and contain osteoblast and osteocyte-like cells.

Animals↗
PubMed1994

Characterization of the stromal osteogenic cell line MN7: mRNA steady-state level of selected osteogenic markers depends on cell density and is influenced by 17 beta-estradiol.

The steady-state mRNA levels of different osteogenic markers and their modulation by 17 beta-estradiol in the murine osteogenic cell line MN7 during proliferation and differentiation in vitro were examined. mRNA of collagen type I, osteopontin, bone morphogenetic protein 2, plasminogen activator inhibitor 1, alkaline phosphatase, and osteocalcin were isolated from MN7 cultures grown for 7, 11, 14, and 17 days. Northern blot analysis revealed steady-state transcript levels depending on MN7 cell density. The order of appearance of Col I, OP, ALP, and OC resembled the pattern of gene expression observed during osteoblast maturation in vitro. Furthermore, PAI-1 steady-state transcript levels peaked during subconfluence (day 11) but BMP-2 RNA levels reached their maximum after the culture had become confluent. 17 beta-Estradiol showed a dose-dependent stimulation of the different osteoblast-related transcripts present in a subconfluent MN7 culture at the time of analysis. Furthermore, the effects of 17 beta-estradiol (17 beta E2) at different time points of MN7 growth varied according to cell density. 17 beta E2 added to subconfluent MN7 cultures modulated the transcript level in a negative way, but RNA levels of the investigated osteogenic markers in confluent cultures were stimulated with 100 nM 17 beta-estradiol. No effect of 17 beta-estradiol on proliferation was detected. The present studies have revealed differential osteoblast gene expression related to MN7 cell proliferation and differentiation in vitro and emphasize the importance of 17 beta E2 in the regulation of growth of this preosteoblastic cell line in vitro.

Alkaline Phosphatase↗
PubMed1994

Characterization of the osteogenic stromal cell line MN7: identification of secreted MN7 proteins using two-dimensional polyacrylamide gel electrophoresis, western blotting, and microsequencing.

Proteins secreted by the osteogenic stromal cell line MN7 were analyzed using two-dimensional polyacrylamide gel electrophoresis (PAGE), western blotting, immunodetection, and microsequencing. Trichloroacetic acid-precipitated proteins from the conditioned medium of MN7 cell cultures, harvested at different times of growth, were dissolved in denaturing and reducing sample buffer and separated in the first dimension according to isoelectric point and in the second dimension according to molecular weight. Protein patterns were visualized using silver staining. Among the 350 separated protein spots, we identified type I collagen, bone sialoprotein, osteonectin, and cathepsin B by western blotting and immunodetection using polyclonal antibodies. Osteocalcin could not be detected in the conditioned medium of MN7 cells. Furthermore, 15 MN7-specific protein spots were localized after comparison with two-dimensional PAGE patterns from the conditioned medium of the nonosteogenic stromal cell lines MM1 and MV1. Microsequencing of the internal peptides of five selected spots revealed three known proteins, namely the carboxyl-terminal propeptide of the alpha 2 chain of collagen type I, cathepsin L, and the tissue inhibitor of metalloproteinases-2, an 18 kilodalton peptide fragment from osteopontin that has not previously been described, and a novel glycosylated 85 kD protein with an average isoelectric point of 5.7. All identified proteins did not vary in presence between the different time points analyzed by two-dimensional PAGE. The use of two-dimensional PAGE to investigate the secreted proteins of MN7 cells will enable us to establish a complete protein data base of extracellular osteoblast-specific proteins. Furthermore, two-dimensional PAGE in combination with other techniques is a fast and accurate method for the identification of novel proteins that could function as markers in osteoblast differentiation and/or bone formation.

Amino Acid Sequence↗
PubMed1994

[Portal vein thrombosis: a rare complication of Crohn disease].

Among usual extra digestive manifestations of inflammatory bowel diseases, vascular injuries are rare, but often of excessive gravity. The emergence of a portal thrombosis, often accelerated by infection or traumatism, particularly surgical, remains exceptional. We report the case of a young patient who is followed for a severe crohn's disease and affected with a brutal portal vein thrombosis. Although initial prognosis was bad, the patient now lead a normal life ten months after this episode. It's the third described case of portal thrombosis in crohn's disease. We discuss the predisposing role of an acquired protein S deficiency during a estroprogestative treatment.

Adult↗
PubMed1994

Combined analysis of islet cell antibodies that cross-react with mouse pancreas, antibodies to the M(r) 64,000 islet protein, and antibodies to glutamate decarboxylase in type I diabetic patients.

OBJECTIVE: A combined analysis of whether islet cell autoantibodies (ICAs) are cross-reactive with mouse pancreas, with glutamate decarboxylase (GAD) antibodies, and with 64K antibodies was performed in a large sample of recently diagnosed type I diabetic patients. The disappearance rates of these different autoantibodies were compared in some patients after onset of the disease. The aims were to determine patterns in GAD/64K antibodies with regard to cross-species reaction of ICA and to assess whether GAD could contribute to ICA positivity in mouse and human pancreases and whether the simultaneous search for all the antibody specificities enhances the detection of autoimmune stigma. RESEARCH DESIGN AND METHODS: ICA detected by immunofluorescence in human and mouse pancreases, antibodies immunoprecipitating the 64K rat islet antigen, and antibodies immunotrapping brain GAD activity were quantified at diagnosis of diabetes in 95 patients and in sequential samples during 1 year after diagnosis in 13 patients. The contribution of GAD to ICA positivity in mouse and human pancreases was evaluated by the analysis of correlations between tests and by the ability of brain homogenate to block ICA reactivity in pancreases from both species. RESULTS: ICAs were detected in human pancreases in sera from 63 (66%) patients, among which 61% bound also to a mouse pancreas. GAD and 64K antibodies were strongly correlated (P < 0.0001) and were detected in 69 and 73% of the patients, respectively. All but two patients with ICA in human pancreas also displayed either ICA in mouse pancreas or GAD/64K antibodies. Among 32 patients without ICA in human pancreas, 54% displayed either GAD/64K antibodies or ICA in mouse pancreas. Only 16% of the patients displayed neither ICA nor GAD/64K antibodies. A correlation (P < 0.005) was found between ICA in human and mouse pancreases. GAD or 64K antibodies were strongly correlated with ICA in human pancreas (P < 0.0001), but not with ICA in mouse pancreas. After preincubation of six sera with GAD-containing brain homogenate, ICA titers were unaffected in mouse pancreas but reduced in human pancreas. ICA titers in mouse pancreas were decreased after 3 months (P < 0.01) in diabetic patients, contrasting with the stability of ICA in human pancreas and GAD antibodies by 1 year after diagnosis. CONCLUSIONS: According to cross-species reaction, we confirm the heterogeneity of ICA in a large series of type I diabetic patients, ICAs that cross-reacted with mouse pancreas being more frequent than ICAs without cross-species reactivity. GAD and 64K antibodies were also present in a majority of patients. The simultaneous search for all the antibody specificities enhances the detection of autoimmune stigma so that only a few patients did not display any autoantibody at diagnosis. GAD is not the target of ICAs in mouse pancreas, whereas GAD accounts for ICA positivity in human pancreas. The conclusion that ICAs in mouse pancreas are not GAD-reactive is reinforced by the fact that they are more transient after onset of diabetes than are GAD antibodies or the complex mixture of ICAs in human pancreas.

Adolescent↗
PubMed1993

Hydrophobic cluster analysis of G protein-coupled receptors: a powerful tool to derive structural and functional information from 2D-representation of protein sequences.

Current methods for comparative analyses of protein sequences are 1D-alignments of amino acid sequences based on the maximization of amino acid identity (homology) and the prediction of secondary structure elements. This method has a major drawback once the amino acid identity drops below 20-25%, since maximization of a homology score does not take into account any structural information. A new technique called Hydrophobic Cluster Analysis (HCA) has been developed by Lemesle-Varloot et al. (Biochimie 72, 555-574), 1990). This consists of comparing several sequences simultaneously and combining homology detection with secondary structure analysis. HCA is primarily based on the detection and comparison of structural segments constituting the hydrophobic core of globular protein domains, with or without transmembrane domains. We have applied HCA to the analysis of different families of G-protein coupled receptors, such as catecholamine receptors as well as peptide hormone receptors. Utilizing HCA the thrombin receptor, a new and as yet unique member of the family of G-protein coupled receptors, can be clearly classified as being closely related to the family of neuropeptide receptors rather than to the catecholamine receptors for which the shape of the hydrophobic clusters and the length of their third cytoplasmic loop are very different. Furthermore, the potential of HCA to predict relationships between new putative and already characterized members of this family of receptors will be presented.

Amino Acid Sequence↗
PubMed1992

Establishment of an osteogenic cell line derived from adult mouse bone marrow stroma by use of a recombinant retrovirus.

In order to characterize fibroblastic colony-forming units (CFU-F) from murine bone marrow in relation to osteogenesis, adherent cells of 7-day-old BALB/c mouse bone marrow cultures were infected with a recombinant retrovirus (N2/ delta fosB) containing the bacterial neomycin resistance gene. One of the G418-resistant clones, MN7, was selected for further analysis on the basis of its high expression of the bone-specific alkaline phosphatase. The cells have now been in culture for more than 1 year and maintain a stable phenotype. The osteogenic nature of the immortalized clone MN7 was demonstrated as follows: (1) Mineralization was detected by 85Sr uptake and with the Von Kossa staining method only after in vitro cultivation on a collagen type I matrix. (2) Osteoblastic phenotype markers, including the synthesis of type I collagen, osteonectin, and the bone-specific isoenzyme of alkaline phosphatase were expressed in vitro. (3) MN7 cells responded to bone effectors such as parathyroid hormone and 1,25-dihydroxyvitamin D3. (4) Intraperitoneal injection of MN7 cells into 1-day-old BALB/c mice produced typical osteosarcomas in all animals. We conclude that MN7, derived entirely in vitro from a stromal CFU-F colony, represents a stable murine osteosarcoma cell line expressing the osteoblastic phenotype and provides the first direct evidence needed to establish adult mouse marrow-derived, nonhematopoietic stromal cells as osteoprogenitors.

Alkaline Phosphatase↗
PubMed1992

Striatal protection induced by lesioning the substantia nigra of rats subjected to focal ischemia.

Unilateral 6-hydroxydopamine lesion of the substantia nigra reduced the volume of striatal necrosis and suppressed the increase in extracellular glutamate concentration in the striatum induced by middle cerebral artery occlusion in rats. These results indicate that the dopaminergic nigrostriatal pathway is highly involved in the vulnerability of the striatum to ischemia and suggest that glutamate-dopamine interactions may play a key role in the striatal ischemic insult.

Animals↗
PubMed1991

Stromal cells from murine developing hemopoietic organs: comparison of colony-forming unit of fibroblasts and long-term cultures.

The adherent stromal layer in long-term marrow cultures is essential to the proliferation and differentiation of hemopoietic cells. Adhering cells are heterogeneous and morphologically not adequately characterized. Comparative morphological studies were conducted on adherent cells in short-term clonal assays and long-term cultures derived from liver and bone marrow. Liver and bone marrow at different developmental ages have different hemopoietic activities in vivo and in vitro, as tested via CFU-GM recovery in long-term cultures. Adherent cells from each organ were recovered at an age with high hemopoietic activity (fetal liver and adult bone marrow) and at an age with low hemopoietic activity (neonatal liver and bone marrow). The presence of macrophages, alkaline phosphatase, acid phosphatase, myeloperoxidase, sulfated and non-sulfated glycosaminoglycans (GAGs) and fibronectin was compared. For a given organ, CFU-f colonies showed characteristics similar to those of the confluent adherent stromal layer in long-term cultures. The presence of macrophages and GAGs (sulfated and non-sulfated) in the adherent layer were directly related to the hemopoietic activity. The amount of alkaline phosphatase-positive cells and the amount of fibronectin showed no correlation with the hemopoietic activity of the cultures.

Alkaline Phosphatase↗