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Biomedical subjects

C Legrand

Publications and source records attributed to C Legrand.

At least 91 records · Page 5Linked to original sources
PubMed1995

Regulation of myometrial Gi2, Gi3, and Gq expression during pregnancy. Effects of progesterone and estradiol.

The possibility that progesterone or estradiol may regulate expression of G protein in the rat myometrium during the course of pregnancy has been investigated using 1) immunoblot analysis of Gi2 alpha, Gi3 alpha, and Gq alpha subunits and 2) hybridization blot analysis of subunit mRNA. Eighteen hours after administration, estradiol had significantly increased the levels of both Gi2 alpha subunit and Gi2 alpha mRNA (by 40% and 32%, respectively). In control pregnant rats, we observed similar changes at the end of pregnancy, when myometrial concentrations of estradiol had increased, i.e., a 41% increase in immunoreactive Gi2 alpha subunit that correlated with a parallel 45% increase in mRNA levels. In contrast, levels of immunoreactive Gi3 alpha subunit and mRNA, which decreased with advancing gestation, were not influenced by estradiol or progesterone administration. Progesterone administration resulted 30 h later in a significantly decreased level of Gq alpha immunoreactivity (32%) and Gq alpha mRNA (30%). In control rats, Gq alpha protein and mRNA were also significantly lower at midpregnancy under progesterone dominance vs. term. At this stage, a twofold increase in Gq alpha subunit correlated with a 40% increase in mRNA levels. These results demonstrate that myometrial Gi2 alpha and Gq alpha subunits are physiological targets for estradiol and progesterone, respectively, in vivo. Alterations of these G protein levels are discussed in relation to their mediating effects on adenylyl cyclase activity or the phospholipase C pathway during the course of pregnancy.

Adenylyl Cyclases↗
PubMed1995

Cross-reactivity of human molecular markers for detection of prethrombotic states in various animal species.

The aim of the present study was to investigate the reactivity of immunoreagents developed for clinical applications in humans in different animal species (hen, mouse, rat, rabbit, guinea-pig, dog, pig, sheep, baboon). Prothrombin fragment 1 + 2, thrombin-antithrombin III complex and fibrinopeptide A were tested for coagulation, platelet factor 4 and beta-thromboglobulin for platelet activation, glycoprotein IIb-IIIa, glycoprotein Ib and P-selectin for platelet membrane glycoproteins, D-dimers for fibrinolysis, thrombomodulin for activation of endothelial cells and thrombospondin and von Willebrand factor for adhesive proteins. Prothrombin fragment 1 + 2, platelet factor 4, beta-thromboglobulin and D-dimers were revealed only in baboons. Fibrinopeptide A was well detected in baboons but weakly in mice, dogs, pigs and sheep. Whereas glycoprotein IIb-IIIa was revealed on guinea-pig, dog and sheep platelets and glycoprotein Ib on rabbit and dog platelets, P-selectin and thrombomodulin were never detected. Thrombospondin was revealed in hens, mice, rats, guinea-pigs, pigs, sheep and baboons and von Willebrand factor in mice, rats, guinea-pigs, dogs, pigs, sheep and baboons. Interestingly, thrombin-antithrombin III complex (TAT) was detected in all species tested except the hen. A time- and dose-dependent increase in TAT was observed when rats, dogs or pigs were infused with thromboplastin (4.5-450 microliters/kg/h), while administration of hirudin (1 mg/kg) abolished this TAT generation. Thus, the TAT immunoassay could provide a tool for the screening of antithrombotic drugs in a number of animal species. However, the possibility of using a wider panel of human immunoreagents would appear to be restricted to baboons which display good species cross-reactivity.

Animals↗
PubMed1995

Increased platelet CD36 constitutes a common marker in myeloproliferative disorders.

The distribution of the major platelet membrane glycoproteins (GP), Ib, IX, IIb-IIIa and IV (or CD36), which play important roles as receptors for adhesive molecules in haemostasis and thrombosis, was studied in 34 patients with myeloproliferative disorders (MPD): 13 had essential thrombocythaemia (ET), 12 had polycythaemia vera (PV) and nine had chronic myelogenous leukaemia (CML). Only occasionally were modifications of the numbers of GPIb or GPIIb-IIIa measured using the binding of specific radiolabelled antibodies to platelets. In contrast, 2-3-fold increases of the total CD36 content and the surface CD36 expression were measured in almost all patients studied, using a radioimmunoassay and the direct binding of the radiolabelled antibody, FA6-152, to the platelet surface, respectively. These results indicate that the abnormality affected both the external and internal CD36 pools. Therefore platelet CD36 may be a useful tool for the diagnosis and the follow-up of MPD patients. Surface CD36 has been proposed as a platelet receptor for thrombospondin, an adhesive glycoprotein that is released from platelets upon activation and promotes aggregate formation. Despite a 2-fold increase of CD36 molecules, resting and thrombin-activated platelets from ET patients expressed the same amount of thrombospondin as normal platelets, suggesting that there is not a direct correlation between the CD36 expression and thrombospondin binding either spontaneously or after activation.

Adult↗
PubMed1994

Characterization of a novel monoclonal antibody (V58A4) raised against a recombinant NH2-terminal heparin-binding fragment of human endothelial cell thrombospondin.

We report herein the characterization of a mouse monoclonal antibody (Mab) raised against the recombinant NH2-terminal heparin-binding domain (rHBD) of human endothelial cell thrombospondin (TSP). The antibody, a IgG1 (kappa), hereafter referred to as V58A4, reacted with two rHBD, TSPN18 and TSPN28 (i.e. 18 kDa and 28 kDa, respectively) with an affinity constant of 1.33 x 10(-8) M. However, V58A4 failed to recognize native or deglycosylated forms of TSP purified from platelets or endothelial cells, as well as a 25-30 kDa HBD fragment produced by limited proteolysis of native TSP. In contrast, Mab V58A4 was shown to react with larger HBD fragments (50-60 kDa) that were present in platelet or endothelial cell extracts and could be retained on a heparin-Sepharose column at low salt concentrations. These fragments also reacted with MA-II, a mouse Mab (IgG1), which recognizes both rHBD and HBD as well as intact TSP. Thus, V58A4 Mab appears to selectively recognize naturally occurring HBD fragments of TSP and may thus prove to be useful for detecting TSP proteolysis in situ under various physiopathological conditions.

Animals↗
PubMed1994

Effect of L-carnitine and acylcarnitine derivatives on the proliferation and monoclonal antibody production of mouse hybridoma cells in culture.

The effect of L-carnitine (Cn) on cell growth metabolism and antibody production rates was investigated using the murine hybridoma cell line, Mark3, in batch and fed harvest cultures. Two acylcarnitine derivatives were also tested: palmitoyl L-Cn and acetyl-DL-Cn. The addition of 20 microM L-Cn to cultures of Mark3 hybridoma cells that had been adapted to L-Cn significantly stimulated monoclonal antibody (mAb) production without affecting cell growth. In contrast, mAb secretion slightly decreased when L-Cn was added to culture of cells that had not been adapted to L-Cn. Palmitoyl L-Cn also stimulated mAb production by adapted cells, whereas the acetyl-DL-Cn, reduced mAb secretion. The presence of L-Cn in the medium did not affect glucose consumption or lactate production, but the metabolism of some amino acids was altered. The medium concentrations of valine, leucine, isoleucine and lysine were enhanced from 22% to 41% according to amino acids, whereas those of alanine, glycine and proline decreased. The mechanism by which L-carnitine affects mAb production and the metabolism of some amino acids is unknown. This effect is likely to be indirect, since there was no net entry of L-[3H]carnitine into the cells.

Acylation↗
PubMed1994

Proinflammatory cytokines (interleukin-1 beta and tumor necrosis factor-alpha) down regulate synthesis and secretion of thrombospondin by human endothelial cells.

We examined the effects of proinflammatory cytokines on the expression of two extracellular matrix proteins, e.g., thrombospondin (TSP) and fibronectin (FN) b cultured human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with human recombinant interleukin-1 beta (IL-1 beta) or human tumor necrosis factor-alpha (TNF-alpha) caused a time-and dose-dependent decline in TSP production whereas FN production was not modified. At low concentrations, IL-1 beta and TNF-alpha in combination ha a greater effect than either agent alone. Interferon-gamma (IFN-gamma) was without effect. The decline in TSP synthesis resulted in a decreased secretion of this glycoprotein into the extracellular matrix. Endothelial cell monolayers cultured on porous filters were used to study the polarity of TSP secretion. Approximately two thirds of the synthesized protein was secreted to the apical side medium and one third to the basal side medium and both types of secretion were inhibited to a similar extent by cytokine treatment. Immunoprecipitation experiments revealed no apparent degradation of secreted TSP, either in the apical or in the basal compartment. Treatment of HUVECs with lL-1 beta, either alone or in combination with TNF-alpha, had no significant effect on the steady-state TSP mRNA levels, suggesting a posttranscriptional regulation. Our results indicate that IL-1 beta decreasing TSP deposition and suggest different regulatory mechanisms for the expression of various secreted proteins by endothelial cells.

Cells, Cultured↗
PubMed1994

High gelsolin content of developing oligodendrocytes.

The actin-binding protein gelsolin that severs and caps the actin microfilaments under the control of the cytoplasmic free calcium and the membranous phosphatidylinositol 4,5-bisphosphate, is essentially restricted to the oligodendroglia in the central nervous system. Immunocytochemistry showed that gelsolin is an early marker of oligodendrocytes, both in vivo, in the rat cerebellum, and in vitro, in oligodendrocyte culture. We report the early appearance of gelsolin in A2B5-positive precursor oligodendrocyte cells and the specific expression of gelsolin in OL-1-, GC-, and MBP-positive oligodendrocytes in culture. The protein was distributed throughout the cell body and in the branched cell processes of cultured oligodendrocytes, but not in the MBP-positive membrane sheets. Gelsolin is thus cytosolic and not a myelin component. The quantitative study demonstrated that that the cerebellar gelsolin content changes significantly with age, with the maximal value at the age of 21 days, confirming that large amounts of gelsolin are transiently synthesized during development, especially from the first events of myelinogenesis. The results are consistent with gelsolin being involved, through its effects on the actin cytoskeleton, in the motile events occurring during the growth of the oligodendroglial processes towards the axons and the wrapping of the myelin sheaths around the axons.

Animals↗
PubMed1994

Quantitative evaluation of an experimental inflammation induced with Freund's complete adjuvant in dogs.

A chronic inflammation model in dogs was induced by intraarticular injection of Freund's Complete Adjuvant in the stifle. After a primary, acute response during the first 24 hr, a secondary subacute response was observed after a delay of approximately 3 weeks and persisted for several weeks. To evaluate the time course of the inflammatory process quantitatively, we tested more than 100 different parameters. Finally, only four parameters were selected based on practicability and metrological properties, namely, the body temperature, difference in skin temperature, difference in stifle diameter and vertical force exerted by arthritic hind limb measured using a force plate. The main results of the experimentation were the demonstration that these four parameters were sufficiently repeatable, reproducible, and appropriate to be used for quantitative evaluation of the inflammatory process, and that training of both animals and investigators was required. Finally, it was illustrated that an adjuvant periarthritis in dogs can be used to carry out a pharmacokinetic/pharmacodynamic modelling of an antiinflammatory drug.

Administration, Oral↗
PubMed1994

Plasma concentrations and therapeutic efficacy of phenylbutazone and flunixin meglumine in the horse: pharmacokinetic/pharmacodynamic modelling.

The purpose of the present study was to establish in the horse the relationship between plasma concentration profiles of phenylbutazone (PBZ) and flunixin meglumine (FM) and their pharmacological effects in order to build a predictive pharmacokinetic/pharmacodynamic (PK/PD) model. In five horses, an experimental arthritis was induced by injecting Freund's adjuvant into a carpal joint. PBZ (4 mg/kg) and FM (1 mg/kg) were injected by the intravenous route as a single intravenous dose in two different trials. Five pharmacodynamic end-points were regularly measured after test article injection using standardized procedures: local skin temperature, stride length, the rest angle flexion and the maximal carpal flexion of the injured leg and circumference of the inflamed joint. Plasma drug concentrations and pharmacodynamic data were analysed according to an integrated PK/PD model; for the stride length, the PBZ EC50, i.e. the plasma concentration for which half the maximum effect could be obtained, was 3.6 +/- 2.2 micrograms/ml and the maximum potential effect was 10.7 +/- 9.4% above the control value. For FM, the corresponding values were 0.93 +/- 0.35 micrograms/ml and 16.3 +/- 4.6%. EC50 values for rest angle flexion and local skin temperature were similar to that obtained for stride length. Maximal carpal flexion was an unreliable end-point, and circumference of the joint did not display significant response to the drugs. Using these experimental parameters, a dose-effect relationship was simulated for both drugs; it was shown for PBZ that the model predicts an absence of effect for a 1 mg/kg dose and a maximum effect at about 2 mg/kg; at higher PBZ doses, the maximum effect was not modified, but its duration was increased from 8 h with a 2 mg/kg dose to about 24 h with an 8 mg/kg dose. For FM the model predicts that a dose of 0.5 mg/kg will be without significant effect, whereas a 1 mg/kg dose allows a nearly maximal effect with a return to the control value after a delay of 16 h. A 2 mg/kg dose allows the effect to be maintained for 24 h. It is concluded that PK/PD is a tool of potential value for the preclinical screening of a dosage regimen.

Animals↗
PubMed1994

Selective inhibition of platelet macroaggregate formation by a recombinant heparin-binding domain of human thrombospondin.

Thrombospondin (TSP) is a platelet alpha-granule adhesive protein that plays a critical role in the stabilization of thrombus by promoting the formation of platelet macroaggregates. We have recently shown that a monoclonal antibody (mAb) to the NH2-terminal heparin-binding domain of TSP, MAII, inhibits platelet aggregation induced by thrombin in a dose-dependent manner. In this study, we have expressed in Escherichia coli two recombinant proteins comprising residues 1 to 174 (TSP18) and 1 to 242 (TSP28) of TSP. After purification, both proteins reacted equally well with mAb MAII, whereas the reactivity of TSP18 for heparin was lower than that of TSP28 or native TSP. At micromolar concentrations, TSP18 and TSP28 inhibited the second wave of platelet aggregation and the concomitant release of [14C]5-hydroxytryptamine induced by ADP in citrated platelet-rich plasma as well as aggregation and secretion induced by a low concentration of thrombin in washed platelet suspensions. The proteins did not inhibit surface expression of endogenous TSP on activated platelets, as measured by the binding of radiolabeled mAb 5G11, indicating that they did not interfere with the primary binding of TSP to the plasma membrane. In contrast, in a solid-phase binding assay, the proteins inhibited in a dose-dependent manner (IC50, 0.1 and 0.06 mumol/L for TSP18 and TSP28, respectively) the binding of radiolabeled TSP to surface-adsorbed fibrinogen. Furthermore, specific and saturable binding of the proteins to immobilized fibrinogen was demonstrated by enzyme-linked immunosorbent assay. The results suggest that interaction between the heparin-binding domain of TSP and membrane-bound fibrinogen may be critical in the platelet aggregation/secretion process.

Blood Platelets↗
PubMed1994

Tropomyosin isoforms in rat neurons: the different developmental profiles and distributions of TM-4 and TMBr-3 are consistent with different functions.

Antipeptide antisera specific for TM-4 and TMBr-3, the two tropomyosin isoforms in neurons, were used to investigate the concentrations and distributions of these F-actin-binding proteins in neurons in vitro and in vivo. TM-4 and TMBr-3 tropomyosins had different developmental profiles. TM-4 was found mainly in immature stages, while the concentration of TMBr-3 increased with maturation. The two isoforms also had different subcellular distributions. TM-4 was concentrated in the growth cones of cultured neurons and, in vivo, in areas where neurites were growing. Later, when development was complete, TM-4 was restricted to postsynaptic sites in the cerebellar cortex, whereas TMBr-3 was found in the presynaptic terminals. These data suggest that the tropomyosin isoforms have different functions, through their interaction with the actin cytoskeleton. TM-4 may be involved in the motile events of neurite growth and synaptic plasticity, while TMBr-3 could play a role in stabilizing neuronal networks and synaptic functioning.

Amino Acid Sequence↗
PubMed1994

Pregnancy-related modifications of rat myometrial Gs proteins: ADP ribosylation, immunoreactivity and gene expression studies.

Previous studies from our laboratory have suggested that post-receptor events at the level of beta-adrenergic receptor-adenylate cyclase interaction could be altered in myometrium by steroid hormones or pregnancy. In this study, we have addressed this question by performing a direct evaluation of rat myometrial Gs proteins at various stages of pregnancy or 24 h after administration of progesterone. In the 50,000 g myometrial plasma membrane fraction, in the presence of 32P-labelled NAD, cholera toxin ribosylated three predominant proteins with apparent molecular masses of 42, 47 and 55 kDa. Western blot analysis using the RM/1 antibody recognized the 42 and 47 kDa cholera toxin ADP-ribosylated bands but not the 55 kDa band. Thus, the 42 and 47 kDa immunoreactive bands were interpreted as being the small (Gs alpha-S) and large (Gs alpha-L) forms of Gs respectively. With a more purified myometrial plasma membrane fraction (105,000 g) an additional minor band of 44 kDa could be observed with both techniques. Treatment of late pregnant rats with 5 mg progesterone resulted in a significant increase in both Gs alpha subunits: +25% and +30% after ADP-ribosylation, +50% and +60% after Western blot analysis for Gs alpha-L and Gs alpha-S respectively. Pretreatment with the antiprogestin RU 486 completely suppressed the effect of progesterone, suggesting that the expression of Gs alpha subunits may be under the control of progesterone. However, changes in the myometrial content of Gs in progesterone-treated rats were not associated with concomitant variations in the steady-state levels of mRNA as demonstrated by Northern blot analysis. These data suggest a post-translational regulation of Gs expression by progesterone. Amounts of ADP-ribosylated Gs showed characteristic changes during the course of pregnancy with a fourfold or threefold increase (P < 0.05) on day 15 versus day 12 or delivery respectively. During pregnancy, or after progesterone administration, myometrial alterations of Gs strongly correlated (r = 0.913, P < 0.01) with the cholera toxin-stimulated adenylate cyclase activity. These findings provide evidence that changes in myometrial amounts of functional Gs i) are controlled by the hormonal status of pregnancy and progesterone and ii) play an important role in the transduction pattern of adenylate cyclase activity during the course of pregnancy.

Adenosine Diphosphate Ribose↗
PubMed1993

Involvement of thrombospondin in the adherence of human breast-adenocarcinoma cells: a possible role in the metastatic process.

The attachment of cancer cells to adhesive molecules, such as laminin (LN) and fibronectin (FN) in the extracellular matrix is a critical step in tumor invasion and metastasis. Recent data have suggested a potential role for thrombospondin (TSP), a 420-kDa cyto-adhesive glycoprotein, in the growth and spread of breast cancer. In this study, we have measured the ability of the human breast adenocarcinoma cell line, MDA-MB-231, to synthesize TSP and to use this molecule as an adhesion factor. The level of TSP in cells and secreted into the culture medium were determined by an enzyme-linked immunosorbent assay (ELISA). At pre-confluence, MDA-MB-231 cells were shown to produce a high level of TSP, most of which was retained within the cells. In comparison, FN was almost entirely secreted into the culture medium. An increased secretion of TSP was however measured at low cell density, suggesting that TSP might be required for cell/substratum or cell/cell interactions. As shown by flow cytometry, the cells expressed membrane-bound TSP as well as unoccupied TSP receptors. 125I-TSP bound saturably to 1.2 x 10(6) sites per cell with an apparent dissociation constant of 23 nM. The binding was inhibited by an excess of unlabeled TSP and by heparin, suggesting that the receptor could be a heparan-sulfate proteoglycan or a sulfatide. TSP promoted attachment but not spreading of MDA-MB-231 cells which attached and spread on FN and LN substrates. These results suggest that endogenously synthesized TSP may have a role in the cyto-adherence of tumor cells during the spread of breast cancer.

Blotting, Western↗
PubMed1993

Studies on fibronectin and its domains. I. Novel recombinant cell-binding domain of fibronectin--a modulator of human platelet functions.

The DNA sequences encoding for two proteins of the cell-binding domain (CBD) of human fibronectin (FN), namely a 33-kDa protein (aa 1329-1722) and a 40-kDa (aa 1380-1851) protein, were cloned and expressed in Escherichia coli. The interactions of the resulting rCBD proteins, refolded and purified to homogeneity, with human platelets were studied in comparison with those of the pentapeptide GRGDS. The binding of both the 33-kDa and the 40-kDa proteins to washed platelets appeared to be dependent upon platelet activation. In the case of the 33-kDa protein, binding to stimulated platelets was shown to be saturable, with Kd = 2 microM (thrombin as agonist). Moreover, both the 33-kDa and the 40-kDa proteins inhibited fibrinogen binding (at 0.1 microM) to ADP- or thrombin-stimulated platelets with IC50 values in the same concentration range. Binding seemed therefore to occur mainly at the GPIIb/IIIa receptor, and accordingly monoclonal antibodies against this receptor prevented up to 85% of the binding of the 33-kDa protein to platelets. With most stimuli the 33-kDa and the 40-kDa proteins inhibited platelet aggregation at concentrations 15- to 25-fold lower than those required by GRGDS and, in the case of the 33-kDa protein, this was shown to occur in either platelet-rich plasma, washed platelets, or whole blood. The 33-kDa protein also inhibited platelet aggregation and thromboxane A2 (TXA2) generation on the subendothelial extracellular matrix, whereas the GRGDS peptide inhibited only matrix-induced platelet aggregation, but not TXA2 formation. Furthermore, the 33-kDa protein, which is derived from the human FN CBD, seemed to be highly selective, since it inhibited the aggregation of platelets from primates only, and not from other animals tested. Finally, the 33-kDa protein did not promote fibroblast cell attachment, as was observed for both whole FN and the 40-kDa protein, thus displaying a selectivity toward platelets. In conclusion, the unique properties of the 33-kDa protein, and, in particular, its special affinity directed only toward activated primate platelets, seem to hold a promising potential for the further development of an antithrombotic agent.

3T3 Cells↗
PubMed1993

MVM(p) NS-2 protein expression is required with NS-1 for maximal cytotoxicity in human transformed cells.

The parvovirus-encoded nonstructural (NS) proteins have been implicated in the cytopathogenicity of these agents. Although protein NS-1 of minute virus of mice (MVM) has been shown to be toxic, little is known about the role of NS-2 in this process. In order to determine the contribution of NS-1 and NS-2 to cytotoxicity, we took advantage of an expression system controlled by the mouse mammary tumor virus promoter which responds to glucocorticoid stimulation and which controls the expression of both MVM(p) NS proteins. Different mutations were introduced in NS genes so as to affect the NS-1 or NS-2 protein. Neoplastic human cell lines expressing only NS-1 protein after induction by dexamethasone undergo a smaller lethality compared to lines expressing both wild-type proteins. Mutations that were introduced in NS-1 coding sequence and did not affect NS-2 were found to drastically suppress the cytotoxic effect. It is concluded that the NS-2 protein has little cytotoxic activity by itself but is required for the full expression of the viral cytopathic effect on transformed human cells. Furthermore these results lead us to suggest that the NS-2 cytotoxic domain is localized in the amino-terminal portion of the protein.

Base Sequence↗
PubMed1993

Role of thrombospondin in the adhesion of human endothelial cells in primary culture.

The role of thrombospondin on the adhesion of endothelial cells in primary culture was studied using a serum-free defined medium or thrombospondin-depleted fetal bovine serum. Under these conditions, only 6% of the cells adhered to gelatin-coated dishes, whereas cells adhering to gelatin in the presence of normal fetal bovine serum were considered as 100% adhesion. The percentage of cells attached to fibronectin or thrombospondin-coated dishes in thrombospondin-depleted serum was 66 and 32%, respectively. The addition of purified platelet thrombospondin to thrombospondin-depleted serum increased the adhesion of endothelial cells to gelatin and to thrombospondin, up to 32 and 59%, respectively, and restored the attachment to fibronectin to the same extent as that observed in the presence of normal serum. In contrast to the attachment, the spreading of the adhering cells was not further influenced by the addition of soluble thrombospondin. Subcultured cells did not require any protein for adhering to gelatin substrata. These observations indicate that thrombospondin plays a major role in the adhesion of endothelial cells in primary culture.

Cell Adhesion↗