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Biomedical subjects

C Legrand

Publications and source records attributed to C Legrand.

At least 109 records · Page 6Linked to original sources
PubMed1993

Search for cell proliferation markers suitable for cell count in continuous immobilized cell bioreactors.

The control of hybridoma cell cultures in bioreactors requires the use of convenient indicators to monitor the proliferation of the biomass. In order to select appropriate indications, we followed the variations of several compounds including tumoral markers, polyamines, sialic acids, purine and pyrimidine bases, enzymes and metabolites such as glucose, lactate and amino acids, and the variations of cell density during batch culture. Significant correlations were found between the number of viable cells and alkaline phosphatase, beta-glucuronidase, glucose and lactate measured in the culture medium of hybridoma strains. The correlation calculated from alkaline phosphatase and beta-glucuronidase concentrations in culture medium underestimated cell number. The correlation established with glucose and lactate gave the best indication of cell proliferation in continuous culture with an immobilized cell bioreactor. Finally, the exact quantification of the biomass in these culture conditions can be obtained using the mean of glucose and lactate correlations.

Animals↗
PubMed1993

The expression of tropomyosin genes in pure cultures of rat neurons, astrocytes and oligodendrocytes is highly cell-type specific and strongly regulated during development.

Transcripts from the alpha-, beta- and delta-tropomyosin genes were studied during development of pure cultures of rat neurons, astrocytes and oligodendrocytes. The three cell types contained five alpha-tropomyosin messengers, produced using both alternative promoters and splicing; one was specific for mature neurons. The beta-tropomyosin gene is expressed only in astrocytes and the delta-tropomyosin gene in all three cell types, especially in immature cells. Most of the tropomyosin isoforms are highly cell-specific. Their developmental regulation involves either differential expression of genes, in neurons and oligodendrocytes, and/or changes in alternative splicing, in astrocytes, delta-Tropomyosin (TM-4) may be important during the growth of neuronal and glial cell processes, while specialized isoforms such as the neuron-specific alpha-tropomyosin TMBr-3 may be involved in the function or plasticity of the mature cells.

Animals↗
PubMed1993

Importance of 3' non-coding sequences for efficient retrovirus-mediated gene transfer in avian cells revealed by self-inactivating vectors.

Avian leukosis virus-derived vectors were constructed with an internal transcriptional promoter and various 3' non-coding sequences. Deletions were introduced into the downstream U3 long terminal repeat (LTR) to obtain self-inactivation of LTR-mediated transcription after one round of replication. However, 3' non-coding sequences appeared to determine not only self-inactivation of the vectors but also gene transfer efficiency. Further analysis revealed the influence of these sequences on both internal gene expression and RNA packaging. One construct permitted gene transfer while inactivating 5' LTR-promoted transcription.

Animals↗
PubMed1993

Molecular basis for fibrinogen Dusart (A alpha 554 Arg-->Cys) and its association with abnormal fibrin polymerization and thrombophilia.

The molecular defect in the abnormal fibrinogen Dusart (Paris V) that is associated with thrombophilia was determined by sequence analysis of genomic DNA that had been amplified using the polymerase chain reaction. The propositus was heterozygous for a single base change (C-->T) in the A alpha-chain gene, resulting in the amino acid substitution A alpha 554 Arg-->Cys. Restriction analysis of the amplified DNA derived from the family members showed that his father and his two sons were also heterozygous. Electron microscopic studies on fibrin formed from purified fibrinogen Dusart demonstrated fibers that were much thinner than in normal fibrin. In contrast to the previously observed defective binding of plasminogen, the binding of thrombospondin to immobilized fibrinogen Dusart was similar to that of normal fibrinogen. Immunoblot analysis of plasma fibrinogen demonstrated that a substantial part of the fibrinogen Dusart molecules were disulfide-linked to albumin. The plasma of the affected family members also contained fibrinogen-albumin complexes. Furthermore, small amounts of high molecular weight complexes containing fibrinogen were detected in all the heterozygous individuals. These data indicate that the molecular abnormality in fibrinogen Dusart (A alpha 554 Arg-->Cys) results in defective lateral association of the fibrin fibers and disulfide-linked complex formation with albumin, and is associated with a family history of recurrent thrombosis in the affected individuals.

Adult↗
PubMed1993

Heterogeneity of alpha-2 adrenoceptors in pregnant rat uterus: identification of subtypes and autoradiographic distribution.

Pharmacological characterization of alpha-2 adrenoceptors of the pregnant rat myometrium was assessed using the ability of various alpha adrenoceptor agonists and antagonists to inhibit [3H]rauwolscine or [3H]idazoxan binding to myometrial 50,000 x g fraction or to slide-mounted sections of the whole pregnant uterus. Saturation binding studies with both radioligands showed that the number of myometrial alpha-2 adrenoceptors is greatly increased on days 10 to 12 of pregnancy vs. cyclic rats. It then decreased from midpregnancy to term (about -75%; P < .01) with no change of the equilibrium dissociation constant (between 7-11 nM). Chemical sympathectomy by 6-hydroxydopamine significantly decreased (P < .01) the density of alpha-2 adrenoceptors at days 8 and 12 of pregnancy. Later, 6-hydroxydopamine administration did not alter Bmax or Kd values suggesting that the pregnancy decrease of alpha-2 adrenoceptors may be related to a loss of presynaptic receptors. In order to identify myometrial postsynaptic alpha-2 adrenoceptor subtypes, the inhibition of [3H]rauwolscine or [3H]idazoxan binding by oxymetazoline, prazosin and chlorpromazine was studied on days 20 and 21 of pregnancy. All inhibition curves were consistent with a model of two classes of binding sites: about 55% of the myometrial alpha-2 adrenoceptors, which had a higher affinity for oxymetazoline, may represent the alpha-2A subtype whereas the other 45% of the sites, which had a higher affinity for prazosin and chlorpromazine, may represent the alpha-2B subtype. Autoradiographic studies using [3H]rauwolscine revealed that both subtypes are colocalized in the longitudinal muscle. A high density of alpha-2A and alpha-2B subtypes was also detected in the chorioallantoic and yolk sac placenta and in the embryonic nervous system.

Adrenergic alpha-Antagonists↗
PubMed1992

Quantitation of platelet glycoprotein IV (CD36) in healthy subjects and in patients with essential thrombocythemia using an immunocapture assay.

Glycoprotein IV (GPIIb, CD36) is a major platelet membrane glycoprotein which is thought to participate in a number of adhesive reactions and to mediate signal transduction. In order to measure the total content of GPIV in human platelets, we have developed a simple and sensitive solid-phase radioimmunoassay based on the immunocapture of GPIV from Triton X-100-solubilized platelets. FA6-152, a monoclonal antibody to GPIV was coated on microtiter plates and bound antigen was quantified with a radiolabeled polyclonal antibody to GPIV. Using purified GPIV as a standard, the coefficients of variation of the assay were found to be less than 10% at concentrations of GPIV ranging from 0.15 to 0.75 micrograms/ml. The assay was validated by the parallelism obtained between purified GPIV dose-response curves and those obtained with platelet lysates, indicating a similar antigenic activity for GPIV in both samples. The level of GPIV in platelets from healthy donors was 0.23 +/- 0.05 (mean +/- SD, n = 15) micrograms per 100 micrograms of platelet proteins and a mean value of 27,440 +/- 6,200 (SD) molecules per platelet was calculated. The radioimmunoassay could be used to discriminate between the high level of platelet GPIV in patients with essential thrombocythemia (mean +/- SD = 81,850 +/- 27,780 molecules/platelet; n = 8) and the normal GPIV level in patients with secondary thrombocytosis (mean +/- SD = 26,810 +/- 4,030 molecules/platelet; n = 5), thereby demonstrating the clinical usefulness of the assay. The specific increase in platelet GPIV in patients with essential thrombocythemia was confirmed by immunoblot analysis whereas no increase in platelet GPIb or GPIIb-IIIa was observed by this technique.

Adult↗
PubMed1992

A defect of platelet aggregation associated with an abnormal distribution of glycoprotein IIb-IIIa complexes within the platelet: the cause of a lifelong bleeding disorder.

A young Italian man (A.P.) has a lifelong history of bleeding from gums and mucocutaneous tissue. Electron microscopy showed a wide diversity of platelet size including giant forms. In citrated platelet-rich plasma (PRP), platelet aggregation induced by adenosine diphosphate (ADP) and other agonists was much reduced. Both secretion and clot retraction were normal. The aggregation of washed platelets with ADP was improved but remained subnormal, as was aggregation with collagen and thrombin. Fibrinogen-binding was analyzed by flow cytometry using platelets in whole blood or PRP and was markedly decreased. Crossed immunoelectrophoresis of Triton X-100 extracts of (A.P.) platelets showed that GP IIb-IIIa levels were 40% to 50% of normal. Glycoprotein (GP) IIb and GP IIIa were of usual migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but their labeling was much reduced during lactoperoxidase-catalyzed iodination. Binding to (A.P.) platelets of four different 125I-labeled monoclonal antibodies to GP IIb-IIIa complexes was reduced to 12% to 20% of normal levels. However, when the patient's platelets were stimulated with alpha-thrombin, monoclonal antibody binding showed the same increase (approximately 20,000 sites) as normal platelets. Both flow cytometry and immunocytochemical studies showed that the distribution of residual surface GP IIb-IIIa within the total (A.P.) platelet population was heterogeneous and not related to platelet size. Staining of ultrathin sections confirmed the presence of an internal pool of GP IIb-IIIa. Monoclonal antibodies to other membrane glycoproteins bound normally to (A.P.) platelets. The patient has a selective deficiency of the surface pool of GP IIb-IIIa complexes that is manifested clinically by a mild Glanzmann's thrombasthenia-like syndrome.

Adenosine Diphosphate↗
PubMed1992

Progesterone transcriptionally regulates the beta 2-adrenergic receptor gene in pregnant rat myometrium.

Administration of 5 mg of progesterone to late pregnant rats induced an increase in myometrial beta-adrenergic receptors density detected by 125I-cyanopindolol binding. This increase was significant after 24 h (1.4-fold; p less than 0.05) and reached 1.6-fold (p less than 0.05) after 36 h. The antiprogestin RU 486 or cycloheximide completely suppressed the effect of progesterone injection. Quantification of the beta 1- and beta 2-receptor subtypes revealed that progesterone selectively up-regulated the beta 2-subtype. The increase in beta 2-adrenoreceptors was preceded by an elevation of their mRNA (2.3 kilobases) levels as determined by Northern blot hybridization with a rat heart beta 2-adrenoreceptor cDNA probe. This increase was significant after 18 h of exposure to progesterone (2.1-fold; p less than 0.05) and reached a maximum after 24 h (3.4-fold; p less than 0.01). The rate of beta 2-adrenergic gene transcription evaluated by nuclear run-on transcription assays, increased by 2.5-fold in myometria exposed for 4 h to progesterone. This study indicates that progesterone regulates myometrial beta 2-adrenergic receptor expression by controlling the rate of transcription of the gene.

Animals↗
PubMed1992

Molecular requirements for the interaction of thrombospondin with thrombin-activated human platelets: modulation of platelet aggregation.

We have investigated the molecular requirements for thrombospondin (TSP) to bind to the platelet surface and to support the subsequent secretion-dependent platelet aggregation. For this, we used two distinct murine monoclonal antibodies (MoAbs), designated MAI and MAII, raised against human platelet TSP, and three polyclonal antibodies, designated R3, R6, and R5, directed against fusion proteins containing the type 1 (Gly 385-Ile 522), type 2 (Pro 559-Ile 669), and type 3 (Asp 784-Val 932) repeating sequences, respectively. Among them, R5 and R6, but not R3, inhibited thrombin-induced aggregation of washed platelets and the concomitant secretion of serotonin. These antibodies, however, did not inhibit the expression of TSP on thrombin-activated platelets, as measured by the binding of a radiolabeled MoAb to TSP, suggesting that they may inhibit platelet aggregation by interfering with a physiologic event subsequent to TSP binding. In contrast, MoAb MAII, which reacts with an epitope located within the heparin-binding domain of TSP, inhibited both TSP surface expression and platelet aggregation/secretion induced by thrombin. In addition, this MoAb inhibited in a dose-dependent manner (IC50 approximately 0.5 mumol/L) the interaction of 125I-TSP with immobilized fibrinogen and platelet glycoprotein IV, both potential physiologic receptors for TSP on thrombin-activated platelets. These results indicate that the interaction of TSP with the surface of activated platelets can be modulated at the level of a specific epitope located within the amino terminal heparin-binding domain of the molecule. Thus, selective inhibition of the platelet/TSP interaction may represent an alternative approach to the inhibition of platelet aggregation.

Antibodies↗
PubMed1992

Lactate dehydrogenase (LDH) activity of the cultured eukaryotic cells as marker of the number of dead cells in the medium [corrected].

One significant problem in monitoring a culture's evolution is to assess change in cell viability. We have demonstrated that LDH release could be a good indicator of cellular damage of many cell lines, especially during shear stress or sonication. Moreover, we have found a significant correlation between the number of dead cells, determined by Trypan Blue staining, and LDH activity measurements in the supernatant of hybridoma strains, whatever the culture conditions. We have also shown that when viability is still near 100% no LDH is released even at high cell concentrations. Therefore, LDH should serve as a potential marker of cell injury and death.

Animals↗
PubMed1992

Bacteremia due to Achromobacter xylosoxidans in patients with cancer.

Bacteremia due to Achromobacter xylosoxidans is rare, and little information on treatment is available. Between 1983 and 1988, A. xylosoxidans was recovered from 26 cultures of blood from 10 patients with cancer and clinical signs of infection, including one patient with septic shock and two with pneumonia. Neutropenia did not seem to be a predisposing factor. The infection may have been catheter related in four patients and associated with gastrointestinal pathology in four others. Probable cause was not determined in the remaining two. In vitro studies of susceptibility showed that the isolates were susceptible to trimethoprim-sulfamethoxazole (TMP-SMZ), the antipseudomonal penicillins, ceftazidime, cefoperazone, and imipenem; moderately susceptible to ciprofloxacin; and resistant to ceftriaxone, cefotaxime, cefoxitin, ceftizoxime, aztreonam, and amikacin. All patients receiving therapy recovered, including those six who received TMP-SMZ or a beta-lactam antibiotic as a single agent. A. xylosoxidans bacteremia is a significant infection and may be catheter related or associated with gastrointestinal pathology. The infection usually responds to therapy with TMP-SMZ or an appropriate beta-lactam antibiotic.

Adolescent↗
PubMed1992

Cooperation of oncogenes in cell transformation and sensitization to killing by the parvovirus minute virus of mice.

The established line of normal Fisher rat fibroblasts (FR3T3) is naturally resistant to the parvovirus minute virus of mice (MVM), and was used as a model system to study the influence of stepwise transformation on the susceptibility of cells to this virus. When transformed with genes encoding the class I nuclear oncoproteins large T antigen of polyomavirus (PyLT) or v-myc, cells retained a normal appearance, but acquired some ability to form colonies in soft agar. On the other hand, the class II transforming oncogenes encoding the middle T antigen of polyomavirus (PyMT) and c-Ha-ras-1 induced both morphological alterations and a high capacity for anchorage-independent growth in transfected cells. The concomitant expression of oncogenes from both classes (PyLT-(+)PyMT; v-myc+c-Ha-ras-1) induced a supertransformed phenotype characterized by the piling-up of cells into poorly adherent foci, even in low density cultures. The progressive transformation of this cellular system was found to coincide with a gradual increase in its susceptibility to MVMp (MVM prototype strain) infection. Compared to parental cells, class I, class II and double transformants proved to be sensitized to killing by MVMp to a low, moderate and large extent, respectively. Thus, oncogenes from different functional classes appeared to cooperate in the responsiveness of cells to parvovirus attack. Interestingly, this cooperation exacerbated both the killing of infected cells and their capacity to produce viral non-structural (NS) proteins, in agreement with the reported cytotoxic activity of NS polypeptides. Therefore, in this system, parameters of the parvovirus life cycle may serve as indications of the overall progression of the transformation process.

Animals↗
PubMed1992

Lack of activity of amphotericin B in systemic murine fusarial infection.

Systemic fusarial infections have emerged as a significant cause of mortality in cancer patients. Yet, little is known about the management of these infections. The in vivo antifungal activity of amphotericin B in CF1 mice with disseminated fusarial infections was studied. Two pathogenic strains of Fusarium solani were used. Intraperitoneal administration of amphotericin B in daily doses of 0.5, 1, and 2 mg/kg for less than or equal to 10 days did not prolong survival of treated animals. Clearance of F. solani from kidneys was similar in mice treated with 1 mg/kg per day of amphotericin B and in untreated animals. These results are in agreement with the known in vitro and in vivo resistance of Fusarium species to amphotericin B.

Amphotericin B↗
PubMed1991

A novel mechanism of action for v-ErbA: abrogation of the inactivation of transcription factor AP-1 by retinoic acid and thyroid hormone receptors.

Ligand-activated retinoic acid receptor alpha (RAR alpha) and c-ErbA alpha repress the AP-1-mediated transcriptional activation of the interstitial collagenase gene promoter by specifically decreasing the activity of the AP-1 transcription factor. On the other hand, the v-ErbA oncoprotein fails to repress the AP-1 activity and acts as a dominant negative oncoprotein by overcoming the repression of the AP-1 activity induced by RAR alpha and c-ErbA alpha. This maintenance by v-ErbA of a fully active AP-1 complex is correlated with the abrogation by this same oncogene product of the growth-inhibitory response of chicken embryo fibroblasts to retinoic acid treatment. This new mechanism of action of v-ErbA together with its previously discovered dominant repressor effect on transcription of thyroid hormone-activated target genes may explain the contribution of the v-erbA oncogene to sarcomatogenic and leukemogenic transformation.

Carrier Proteins↗
PubMed1991

Platelet thrombospondin and glycoprotein IV abnormalities in patients with essential thrombocythemia: effect of alpha-interferon treatment.

Platelet aggregability and some biochemical parameters were evaluated in seven patients with essential thrombocythemia (ET) compared with seven patients with secondary thrombocytosis (ST). Defective platelet aggregation with one or more agonists was seen in five patients with ET whereas aggregation was increased in two other patients. In addition, three patients with ET demonstrated spontaneous platelet aggregation in citrated plasma. This was associated with increased level of thrombospondin (TSP) in the plasma membrane. Interestingly, the presence of a proteolyzed 160 kDa form of TSP was detected in all patients with ET, whereas it was never found in patients with ST. Furthermore, three patients with ET demonstrated increased levels of platelet surface glycoprotein IV (GP IV), the putative receptor for TSP in the plasma membrane. In two of these patients, this correlated with increased surface expression of TSP and spontaneous platelet aggregation. The results suggest a possible link between the increased number of plasma membrane GP IV molecules, the spontaneous expression of TSP on the platelet surface and platelet hyperaggregability in some ET patients. The levels of plasma membrane GP IV and platelet surface-associated TSP tended to be normalized during alpha-interferon treatment, whereas the presence of an altered form of TSP persisted. This last parameter might be of practical usefulness in the characterization of the disease, permitting a clear distinction from ST.

Aged↗
PubMed1991

Distribution of gelsolin in the retina of the developing rabbit.

The distribution of gelsolin, a calcium-dependent actin-severing and capping protein, in the retina of the developing and adult rabbit was studied. Gelsolin immunoreactivity was found in the photoreceptors and ganglion cells, where it may have a role in neuronal morphogenesis. Only the inner segment of the photoreceptors retained a high gelsolin content in the adult retina, perhaps because the attached outer segment is continuously renewed throughout life. Gelsolin, which is a major component of the rabbit brain oligodendrocytes, was also found in the myelin of the medullary ray region of the rabbit retina. Müller cells in all regions of the rabbit retina also contain gelsolin from early in development to adulthood. Since one of the functions of these cells is to ensheath neuronal elements in the inner plexiform and optic fiber layers, we suggest that gelsolin may play the same role in Müller cells as it does in oligodendrocytes, i.e., sheath formation via its calcium-dependent action on the actin microfilament networks.

Animals↗
PubMed1991

Experimental fusarial hyalohyphomycosis in a murine model.

The pathogenicity of two clinical strains of Fusarium solani was studied in normal and transiently neutropenic outbred CF1 and CD1 male mice. Three inocula (5 x 10(5), 1 x 10(6), and 5 x 10(6) spores/animal) were tested. Groups of 10 mice each were injected with a single intravenous dose of one inoculum. Mortality correlated with the dose of inoculum, as survival was significantly shorter in mice injected with 5 x 10(6) cfu/mouse than in mice that received 1 x 10(6) or 5 x 10(5) cfu/mouse (P less than .001). Necrotizing abscesses with acute branching septate hyphae, neutrophil and macrophage infiltration, and hemorrhage were observed. The median survival of neutropenic mice was shorter than that of normal mice (P less than .001). Neutropenic mice did not show evidence of an inflammatory cellular reaction and exhibited significantly higher numbers of fungi per gram of infected tissue (P less than .001). Intact host defenses in normal mice were able to confine the infection to the kidneys after initial dissemination. In contrast, disseminated infection persisted in most organs in immunosuppressed animals.

Animals↗