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Biomedical subjects

J Drouin

Publications and source records attributed to J Drouin.

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

Developmental stage-specific regulation of atrial natriuretic factor gene transcription in cardiac cells.

Cardiac myocytes undergo a major genetic switch within the first week of postnatal development, when cell division ceases terminally and many cardiac genes are either activated or silenced. We have developed stage-specific cardiocyte cultures to analyze transcriptional control of the rat atrial natriuretic factor (ANF) gene to identify the mechanisms underlying tissue-specific and developmental regulation of this gene in the heart. The first 700 bp of ANF flanking sequences was sufficient for cardiac muscle- and stage-specific expression in both atrial and ventricular myocytes, and a cardiac muscle-specific enhancer was localized between -136 and -700 bp. Deletion of this enhancer markedly reduced promoter activity in cardiac myocytes and derepressed ANF promoter activity in nonexpressing cells. Two distinct domains of the enhancer appeared to contribute differentially to cardiac specificity depending on the differentiation stage of the myocytes. DNase I footprinting of the enhancer domain active in differentiated cells revealed four putative regulatory elements including an A+T-rich region and a CArG element. Deletion mutagenesis and promoter reconstitution assays revealed an important role for the CArG-containing element exclusively in cardiac cells, where its activity was switched on in differentiated myocytes. Transcriptional activity of the ANF-CArG box correlated with the presence of a cardiac- and stage-specific DNA-binding complex which was not recognized by the c-fos serum response element. Thus, the use of this in vitro model system representing stage-specific cardiac development unraveled the presence of different regulatory mechanisms for transcription of the ANF gene during cardiac differentiation and may be useful for studying the regulatory pathways of other genes that undergo switching during cardiac myogenesis.

Animals↗
PubMed1993

Role of platelet membrane glycoproteins Ib/IX and IIb/IIIa, and of platelet alpha-granule proteins in platelet aggregation induced by human osteosarcoma cells.

We have previously shown that the platelet-aggregating activity of human MG-63 and HOS osteosarcoma cells depends at least in part upon tumor cell surface-associated thrombospondin, and suggested that platelet-osteosarcoma cell interactions could occur through interactions with specific platelet membrane receptors. In this study, the platelet-aggregating activity of MG-63 and HOS cells was studied by using a variety of platelet disorders. Both osteosarcoma cell lines induced a biphasic platelet aggregation response when added to normal platelet-rich plasma, while the second phase of aggregation was absent when added to gray platelets (deficiency in alpha-granule proteins) and to aspirin-treated platelets. Platelets from two unrelated patients with type I Glanzmann's thrombasthenia (deficiency in glycoprotein (GP) GPIIb/IIIa) did not aggregate at all with osteosarcoma cells. Using giant platelets from three patients with Bernard-Soulier syndrome (deficiency in GPIb/IX), the aggregation response induced by MG-63 and HOS cells was monophasic and reversible when compared to normal-sized platelets and to giant platelets from a patient with May-Hegglin anomaly (no membrane GP defect). Because GPIb serves as a receptor for von Willebrand factor during hemostasis, aggregation experiments were also conducted with the platelet-rich plasma of two patients with a low plasma von Willebrand factor concentration (type I von Willebrand's disease) before and after the infusion of deamino-D-arginine vasopressin. MG-63 and HOS cells induced biphasic platelet aggregation both before and after deamino-D-arginine vasopressin treatment, while the ristocetin-dependent binding of von Willebrand factor to platelets only occurred after deamino-D-arginine vasopressin treatment. Preincubation of normal platelet-rich plasma with monoclonal antibody SZ-2 directed against the von Willebrand binding domain of GPIb did not inhibit the platelet-aggregation activity of osteosarcoma cells, whereas anti-GPIb antibody SZ-2 did inhibit ristocetin-induced platelet agglutination. In addition, anti-GPIX antibodies did not affect platelet-osteosarcoma cell interactions. In conclusion, our data demonstrate that the first phase of the platelet-aggregating activity of human osteosarcoma cells is initiated by the interaction of these tumor cells with platelet membrane GPIIb/IIIa, whereas the second phase, even if plasma von Willebrand factor is deficient, involves platelet membrane GPIb and the participation of platelet alpha-granule proteins in membrane-mediated events, making aggregation irreversible.

Antibodies, Monoclonal↗
PubMed1993

Novel glucocorticoid receptor complex with DNA element of the hormone-repressed POMC gene.

Previous studies defined a DNA element necessary for glucocorticoid repression of the pro-opiomelanocortin (POMC) gene. The glucocorticoid receptor (GR) binds this negative glucocorticoid response element (nGRE) with an in vitro affinity similar to that of GR for positive GREs. However, whereas GR binds GREs as homodimers, a novel GR complex which forms with nGRE appears to contain three GR molecules. Biochemical characterization of this complex as well as equilibrium binding studies suggest that it is formed by sequential binding of a GR homodimer followed by binding of a GR monomer on the opposite side of the double helix. The DNA-binding domain (DBD) of GR is sufficient for differential binding of GRE and nGRE, as bacterially-expressed DBD formed unique nGRE complexes that contain three GR polypeptides. Thus, the POMC nGRE provides the first example of an interaction between GR and DNA in which GR binds otherwise than as a homodimer. Despite its high affinity for GR, the nGRE differs significantly from GREs in that it does not activate transcription in any context. As the nGRE appears insufficient on its own to confer hormone responsiveness, other POMC promoter elements are likely to be required to mediate glucocorticoid repression.

Animals↗
PubMed1993

Cell-specific helix-loop-helix factor required for pituitary expression of the pro-opiomelanocortin gene.

Pro-opiomelanocortin (POMC)-expressing cells appear to be the first pituitary cells committed to hormone production. In this work, we have identified an element of the POMC promoter which confers cell-specific activity. This element did not exhibit any activity on its own and required at least one other element of the promoter to manifest its cell-specific activity. Fine mutagenesis of this element indicated that a CANNTG motif is responsible for activity. This E-box motif is typical of binding sites for helix-loop-helix (HLH) transcription factors; however, the POMC cell-specific E box cannot be replaced by other E boxes like the kappa E2 site of the immunoglobulin gene or a muscle-specific E box. Similar E boxes which are present in the insulin gene promoter were shown to contribute to the pancreatic specificity of the insulin promoter. However, E-box-binding proteins found in nuclear extracts from POMC-expressing AtT-20 cells and from insulin-expressing cells have different electrophoretic mobilities. The AtT-20 proteins were named CUTE (for corticotroph upstream transcription element-binding) proteins, and they were not found in any other cells. CUTE proteins have DNA-binding properties characteristic of HLH transcription factors. Overexpression of the dominant negative HLH protein Id or of the ubiquitous positive HLH factor rat Pan-2 decreased or augmented POMC promoter activity, respectively. These observations are consistent with the hypothesis that CUTE factors might be heterodimers. This hypothesis was further supported by antibody shift experiments and by abrogation of DNA binding in the presence of bacterially expressed Id protein. Thus, the cell-specific CUTE proteins and their binding site in the POMC promoter appear to be important determinants for cell specificity of this promoter. The requirement for HLH factors in POMC transcription also presents the possibility that these factors are involved in differentiation of pituitary cells, in analogy with the role of HLH factors in muscle development.

Animals↗
PubMed1993

Phospholipase C activity in platelets from Bernard-Soulier syndrome patients.

The levels of glycoprotein (GP) Ib and GPV and phospholipase C activity were measured in platelets from three Bernard-Soulier syndrome patients. The patients' platelets had 46%, 46%, and 24% of control levels of GPIb alpha and 43%, trace, and 13% of control levels of GPV as determined by immunoblot analysis. Stimulation by thrombin, trypsin, the thromboxane analogue U46619, and the combination of U46619 and trypsin caused the formation of [32P]phosphatidic acid, an index of phospholipase C activity, in [32P]orthophosphate-prelabeled platelets. With all agonists, however, the formation of [32P]phosphatidic acid was markedly reduced in Bernard-Soulier syndrome platelets compared with control platelets. These data indicated a postreceptor defect in phospholipase C activation in Bernard-Soulier syndrome platelets and confirmed earlier observations of potential proteolytic and nonproteolytic mechanisms of platelet activation.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1993

[The effects of Arnica Montana on blood coagulation. Randomized controlled trial].

The purpose of this study, which took the form of a two-period cross-over clinical trial, was to determine whether a homeopathic substance, Arnica Montana, significantly decreased bleeding time (Simplate II) and to describe its impact on various blood coagulation tests. It was not shown that this substance had a significant impact on various parameters of blood coagulation in healthy volunteers in the period immediately following administration [corrected].

Adult↗
PubMed1992

Homeopathic treatment of plantar warts.

OBJECTIVE: To evaluate the efficacy of a homeopathic treatment of plantar warts. DESIGN: Randomized double-blind placebo-controlled trial. SETTING: Hospital-based family medicine unit. PATIENTS: Patients were recruited from the unit, through advertisements in the local media and through personal contacts with colleagues. Of the 853 people screened between December 1987 and January 1989, 174 met the eligibility criteria (age 6 to 59 years and presence of one or more plantar warts untreated during the previous 3 months) and agreed to participate; 162 (93%) completed the 18-week follow-up. INTERVENTIONS: The 6-week homeopathic treatment consisted of thuya 30 "centésimal hahnemannien" (CH) (one tube containing 200 pellets weekly), antimonium crudum 7 CH (5 pellets daily) and nitricum acidum 7 CH (one tube containing 200 pellets daily). The placebo pellets were identical to the treatment pellets in appearance and taste. MAIN OUTCOME MEASURE: The proportion of healed patients; a patient was considered healed if all of the warts had disappeared. MAIN RESULTS: The rates of healing at 6, 12 and 18 weeks were 4.8%, 13.4% and 20.0% respectively in the homeopathic treatment group and 4.6%, 13.1% and 24.4% in the placebo treatment group. CONCLUSION: The homeopathic treatment was no more effective than the placebo treatment of plantar warts.

Adolescent↗
PubMed1992

Homodimer formation is rate-limiting for high affinity DNA binding by glucocorticoid receptor.

The glucocorticoid receptor (GR) is a hormone-inducible transcription factor which activates transcription of specific genes by binding to a DNA sequence present in the promoters of inducible genes. These glucocorticoid response elements (GREs) have a conserved palindromic sequence. Each half-GRE palindrome binds one subunit of GR. We have assessed the relative affinity of GR monomers and homodimers for GRE and determined whether homodimer formation is rate-limiting for high affinity GRE binding. The in vitro affinity of GRE binding by GR homodimers was approximately 2 x 10(-10) M, whereas it was approximately 1 nM for GR monomers. While homodimer:GRE complexes were very stable, monomer:GRE complexes appeared less stable in vitro. At low receptor concentration, GR preferentially bound GRE as a homodimer. Prior dilution of GR (equilibrium shifted to monomers) before addition to a GRE binding reaction resulted in slower kinetics of binding by comparison to parallel reactions in which concentrated (largely homodimeric) GR was added first. Taken together, these experiments suggest that homodimer formation is rate-limiting for high affinity GRE binding. A GRE mutant which contained only a half-binding site and which was unable to bind GR homodimers was also unable to confer glucocorticoid-inducible transcription. Taken together with previous work, these experiments support the model that GR homodimers are required for hormone-dependent activation of transcription and that receptor homodimer formation is rate-limiting for GRE binding.

Base Sequence↗
PubMed1992

Rearrangement of BCR genes in malignant lymphoma.

The Philadelphia chromosome, originally thought to be associated solely with chronic myelogenous leukemia (CML), has since been identified in acute leukemias and in some cases of lymphoma. The Philadelphia chromosome results from reciprocal translocation of genetic material between chromosome 9 and 22 involving the c-abl and BCR genes respectively. Southern blot analysis of the BCR genes was carried out on biopsy specimens from 49 patients presenting with malignant lymphoma without a previously documented CML phase. In two patients, BCR gene rearrangements were detected in the malignant lymph nodes but not in the bone marrow samples. A third patient showed BCR gene rearrangements in the bone marrow but not in the lymph node. From this limited study, it seems that the overall incidence of BCR gene rearrangement in malignant lymphoma is similar to that observed in adult AML.

Blotting, Southern↗
PubMed1991

Distal cis-acting promoter sequences mediate glucocorticoid stimulation of cardiac atrial natriuretic factor gene transcription.

Although receptors for most steroid hormones are present in the heart, few cardiac-specific target genes have been identified and studied at the molecular level. Transcription of the atrial natriuretic factor (ANF) gene, which encodes the major secretory product of the heart, is induced by glucocorticoids. In both atrial and ventricular cardiac cells in primary cultures, ANF mRNA levels are increased 3-4-fold after dexamethasone treatment in a time- and dose-dependent manner. This response to glucocorticoids is completely abolished by the antagonist RU486. Interestingly, ventricular myocytes appear to be more sensitive to glucocorticoids than atrial myocytes. DNA-mediated gene transfer studies indicate that glucocorticoids affect ANF gene transcription via a glucocorticoid response element located in the distal 5'-flanking sequences of the rat ANF gene between -697 and -1,029 base pairs. In vitro DNase I footprinting experiments reveal the presence of two binding sites for purified glucocorticoid receptor within this region. Mobility shift assays and competition experiments show that binding of the glucocorticoid receptor to both ANF sites results in a DNA-protein complex similar in affinity and specificity to that of the well characterized mammary tumor virus glucocorticoid response element. Since glucocorticoid activation of the ANF promoter appears specific to cardiac cells, the interaction between the glucocorticoid receptor binding sites and cardiac-specific regulatory elements of this promoter could provide a model to study a mechanism of hormone-dependent signal transduction in the heart.

Animals↗
PubMed1991

Thrombospondin binds to the surface of human osteosarcoma cells and mediates platelet-osteosarcoma cell interaction.

We have previously shown that thrombospondin (TSP) is synthesized and secreted by human MG-63 osteosarcoma cells. In this study, the secretion and cell surface expression of TSP by two different human osteosarcoma cell lines (MG-63 and TE-85) as well as the involvement of TSP in the platelet-aggregating activity of these tumor cells were studied. Using a sandwich enzyme-linked immunosorbent assay, MG-63 cells secreted 3-fold as much TSP as TE-85 cells at 48 h (0.17 +/- 0.01 (SD) versus 0.06 +/- 0.006 micrograms/10(6) cells, P = 0.007). Binding of exogenous 125I-TSP to MG-63 and TE-85 cells in monolayer indicated that binding was time and concentration dependent, saturable, and inhibited by excess cold TSP. However, despite a similar affinity, MG-63 cells had 10-fold more TSP-binding sites than TE-85 cells (402,394 +/- 130,346 versus 36,748 +/- 7,708 TSP-binding sites/cell; P = 0.002). Similar binding differences of 125I-TSP were observed with both osteosarcoma cell lines in suspension. A fluorescence-activated cell-sorting analysis was used in conjunction with an anti-TSP polyclonal antibody, and binding of endogenous TSP to MG-63 and TE-85 cells in suspension was investigated. Addition of an anti-TSP antibody to MG-63 and TE-85 cells in suspension increased the mean fluorescence intensity 50-fold when compared to an irrelevant antibody. Moreover, the fluorescence intensity of MG-63 cells with an anti-TSP polyclonal antibody was increased by 40% when compared to TE-85 cells. Since TSP was expressed on the surface of osteosarcoma cells, the involvement of this glycoprotein in the platelet-aggregating activity of MG-63 and TE-85 cells was therefore investigated using an anti-TSP polyclonal antibody and two monoclonal antibodies (P10 and MA-II), the epitopes of which lie within the Mr 140,000 non-heparin-binding fragment and the Mr 25,000 heparin-binding fragment of TSP, respectively. Preincubation of MG-63 cells (1 x 10(6) cells/ml) with either an anti-TSP polyclonal antibody (100 micrograms/ml) or monoclonal antibody P10 (15 micrograms/ml) inhibited by 80% other platelet-aggregating activity of these tumor cells, while anti-TSP monoclonal antibody MA-II (15 micrograms/ml) had no effect. In sharp contrast, the anti-TSP polyclonal antibody (100 micrograms/ml) only exhibited a slight inhibitory effect on platelet aggregation induced by TE-85 cells when using a low concentration of tumor cells (0.6 x 10(6) cells/ml).(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies↗
PubMed1991

Characterization of novel platelet and endothelial cell target antigens in a family with genetic susceptibility to autoimmunity.

This report describes a French Canadian family whose members exhibit a high incidence of allo- and autoantibodies to antigens present on both platelets and endothelial cells. This is correlated with various HLA specificities known to be associated with autoimmunity, such as A1, B8, DR3, and, in some cases, with clinical disorders, including nephritis, hypertension, and thrombocytopenia. Immunoblot analysis using platelet and endothelial cell lysates showed serum antibodies to a 75 kDa endothelial cell surface polypeptide and to polypeptides with apparent mass of 115 kDa and 26 kDa found on both platelets and endothelial cells. This 115 kDa internal platelet protein was also found in a variety of other cell types, such as mononuclear cells, and increased following cell activation. Monoclonal antibody immunobilization assays were used to characterize the 26 kDa polypeptide; in three of the four patients tested, an antibody to leukocyte differentiation antigen CD9 was identified. The asymptomatic child of the propositus also exhibited an autoantibody against an 80 kDa platelet protein which was sensitive to thrombin digestion, suggesting that this polypeptide may be platelet glycoprotein V. In addition, P1A1 alloantibody was identified in one sister who had given birth to a severely thrombocytopenic boy and who herself had a severe vascular rejection to a cadaver kidney 2 years prior to this study. The propositus also developed hypertensive renal disease following a pregnancy and became dialysis dependent. Thus, members of this family have developed a variety of antibodies, particularly to platelet and endothelial cell antigens. Some subjects have remained asymptomatic in spite of having autoantibodies. However, others have been seriously ill, and their immune response to these antigens is believed to have played a role in the pathogenesis of their neonatal alloimmune thrombocytopenic purpura, hypertensive renal disease, renal graft rejection, and thrombocytopenia.

Antigens, CD↗
PubMed1991

Pituitary pro-opiomelanocortin gene expression requires synergistic interactions of several regulatory elements.

The pro-opiomelanocortin (POMC) gene is expressed very early during pituitary development, before expression of the other pituitary hormone genes, growth hormone and prolactin, and before expression of the Pit-1/GHF-1 transcription factor which activates those genes. Thus, analysis of the POMC promoter should provide markers of the early stages of pituitary development at the time when cells are being committed to expression of one or the other pituitary hormone. We have previously localized the rat POMC promoter to a 543-bp 5'-flanking DNA fragment of the gene using transfection and transgenic mice experiments. We have now used mutagenesis and in vitro protein-DNA binding studies to define three domains of the promoter which have distinct and complementary activities. Within these domains which require each other for full activity, at least nine regulatory elements were defined by in vitro footprinting and replacement mutagenesis. Each element appeared equally important for promoter activity, as mutagenesis of any element had similar effect on promoter activity. Most of the elements bound different AtT-20 nuclear proteins in gel mobility shift experiments. Whereas only two elements appeared to be binding sites for the known transcription factors AP-1 and chicken ovalbumin upstream promoter, the seven other elements appeared to bind nuclear proteins with novel properties. Thus, in contrast to the predominant role of Pit-1/GHF-1 in transcription of the growth hormone and prolactin genes, the control of an early pituitary gene, POMC, appears to depend on the synergistic interaction of several regulatory elements which bind different nuclear proteins.

Animals↗
PubMed1991

Differential expression of natriuretic peptide genes in cardiac and extracardiac tissues.

Cardiac myocytes secrete a family of natriuretic and diuretic peptides, including atrial natriuretic factor (ANF) and brain natriuretic peptide or the rat hormone isoANF. These peptides share structural and functional similarities, but their respective physiological roles have yet to be elucidated. Differential expression of natriuretic peptide genes may reflect distinct physiological or pathophysiological functions for these peptides. To test this hypothesis, we have determined the sites of expression of the ANF and isoANF genes in rat tissues using polymerase chain reaction amplification of ANF and isoANF transcripts. Like ANF mRNA, isoANF mRNA was detected in all heart compartments, and the transcription initiation sites of the isoANF gene, determined from primer extension experiments, were identical in atria and ventricles. In the adult heart, the ventricular isoANF mRNA concentration is only 3 times lower than in atria, and in sharp contrast to ANF, the isoANF gene is constitutively expressed in ventricles during postnatal development. Since ventricles are at least 20 times larger than atria, this implies that isoANF is mostly a ventricular hormone, whereas ANF is essentially an atrial hormone in normal adult hearts. Low levels of isoANF mRNA were found in few extracardiac tissues, including hypothalamus, brain, lung, and aorta. IsoANF transcripts were more abundant than ANF transcripts in aorta, whereas ANF and isoANF mRNA levels were similar in lung. In brain and hypothalamus, ANF transcripts were only 7- and 2-fold greater than isoANF transcripts. The presence of isoANF transcripts in brain and hypothalamus suggests that isoANF is the rat homolog of the human hormone brain natriuretic peptide. Thus, the expression of these two natriuretic peptide genes is not coordinated, suggesting that ANF and isoANF may play different physiological roles in cardiac and extracardiac tissues.

Animals↗
PubMed1990

Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction.

Glucocorticoids are potent inhibitors of collagenase induction by phorbol esters and inflammatory mediators. The target for this negative effect is the AP-1 site within the collagenase promoter, which also mediates its induction. Negative regulation is due to repression of AP-1 activity by the glucocorticoid receptor (GCR). While the GCR is a potent inhibitor of AP-1 activity (Jun/Fos), both c-Jun and c-Fos are potent repressors of GCR activity. In vitro experiments using purified GCR and c-Jun proteins suggest that mutual repression is due to direct interaction between the two. Direct interaction between GCR and either c-Jun or c-Fos is demonstrated by cross-linking and coimmunoprecipitation. These findings reveal a cross talk between two major signal transduction systems used to control gene transcription in response to extracellular stimuli, and a novel mechanism for transcriptional repression.

Animals↗