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S Chevalier

Publications and source records attributed to S Chevalier.

At least 73 records · Page 4Linked to original sources
PubMed1996

Phosphotyrosine antibodies preferentially react with basal epithelial cells in the dog prostate.

PURPOSE: Considering the hypothesis that androgen-independent but growth factor dependent epithelial cell division may be important in the development and progression of prostate cancer and that protein tyrosine kinases and phosphotyrosine protein phosphatases are key enzymes modulating the levels of specific phosphotyrosylated proteins implicated in several growth factor regulated signal transduction pathways, our aim was to study the cellular distribution of phosphotyrosine proteins in normal and hyperplastic dog prostates as well as in those of castrated dogs supplemented with either androgens or estrogens in order to modify the relative proportion of basal versus secretory epithelial cells. MATERIALS AND METHODS: Following the determination of optimal conditions to specifically detect phosphotyrosine proteins by a rabbit polyclonal antibody directed against phosphotyrosine, immunohistochemistry was performed on prostate tissue sections from these experimental animals. In addition to morphological criteria, an antibody to high molecular weight cytokeratins and antisera against arginine esterase were used to selectively identify basal and secretory cells. Since prostatic acid phosphatase may be involved in the local regulation of phosphotyrosine proteins, its distribution was also evaluated with a human prostatic acid phosphatase antiserum. RESULTS: In all prostatic tissues examined, basal epithelial cells were preferentially and specifically stained with antiphosphotyrosine. The staining intensity per basal cell was highest in the estrogen-supplemented dogs. In addition, basal cells were numerically increased and all were highly immunoreactive for high molecular weight cytokeratins. In prostates displaying a well-differentiated glandular epithelium, the number of positive basal cells and their staining intensity varied in the following order: normal < hyperplastic < androgen-supplemented dogs. At all times, the levels of phosphotyrosine proteins in prostatic acid phosphatase and arginine esterase positive cells (secretory) remained low. Fibroblasts and smooth muscle cells were unreactive to antiphosphotyrosine, even though estrogen supplementation increased the prostatic stromal volume. CONCLUSIONS: The preferential localization of phosphotyrosine proteins in basal cells, their increased level per cell and the number of positive cells in the different experimental animals support the concept that basal cells represent the stem cells of the prostate. The sex steroid-mediated up-regulation of protein phosphorylation on tyrosine residues in these cells suggests that their proliferation is likely to involve growth factor regulated signal transduction pathways. In this respect, the lack of maturation of basal cells and the differentiation of secretory cells induced by androgen deprivation, combined with estrogen stimulation, favors the activation of these pathways and cell growth. On the other hand, the activation of glandular cell differentiation and the increase of stromal volume do not alter the threshold level of protein tyrosine kinase and phosphatase activities in secretory cells, fibroblasts and smooth muscle cells.

Androgens↗
PubMed1995

Activation of a serine/threonine kinase signaling pathway by transforming growth factor type beta.

Transforming growth factor type beta (TGF-beta) is a multifunctional factor that regulates proliferation and differentiation of many cell types. TGF-beta mediates its effects by binding to and activating cell surface receptors that possess serine/threonine kinase activity. However, the intracellular signaling pathways through which TGF-beta receptors act remain largely unknown. Here we show that TGF-beta activates a 78-kDa protein (p78) serine/threonine kinase as evidenced by an in-gel kinase assay. Ligand-induced activation of the kinase was near-maximal 5 min after TGF-beta addition to the cells and occurred exclusively on serine and threonine residues. This kinase is distinct from TGF-beta receptor type II, as well as several cytoplasmic serine/threonine kinases of similar size, including protein kinase C, Raf, mitogen-activated protein kinase kinase kinase, and ribosomal S6 kinase. Indeed, these kinases can be separated almost completely from p78 kinase by immunoprecipitation with specific antibodies. Furthermore, using different cell lines, we demonstrate that p78 kinase is activated only in cells for which TGF-beta can act as a growth inhibitory factor. These data raise the interesting possibility that protein serine/threonine kinases contribute to the intracellular relay of biological signals originating from receptor serine/threonine kinases such as the TGF-beta receptors.

3T3 Cells↗
PubMed1995

DNA replication. Almost licensed.

Exact duplication of all the DNA in a cell occurs during each S phase, and only once in each cell cycle. Recent results show that conserved proteins of the MCM family contribute to these precisely regulated events.

Animals↗
PubMed1995

Overview of the regulation of S-phase in Xenopus egg extracts.

Recently, several components required for nuclear DNA replication and involved in the regulation of S-phase within the cell cycle have been identified. Though detailed models of these events are not available, we focus on recent developments arisen from analysis in Xenopus egg extracts.

Animals↗
PubMed1995

Early events in DNA replication require cyclin E and are blocked by p21CIP1.

Using immunodepletion of cyclin E and the inhibitor protein p21WAF/CIP1, we demonstrate that the cyclin E protein, in association with Cdk2, is required for the elongation phase of replication on single-stranded substrates. Although cyclin E/Cdk2 is likely to be the major target by which p21 inhibits the initiation of sperm DNA replication, p21 can inhibit single-stranded replication through a mechanism dependent on PCNA. While the cyclin E/Cdk2 complex appears to have a role in the initiation of DNA replication, another Cdk kinase, possibly cyclin A/Cdk, may be involved in a later step controlling the switch from initiation to elongation. The provision of a large maternal pool of cyclin E protein shows that regulators of replication are constitutively present, which explains the lack of a protein synthesis requirement for replication in the early embryonic cell cycle.

Amino Acid Sequence↗
PubMed1995

Both cdc2 and cdk2 promote S phase initiation in Xenopus egg extracts.

Xenopus egg extracts induce S phase DNA replication in added sperm pronuclei in a highly regulated manner, similar to events in vivo. Removal of cyclin-dependant kinases (cdks) or cdk2 from these extracts using affinity matrices severely inhibits initiation of S phase. We have used p13suc1 beads to remove both cdk2 and cdc2 proteins from egg extracts and developed a method to replace either protein alone to assess their capacity to initiate DNA replication. Re-addition of either cdk2 or cdc2 proteins to depleted extracts, through translation of their respective mRNAs, restimulated replication, judged by both total synthesis and labelling index. An ATP-binding-site mutant cdk2 mRNA (cdk2.R33) failed to stimulate replication and inhibited S phase initiation in mock-depleted extracts. Both human and Xenopus cdc2 mRNAs rescued replication in this system. Human mutant mRNAs have been used to show that the stimulation induced requires cdc2 catalytic activity, though not its mitotically active form. Rescue of replication by p34cdc2 is also observed in extracts depleted of cdks with a cdk2 antibody, which still retain much of their endogenous cdc2 protein. We conclude that newly synthesised p34cdc2, but not the inherited 'old' form, can induce S phase and in this form may overlap in function with p33cdk2.

Animals↗
PubMed1994

Transforming growth factor beta down-regulates Src family protein tyrosine kinase signaling pathways.

Transforming growth factor beta (TGF beta) inhibits the proliferation of a wide range of cell types through interaction with its cell surface receptor (R-TGF beta). R-TGF beta possesses serine/threonine kinase activity rather than the tyrosine kinase activity normally associated with peptide growth factor receptors; nevertheless, TGF beta triggers a signaling pathway that leads to the repression of transcription factors, which appear to mediate the action of receptor tyrosine kinases within the nucleus. Accumulating evidence has also shown that the nonreceptor protein tyrosine kinases of the Src family play essential roles in the signal transduction pathways that regulate cell proliferation, differentiation, and function. Here, we investigate whether signals initiated by R-TGF beta are transduced, at least in part, through members of the Src family of tyrosine kinases. Treatment of the responsive human prostate carcinoma cell line PC3 with TGF beta induces a rapid and specific decrease in cellular levels of pp60Src and pp53/56Lyn and a corresponding decrease in their protein kinase activity when the assays were performed in vitro using the exogenous substrate enolase. Consistent with suppression of pp60Src and pp53/56Lyn kinase activity, TGF beta also caused a substantial intracellular accumulation of the unphosphorylated form of SH2-containing protein (SHC), a substrate of the Src family kinases. This was paralleled by decreased formation of a complex between the adaptor protein known as growth factor receptor-bound protein 2 and SHC. These results suggest, for the first time, that TGF beta induces down-regulation of Src family kinases, leading to disruption of the SHC-growth factor receptor-bound protein 2 complex. These events may play a crucial role in the negative regulation of Ras, as well as in the control of downstream effector molecules involved in the regulation of cell growth.

Blotting, Western↗
PubMed1994

Expression and functionality of the trkA proto-oncogene product/NGF receptor in undifferentiated hematopoietic cells.

The expression of the low-affinity NGF receptor (p75) and the trkA proto-oncogene product was analyzed in a series of human hematopoietic cell lines at protein and RNA levels. We did not detect any form of NGF receptor in cell lines displaying a myelomonocytic phenotype (HL60 and U937). In contrast, cells displaying a more immature erythroleukemic phenotype (TF1 and K562) expressed TrkA in the absence of detectable p75. Scatchard analysis showed a single high-affinity site for NGF (kd = 10(-10) mol/L), with a copy number ranging from 300 to 3,000 sites per cell depending on the studied cell line. In addition, NGF induced autophosphorylation of TrkA and could substitute for granulocyte-monocyte colony-stimulating factor to trigger the proliferation of the TF1 cell line, with a half-maximal signal observed at 50 pmol/L, indicating that p75 is not required for DNA synthesis in this cell line. The physiologic relevance of NGF in early hematopoiesis was confirmed by showing that 12% to 15% of progenitor blood cells from mice treated with 5-fluorouracil expressed TrkA and that these cells could be induced to proliferate and differentiate in response to NGF in association with macrophage colony-stimulating factor. Our study demonstrates for the first time that trkA proto-oncogene expression and activation is not restricted to the nervous system, but is also an important element in early hematopoiesis.

Animals↗
PubMed1994

Involvement of gp130/interleukin-6 receptor transducing component in interleukin-11 receptor.

The recently cloned interleukin (IL)-11 displays many biological properties in common with those reported for IL-6. In order to analyze the nature and the functionality of the IL-11 receptor we developed a proliferative assay using the human multifactor-dependent cell line TF1. We showed that a blocking monoclonal antibody GPX7 raised against the gp130/IL-6 receptor transducing subunit was also able to inhibit the IL-11-triggered TF1 line proliferation. In addition, involvement of gp130 in IL-11 signaling was demonstrated by an induction of the transducing protein phosphorylation in response to IL-11, as observed for IL-6. In contrast, the blocking monoclonal antibody B-R6, which recognized the gp80/IL-6 binding subunit failed to interfere with the IL-11 proliferative signal in the TF1 cell line. Similarly, we did not observe any competition between IL-6 and IL-11 for a putative common binding site on the cell surface. These results suggest that the IL-11 binding component is different from the gp80/IL-6 receptor. In conclusion, IL-11, along with IL-6, leukemia inhibitory factor, oncostatin M and ciliary neurotrophic factor, belongs to the same family of cytokines, using gp130 as a transducing protein.

Antibodies, Monoclonal↗
PubMed1994

Detection of phosphotyrosine in glutaraldehyde-crosslinked and alkali-treated phosphoproteins following their partial acid hydrolysis in gels.

Soluble fractions and particulate extracts from human prostate, and extracts from rat-liver membranes were used as a source of kinases to phosphorylate endogenous proteins in the presence of gamma- 32P-labeled ATP. Histone was also added as a substrate in order to compare the direct partial acid hydrolysis of phosphoproteins in gels to an indirect procedure involving partial acid hydrolysis after extraction in sodium dodecyl sulfate followed by precipitation with acetone. These procedures led to recoveries of 32P-labeled material of 90% and 40%, respectively, with a similar proportion of radiolabeled phosphoamino acids. Several 32P-labeled phosphoproteins separated in gels were therefore directly HCl-hydrolyzed and their phosphoamino acids were quantitated either prior to, or after glutaraldehyde crosslinking, with and without alkali treatment. By preventing protein losses occurring in hot alkali, glutaraldehyde crosslinking increased by an average factor of 6.5 the 32P-labeled material available for phosphoamino-acid analyses. For eight phosphoproteins analyzed, the overall effect of combined glutaraldehyde and alkali treatments was a relative decrease in phosphoserine (up to 8-fold), with concomitant relative increases in phosphotyrosine and phosphothreonine (up to 62- and 6-fold, respectively). This method will especially be useful for the detection of pTyr, a less abundant phosphoamino acid, in proteins which suffer from poor transfer efficiency in Western blot, are weakly antigenic towards anti-phosphotyrosine antibodies, can hardly be extracted from a gel and for identification of protein tyrosine kinases renatured in gels.

Acrylic Resins↗
PubMed1994

[Long-term follow-up of patients with complete congenital auriculo-ventricular block treated with a cardiac stimulator].

The congenital complete heart block (CCHB) is a rare affection. In our two centers, in a period of 10 years, 1405 pacemakers were placed and only 10 (0.71%) for CCHB. We review the clinical history, the EKG's, echocardiography and Holter monitoring of these 10 patients. All of them were symptomatic. After pacemaker implantation, symptoms were improved in 90%. No death or other morbidity were observed during the long-term follow-up. So, the clinical outcome of patients with CCHB who underwent prophylactic pacemaker implantation seems to be associated with an excellent prognosis.

Adult↗
PubMed1993

Dietary restriction reduces the incidence of NMU-induced mammary tumors and alters retinoid tissue concentrations in rats.

Previous studies suggested a relationship between dietary restriction (DR) effects on mammary carcinogenesis and DR effects on liver retinoids. Therefore, in this study, retinoid concentrations were measured by high-performance liquid chromatography in the plasma, liver, and peripheral organs of DR rats with chemically induced carcinogenesis. Rats were injected with N-methyl-N-nitrosourea (MNU) and maintained on graded levels of DR (reduction of 10-40% from energy ingested by control animals with free access to food). Mammary tumor incidence and multiplicity induced by MNU were reduced in relation to the degree of DR, with virtual prevention occurring at 30% and 40% DR. Total hepatic retinoid concentrations (retinol + retinyl esters) were significantly greater in rats given MNU and subjected to DR, but liver total retinoid content was comparable between the groups. However, plasma retinol concentrations were significantly lower in DR rats than in controls given the carcinogen without DR. Retinoid concentrations were also elevated in adipose tissue, lungs, and intestine of DR rats, while renal concentrations remained unaltered. Retinoid concentrations in mammary glands and mammary tumors were similar in all groups. Thus, in DR rats, vitamin A concentrations in liver and other target tissues are maintained or increased despite decreases in plasma. It remains to be investigated whether these alterations in retinoid content have any relationship to the cancer-preventive effect of DR.

Adipose Tissue↗
PubMed1993

Protection of islets of Langerhans from antibodies by microencapsulation with alginate-poly-L-lysine membranes.

Microencapsulation of islets has been proposed to prevent their immune destruction following transplantation. An indirect immunofluorescence technique has been developed and used to study the permeability of the alginate-poly-L-lysine microcapsules to antibodies. Wistar rat islets were incubated with the R2D6 monoclonal mouse IgM antibody against rat islets, microencapsulated, and incubated with fluorescein-labeled goat IgG antibodies against mouse IgG and IgM. For the negative controls, the first antibody was omitted or both antibodies were omitted. The positive controls included islets incubated with both antibodies before they were encapsulated. Our study demonstrated that the alginate-poly-L-lysine membranes are not permeable to IgG when poly-L-lysine of molecular weights ranging from 21,000 to 390,000 are used. This simple immunofluorescence technique demonstrated the nonpermeability of the microcapsules to IgG, and could be useful for the initial evaluation of new types of membranes.

Alginates↗
PubMed1993

High circulating leukaemia inhibitory factor (LIF) in patients with giant cell arteritis: independent regulation of LIF and IL-6 under corticosteroid therapy.

Leukaemia inhibitory factor (LIF) is a cytokine which possesses a wide range of biological activities including, like IL-6, the capacity to stimulate acute phase protein (APP) synthesis. We have developed a sensitive and specific ELISA for human LIF, and tested the circulating cytokine levels in various disease states, some of which are associated with inflammation. LIF was detected in 11/20 sera from patients with giant cell arteritis (GCA), a vasculitis syndrome affecting particularly the temporal artery, characterized by panarteritis with inflammatory cell infiltration. LIF levels were considerably elevated in some patients who also displayed elevated levels of IL-6 and C-reactive protein (CRP); however, no correlation was observed between the levels of circulating LIF and levels of IL-6 or CRP. Furthermore, LIF levels were not affected by corticosteroid therapy, whereas IL-6 and CRP decreased rapidly, as clinical symptoms resolved. A putative role for LIF in the persistence of histological lesions is discussed. This is the first report of the presence of circulating LIF in sera. These results are in agreement with the complexity of induced inflammatory cytokines and corticoid regulation of APP synthesis observed in vitro and in vivo.

Administration, Oral↗
PubMed1992

Identification of cytosolic protein tyrosine kinases of human prostate by renaturation after SDS/PAGE.

The identification of protein tyrosine kinases (PTKs) was successfully achieved by renaturation in gels after SDS/PAGE. To this effect, samples were mixed with a PTK substrate, namely the polydispersed co-polymer of glutamic acid and tyrosine [poly(Glu, Tyr), M(r) from 30,000 to 94,000], and were simultaneously submitted to electrophoresis. Following guanidine hydrochloride denaturation, renaturation and phosphorylation with [gamma-32P]ATP, kinase activity was detected by autoradiography. When applied to cytosol from human hyperplastic prostate, eleven protein kinases were detected, among which one major (M(r) 50,000) and two minor proteins (M(r) 40,000 and 38,000) were identified as PTKs by the presence of phosphotyrosine. Incubation of the gel in hot alkali after glutaraldehyde cross-linking almost completely eliminated the detection of non-PTK enzymes. On the other hand, in the absence of poly(Glu,Tyr), no PTK activity was detected. Partial purification of cytosolic PTKs indicates that the native M(r) of the major phosphotransferase was 44,000, as estimated by gel filtration following ammonium sulphate precipitation and anion-exchange chromatography. Upon renaturation after electrophoresis, this fraction showed only one major band active on poly(Glu,Tyr) which was associated with the polypeptide of M(r) 50,000. This enzyme was also identified following two-dimensional electrophoresis and renaturation in the presence of poly(Glu,Tyr), allowing the determination of a pI in the range 7.5-7.8. Thus PTKs can be easily renatured following electrophoresis and rapidly identified on the basis of their M(r) and pI in both crude or partially purified preparations. With the crucial role played by PTKs in the activation of cell function and carcinogenesis, this procedure could be useful in the identification of such enzymes and in distinguishing them from their substrates in gels.

Amino Acids↗