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S Chevalier

Publications and source records attributed to S Chevalier.

At least 109 records · Page 6Linked to original sources
PubMed1992

The major form of protein tyrosine kinase in the dog prostate is expressed by a 50 kDa polypeptide.

We have already reported that the protein tyrosine kinase (PTK) activity in the dog prostate is distributed in cytosolic (75%) and particulate (Triton X-100-solubilized) fractions and that upon gel filtration, both PTKs migrate as entities of Mr 44,000 [(1991) Biochem. Cell. Biol. 69, 146-153]. Herein we demonstrate by immunoprecipitation with anti-phosphotyrosine antibodies that the soluble PTK has the ability to undergo self-phosphorylation. In addition, the polypeptide responsible for that enzymatic activity has been identified by 2 approaches: (1) a two-dimensional electrophoresis, in which the first dimension performed in non-denaturing conditions allowed the localization of the native enzyme, while the second dimension (SDS-PAGE) permitted the analysis of alkali-resistant phosphoproteins corresponding to the activity; (2) protein renaturation after SDS-PAGE followed by in situ phosphorylation (with [gamma-32P]ATP) of polyGT electrophoresed together with the enzyme preparation; the exclusive presence of the radiolabeled phosphotyrosine in the renatured protein confirmed its enzymatic nature. Using these methods, the major form of PTK in the dog prostate was shown to be expressed by a 50 kDa polypeptide which possesses autophosphorylation sites and which is present in the cytosol as an active monomer.

Animals↗
PubMed1992

Identification of RNA-binding proteins specific to Xenopus Eg maternal mRNAs: association with the portion of Eg2 mRNA that promotes deadenylation in embryos.

Maternal Xenopus Eg mRNAs have been previously identified as transcripts that are specifically deadenylated after fertilization and degraded after the mid blastula transition. Destabilizing cis sequences were previously localised in the 3' untranslated region of Eg2 mRNA. In order to characterize possible trans-acting factors which are involved in the post-transcriptional regulation of Eg mRNAs, gel-shift and u.v. cross-linking experiments were performed, which allowed the identification of a p53-p55 RNA-binding protein doublet specific for the 3' untranslated regions of Eg mRNAs. These p53-p55 proteins do not bind to the 3' untranslated regions of either ornithine decarboxylase or phosphatase 2Ac mRNAs, which remain polyadenylated in embryos. These novel RNA-binding proteins are distinct from the cytoplasmic polyadenylation element-binding protein that controls the polyadenylation of maternal mRNAs in maturing Xenopus oocytes, and from previously identified thermoresistant RNA-binding proteins present in oocyte mRNP storage particles. The p53-p55 bind a portion of the Eg2 mRNA 3' untranslated region, distinct from the previously identified destabilizing region, that is able to confer the postfertilization deadenylation of CAT-coding chimeric mRNAs. This suggests that the p53-p55 RNA-binding proteins are good candidates for trans-acting factors involved in the deadenylation of Eg mRNAs in Xenopus embryos.

Animals↗
PubMed1991

Radiation-inactivation size of transformed and non-transformed androgen receptors.

The nucleic acid sequence of the androgen receptor (AR) gene predicts that the protein structure possesses DNA- and steroid-binding domains that show high degrees of sequence similarity with those of other steroid receptors. Since the steroid-binding domain of the AR corresponds to a 30 kDa portion of the protein, and the AR structure may be monomeric or hetero-oligomeric depending on its transformation state, we have herein determined the AR radiation-inactivation size (RIS) in relation to the molecular structure whose binding activity toward methyltrienolone (R1881) is abolished by a radiation 'hit'. Soluble fractions from whole canine prostatic tissue were used as a source of non-transformed AR. The AR transformation was induced by the addition of 0.6 M-KCl, and these preparations were used together with high-salt nuclear extracts as a source of transformed AR. To maximize the binding activity, molybdate and dithiothreitol were included during AR extraction. Receptor transformation was verified by modifications of both the sedimentation coefficients (from 7.5 S to 4.1 S on sucrose gradients) and molecular masses (from 260 kDa to 115 kDa by gel filtration). The RIS values of the non-transformed and transformed ARs were not statistically different: 92 +/- 19 kDa and 110 +/- 25 kDa respectively. In addition, the inactivation of AR binding activity by radiation was attributed to a loss of binding sites, with no significant change in the Kd. When benzoic acid, a free-electron scavenger, was added together with dithiothreitol before and after irradiation, no change in the RIS value was observed. Thus, in the canine prostate, the RIS value of the AR represents the monomeric protein, independently of its association with other proteins, and this value corresponds to that predicted by cloning studies and photoaffinity-labelling of AR.

Animals↗
PubMed1991

Prostatic epithelial cells in culture: phosphorylation of protein tyrosyl residues and tyrosine protein kinase activity.

The ability of dividing canine prostatic epithelial cells in primary monolayers to phosphorylate protein tyrosyl residues was evaluated by metabolic studies performed through incorporation of [32P]-phosphate into alkali-resistant phosphoproteins and by the assay of their tyrosine protein kinase activity. The presence of sodium orthovanadate during cell incubation with [32P]-phosphate greatly enhanced the relative labelling intensity of a 44 kDa alkali-resistant phosphoprotein and the total cellular content of phosphotyrosine in proteins; in this respect, growth factors such as epidermal growth factor, insulin, and insulin-like growth factor I, and the steroids dihydrotestosterone and estradiol were inactive. When the cells were solubilized, sodium orthovanadate stimulated their tyrosine protein kinase activity and inhibited their phosphotyrosine phosphatase activity. To characterize the tyrosine protein kinase of these cultured cells, conditions for optimal activity were established using the substrate poly [Glu80Na, Tyr20]. The subcellular localization of the enzyme was determined upon cell fractionation: 88% of the kinase activity was associated with the particulate fraction and 30% of this activity was partially solubilized with 0.5% Triton X-100; this solubilization was improved to 83% in the presence of 0.25 M KCI. The enzyme directly solubilized from prostatic cells with Triton X-100 (38% of activity) mainly catalyzed the alkali-resistant phosphorylation of pp63, pp59, and pp44, which contained phosphotyrosine. These proteins were also phosphorylated by the major peak of kinase activity which was eluted at an apparent molecular weight of 300-350 kDa upon gel filtration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Involvement of vitronectin and of a natural extracellular matrix in prostatic cell to cell contact and interaction with the substratum in culture.

The kinetics and regulation of attachment, the spreading and initial growth of canine prostatic epithelial cells in primary monolayers, as well as the ability of the cells to produce a natural extracellular matrix (nECM) have been studied. Within 1 day of culture, attachment without spreading occurred in uncoated dishes while spreading was observed in nECM-coated dishes. The attachment was stimulated by dialyzed fetal bovine serum (dFBS) and/or by the nECM and was not affected by the presence of cycloheximide at 10(-6) M. This effect of dFBS on attachment was maximal at a concentration of 10% and increased with cell density and was mainly attributed to vitronectin as demonstrated by gel filtration, selective adsorption of serum proteins to the substratum, and indirect immunofluorescence of prostatic epithelial cells incubated with a rabbit antiserum against bovine vitronectin. Dog serum reduced attachment while bovine serum albumin, phenol red, and steroids were inactive. The maximal effect of the nECM on attachment was observed with matrices produced by canine prostatic epithelial cells cultured in the presence of dog serum for more than 10 days. As revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, several proteins of relative mass (Mr) of 210, 113, 61, 56, 52, 49, 45, and 42 kDa remained in the dish following nECM denudation and could be responsible for its effect on attachment. Their deposition as well as intercell adhesion and cell-to-substratum interactions required an active protein synthesis plus non-steroidal serum components. Once attached and spread in primary monolayers, prostatic epithelial cells divided by the 3rd to 4th day; their growth, stimulated by dFBS at an optimal concentration of 20%, was inhibited by cycloheximide while the nECM was inactive. Thus, vitronectin in serum and the nECM synthesized by canine prostatic epithelial cells are responsible for the attachment and spreading of homologous cells to an in vitro substratum; nECM proteins also contribute to cell-to-cell contact and cell-to-substratum interaction in the monolayers.

Animals↗
PubMed1991

Serum and prostatic growth-promoting factors for steroid-independent epithelial cells of adult dog prostate.

A growth factor-like effect has been observed on canine prostatic epithelial cells when cultured in the presence of their homologous serum and prostatic extracts; the mitogenic activities of both preparations were dose-dependent and not altered by charcoal treatment. The effect of dog serum decreased when the density of the epithelial cell cultures increased and was minimal on canine prostatic fibroblasts. Trace amounts of intracellular sex steroids did not contribute to epithelial cell proliferation since the presence of sex steroid action inhibitors did not alter growth rate; in those conditions, cycloheximide completely prevented cell division. When various hormones and known mitogenic agents were tested alone or in combination with steroids, none elicited an increase in the number of epithelial cells cultured in serum-free medium or altered the proliferative effect of dog serum observed in parallel cultures. On gel filtration, dog serum or tissue cytosol showed a major mitogenic activity at an apparent molecular mass of 150 kDa and a minor one of 1.5 kDa as evaluated by gel filtration of dog serum ultrafiltrate. Acidic extraction of prostatic tissue followed by chromatography on a hydrophobic C-18 column and subsequent gel filtration also led to the detection of the low Mr component. Thus, humoral and/or tissular factors present in vivo and different from known mitogens may be of importance as direct modulators of the basal epithelial cell growth in the adult canine prostate.

Animals↗
PubMed1990

Protein tyrosine kinases in human breast cancer: kinetic properties and evidence for the presence of two forms of native enzyme.

The protein tyrosine kinase (PTK) of human breast tumors classified as positive (TM+) or negative (TM-) according to their estrogen and progestin receptor levels was partially characterized with regard to its distribution, kinetic parameters, molecular size, and ability to phosphorylate endogenous mammary proteins. For both types of tumors, PTK activity depended upon the presence of Mn++ (2-5 mM) and/or Mg++ (10-20 mM). The activities, total (per g of tissue) and specific (per mg of protein), were similar for both types of tumors, and an average of 60% of activity was located in cytosolic fractions. The autoradiographic detection of alkali-resistant phosphoproteins after SDS-PAGE showed very similar patterns between corresponding fractions from both types of tumors. Upon gel filtration, two peaks of activity of apparent Mr 245 kDa (peak I) and 47 kDa (peak II) were observed. Peak II was found in both cytosols and extracts from particulate fractions, while peak I was present only in the latter fraction for both TM+ and TM- tumors. The apparent Km's for ATP ranged from 4.1 to 6.6 microM, and from 11 to 34 micrograms/ml for the synthetic substrate poly [Glu80, Tyr20], at an optimal pH of 6.5-7.5. When endogenous alkali-resistant phosphorylation of peaks I and II was determined by autoradiography after SDS-PAGE, two major mammary proteins of Mr 60 and 45 kDa were phosphorylated by peak II and three, Mr 145, 74, and 62 kDa, by peak I.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Neoplasms↗
PubMed1990

Prostatic acid phosphatase in serum of patients with prostatic cancer is a specific phosphotyrosine acid phosphatase.

We developed an assay to measure at acid pH the phosphotyrosine phosphatase activity in sera from patients with prostatic cancer. The method used quantifies the inorganic phosphate liberated from phosphotyrosine after incubation with serum, followed by the deproteinization of the reaction mixture. A high acid phosphatase (EC 3.1.3.2) activity towards phosphotyrosine was observed in all sera from patients with increased activity of prostatic acid phosphatase. This activity represented 96% of prostatic acid phosphatase and 77% of total acid phosphatase activities. Moreover, it was correlated (r = 0.91) with the amount of serum prostatic acid phosphatase determined by radioimmunoassay. When serum acid phosphatase activity was measured on several phosphorylated substrates, preferential hydrolysis was demonstrated for those in which the phosphate group was esterified on an aromatic ring rather than those presenting an aliphatic chain. Among phosphoamino acids, only phosphotyrosine was a good substrate, with little or no activity observed with phosphoserine and phosphothreonine. Human seminal plasma and partially purified prostatic acid phosphatase, tested for their activity on some of these substrates, gave similar results. On the other hand, sera from patients with above-normal alkaline phosphatase activity and no prostatic disease showed little or no activity on phosphotyrosine at both acid and alkaline pH values. Evidence is presented that the prostatic acid phosphatase in serum is a specific phosphotyrosine acid phosphatase.

Acid Phosphatase↗
PubMed1990

Inducible production of macrophage colony-stimulating factor (CSF-1) by malignant and normal human T cells.

The CEM-ON malignant T cell line and long-term cultured normal T cells can be induced to release CSF-1 in their culture supernatants. Chemical inducers (PMA + A23187) and, more interestingly, cytokines (tumor necrosis factor alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha)), as well as physiological (antigen + IL-2) or specific (anti-CD3 + IL-2 or PMA) stimuli, lead to rapid transient colony-stimulating factor-1 (CSF-1) gene expression and production of biologically active CSF-1. These data suggest that CSF-1 may play a role in the early phases of immune response.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Tyrosine protein kinase activity of human hyperplastic prostate and carcinoma cell lines PC3 and DU145.

Using the substrate poly[Glu80Na,Tyr20] [poly(GT)] and the autoradiographic detection of alkali-resistant phosphoproteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, tyrosine protein kinase (TPK) has been evidenced in human hyperplastic prostates (BPH) and the prostatic carcinoma cell lines PC3 and DU145. The enzyme was mainly found in the soluble fractions from hyperplastic tissues and in Triton extracts from the cell lines. However, its specific activity in tissues was 1.5- to 4.5-fold times higher in particulate than in soluble fractions and it was of the same order of magnitude as that of neoplastic cells. Under these conditions, no activity was detected in human seminal plasma and in sera from normal adult males or patients with BPH and/or prostatic carcinoma. On the other hand, some TPK activity was associated with human spermatozoa, with a specific activity 4- to 6-fold lower than in BPH tissue fractions and a total activity, per 10(6) cells, 5- to 20-fold lower than that in prostatic carcinoma cells. The activity of prostatic TPK was dependent upon the presence of the divalent cations Mn2+ or Mg2+ and it was completely abolished by heat denaturation. Angiotensin II, casein, and histone H2B were poor substrates compared to poly(GT). The TPK activities towards poly(GT) as well as endogenous proteins were not stimulated by epidermal growth factor and insulin or by dihydrotestosterone and estradiol. The autoradiography of alkali-resistant phosphoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed several bands in both BPH tissues and neoplastic cells (molecular weight ranging from 17,000 to 200,000). Preliminary characterization of TPK by gel filtration on Sephacryl S-300 showed that the soluble enzyme from BPH tissues had a molecular weight of 50,000, while the particulate-associated TPK, when assayed on poly(GT), eluted with proteins of Mr 210,000. When these peak fractions were used for endogenous phosphorylation, several major alkali-resistant phosphoproteins in the range of Mr 40,000-60,000 were evidenced, together with a Mr 110,000 band phosphorylated by the particulate TPK of Mr 210,000. In similar conditions, the TPK solubilized from rat liver membranes and partially purified by gel filtration was associated with a Mr 170,000 alkali-resistant phosphoprotein. Thus, TPKs are expressed in BPH tissues and carcinoma cell lines. In BPH tissues, two forms of TPK are expressed and the predominant enzyme is soluble and of low molecular weight (Mr 40,000-60,000).

Aged↗
PubMed1989

Alkali-resistant protein phosphorylation and tyrosine kinase activity of epithelial cell types from normal and metaplastic canine prostates.

To determine how functional changes such as growth and differentiation in the prostatic epithelium would alter protein phosphorylation on tyrosyl residues, differentiated (secretory) and basal (non-secretory) prostatic epithelial cells from normal and from metaplastic canine prostates were labeled with [32P]phosphate and their alkali-resistant phosphoproteins were analyzed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The tyrosine protein kinase activity of the cells was also assayed by phosphorylation of a synthetic substrate, poly(Glu80-NaTyr20), in the presence of [gamma-32P]ATP. The prostates were characterized by tissue morphology and the cells by their density on Percoll gradients and [3H]thymidine incorporation into DNA. In prostates from intact dogs, tall columnar secretory epithelial cells were predominant and the non-secretory or basal epithelial cells were quiescent and did not incorporate [3H]thymidine. In metaplastic glands obtained from estrogen-treated castrated dogs, most of the isolated cells were non-secretory and synthesized DNA. Alkali-resistant phosphoproteins were present in all cell types. Except for two common phosphoproteins, p44 and p82, the relative phosphoprotein distribution within the gel differed when normal prostatic cells were compared to the metaplastic cells. However, the phosphoprotein patterns were the same in secretory and non-secretory cells from the same prostate tissue. On the other hand, the relative labeling intensity of phosphoproteins was always higher in non-secretory cells compared to corresponding secretory cells. Accordingly, tyrosine protein kinase (TPK) activity, expressed on a cell basis, was also higher in non-secretory cells; the ratios of TPK activities, non-secretory over corresponding secretory cells, was always higher than unity for all preparations but overlapped when cells from metaplastic glands were compared to those isolated from normal prostates. Thus, non-secretory epithelial cells isolated from either normal or metaplastic glands have a higher ability than corresponding secretory cells to phosphorylate endogenous alkali-resistant phosphoproteins and their TPK activity, measured with the synthetic substrate poly(Glu80-NaTyr20), is also higher.

Alkalies↗
PubMed1989

Effect of vanadate on protein phosphorylation and on acid phosphatase activity in the canine prostate.

To evaluate the possible role of intracellular phosphatases in the local regulation of prostatic functions, the effect of sodium orthovanadate (VO4), an inhibitor of phosphotyrosyl protein phosphatases, was studied on both protein phosphorylation and acid phosphatase activity. Secretory and non-secretory epithelial cells were isolated from normal and metaplastic prostates and incubated with [32P]phosphate in the presence and in the absence of VO4; the phosphoproteins were separated by electrophoresis and the gels were either directly submitted to autoradiography or after an alkali treatment to reveal those proteins enriched in phosphotyrosine. Prior to alkali treatment, several phosphoproteins were evidenced and in less than half of the cell preparations a slight increase in labeling intensity under vanadate (less than 75%) was observed in two phosphoproteins, p57 and p44. After alkali treatment: (1) the effect of VO4 on p57 remained in the order of 44-45% and it was restricted to less than half of non-secretory cell preparations; (2) its effect on p44 was intensified (134-207%) and observed in all cell types and in more than 80% of all preparations; and (3) in half of non-secretory cell preparations from metaplastic glands, an effect of VO4 on p35 (127%) became evident. In all instances, with normal and/or metaplastic prostates, protein phosphorylation activity, either total or alkali-resistant and in the presence or in the absence of VO4, was always higher in non-secretory epithelial cells as compared to secretory cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1989

Polyclonal rabbit gamma globulins against a human cytotoxic CD4+ T cell clone. I. Clone characteristics and antiblast globulin preparation.

Antihuman lymphocyte rabbit (or horse) gamma globulins used in recipients of organ transplantation are prepared against thymocytes or immortalized cell lines, the only two sources so far allowing enough antigen preparation. These cells lack, however, the surface determinants characteristic of alloreactive blasts involved in the rejection process. We have derived long-term cultures of a panel of alloreactive (untransformed) clones from a rejected kidney. Among them, clone 1E7 has been chosen as a cytotoxic CD4+ (CD2+ CD3+ TCR alpha beta+) clone proliferating against HLA-DR8 targets. This clone (clonality assessed on T cell receptor genomic rearrangements) has been grown using weekly stimulations with the kidney donor-derived EBV cell line and recombinant IL-2. Clone cultures have been adapted to mass production after optimization of culture conditions satisfying pharmaceutical requirements. This procedure warranted a reproducible source of antigen since the functional and phenotypic characteristics of the immunizing 1E7 cells remained identical through the life span of the culture. In addition, the study of the total growth capacity of 1E7 cells showed consistent expansion until the 40th cell cycle, ensuring a progeny that will satisfy the large-scale requirement for a clinical trial. Rabbits were injected with 100 x 10(6) 1E7 cells (21, 14, and 7 days before bleeding). Sera were depleted of agglutinin by red blood cell absorption and globulin antiblast (GAB) prepared by SO4Na precipitation and ion exchange chromatography; 50% complement-mediated target cell lysis and 50% inhibition of E rosette formation and alloproliferation were obtained at GAB dilutions of about 1:250-1:500. Prescreened on cynomolgus monkeys, GAB could significantly prolong skin grafts when given prophylactically. Finally GAB have been used in human recipients of kidney grafts for prophylaxis of early rejection. Results of this pilot study are given in a separate report in this issue. In conclusion we have, for the first time, set up a large-scale preparation of polyclonal globulin against a normal human alloreactive clone, and this new reagent should present several advantages over classic antilymphocyte or antithymocyte sera because it contains specificities against activation antigens and has less crossreactivity variation among batches.

Animals↗
PubMed1989

Clonal expansion of lymphocytes bearing the gamma delta T-cell receptor in a patient with large granular lymphocyte disorder.

Repeated analysis of peripheral blood lymphocytes (PBLs) from a patient with large granular lymphocytosis, neutropenia, and rheumatoid arthritis revealed that approximately 45% of PBLs displayed the following phenotype: CD3+, CD4-, CD8-, CD16+, HNK-1-, WT31-. This population was purified for further analysis by depletion with anti-CD4 and anti-CD8 monoclonal antibodies (MoAbs). Southern blot analysis showed preferential rearrangements of the V gamma 9 genes. Northern blot demonstrated the presence of V gamma 9 mRNA transcripts. With MoAbs directed against either the V gamma 9 peptide (Ti gamma A) or the delta chain of the gamma delta T-cell receptor (TCR delta 1), we further demonstrated that those cell surfaces expressed both V gamma 9 and a delta gene product. In addition, analysis of the gamma gene rearrangements on six clones derived from this population demonstrated a unique rearrangement on a single chromosome, strongly suggesting the monoclonality of this T-cell population. Significant cytotoxic activity against K562, U937 was observed only after an in vitro culture period with interleukin-2 (IL-2), whereas no specific inhibitory effect on autologous bone marrow (BM) CFU-G was noted.

Agranulocytosis↗