Jacques Marti

Differential expression of the RTP/Drg1/Ndr1 gene product in proliferating and growth arrested cells.

Using a differential display method to identify differentiation-related genes in human myelomonocytic U937 cells, we cloned the cDNA of a gene identical to Drg1 and homologous to other recently discovered genes, respectively human RTP and Cap43 and mouse Ndr1 and TDD5 genes. Their open reading frames encode proteins highly conserved between mouse and man but which do not share homology with other know proteins. Conditions in which mRNAs are up-regulated suggest a role for the protein in cell growth arrest and terminal differentiation. We raised antibodies against a synthetic peptide reproducing a characteristic sequence of the putative polypeptide chain. These antibodies revealed a protein with the expected 43 kDa molecular mass, up-regulated by phorbol ester, retinoids and 1,25-(OH)2 vitamin D3 in U937 cells. It was increased in mammary carcinoma MCF-7 cells treated by retinoids and by the anti-estrogen ICI 182,780 but not by 4-hydroxytamoxifen. The mouse Drg1 homologous protein was up-regulated by retinoic acid in C2 myogenic cells. The diversity of situations in which expression of RTP/Drg1/Ndr1 has now been observed shows that it is widely distributed and up-regulated by various agents. Here we show that ligands of nuclear transcription factors involved in cell differentiation are among the inducers of this novel protein.

Synergistic effect of retinoic acid and 1,25-dihydroxyvitamin D3 on the differentiation of the human monocytic cell line U937

The human-derived leukemia cell lines HL-60 and U937 are known to differentiate into more mature phagocytic cells in the presence of retinoic acid or 1,25-dihydroxyvitamin D3. We studied the effects of combinations of these two agents on cell growth and differentiation. These treatments were found to increase inhibition of cell proliferation. A dramatic enhancement of functional properties was observed in U937, but not HL-60 cells exposed to combinations of the two inducers. We investigated the conditions required to obtain the highest synergistic effects on the differentiation of U937 cells. These effects were found to be highly dose-dependent. We found that synergism required the simultaneous presence of both inducers and did not occur upon sequential exposure to each agent used separately.

Preparation, constantes physiques et constitution chimique de la fetuine d'agneau

Abstract Calf fetuin is a well-known glycoprotein found only in the fetus. A similar protein exists in fetal lamb serum but it had been demonstrated only by immunological methods. In the present investigation, we attempted the isolation and characterization of this protein. 1. 1. Fetal lamb serum was analysed by several electrophoretical methods. Its relative complexity does not allow the unambiguous identification of lamb fetuin. 2. 2. Using the alcohol-metal ion fractionation procedure of Spiro, it was possible to isolate a glycoprotein very similar to calf fetuin but in lower yields. Some contaminant proteins were observed. 3. 3. An alternative method is proposed (chromatography on CM-cellulose, pH 4.6, I = 0.01, followed by gel filtration on Sephadex G-200) leading to higher yields of a purer product. 4. 4. Some physicochemical parameters have been measured. E2781% = 4.05, s20,w0 = 3.50 ± 0.10; pHi = 3.85, 4.05, 4.10, 4.40 for the main variants. The apparent molecular weight measured by gel filtration is very much the same as for calf fetuin. 5. 5. The amino acid analyses revealed the presence of 364 residues per mole, representing approximately 79.8% of the weight of the protein. No methionine could be detected, nor any free -SH group, suggesting the presence of 6 disulfide bonds. 6. 6. The N- and C-terminal residues were both isoleucine. 7. 7. The carbohydrate content (approximately 22.75%) was determined by classical methods. Sialic acid (8.50%), is essentially N-acetylneuraminic acid. Hexosamines (6.75%) are glucosamine and galactosamine in the ratio 8:1. Hexoses are galactose (4.50%) and mannose (3.00%). 8. 8. The molecular weight calculated from chemical composition is 48 500. 9. 9. Calf and lamb fetuin appear to be very similar.

Potentiation of VD-induced monocytic leukemia cell differentiation by retinoids involves both RAR and RXR signaling pathways

Retinoids and vitamin D (VD) cooperate to induce the differentiation and inhibit the proliferation of human myelomonocytic leukemia cells. Two classes of retinoids receptors, the RARs and RXRs, respectively, can mediate these effects. RXR forms heterodimers with a variety of nuclear receptors, including RAR and the VD receptor. We have previously found that VD treatment increases RXRα levels in myelomonocytic leukemia cells. By immunoanalysis, we observed in the present work that the RARα protein is expressed in proliferating U937, HL-60 and THP-1 human leukemia cells and that VD treatment induces alterations of its electrophoretic pattern, although with large differences between cell lines. In the three cell lines, 9-cis RA, an agonist of both RARs and RXRs, cooperated with VD more efficiently than all-trans RA and RAR-specific synthetic ligands, thus suggesting an involvement of both RAR and RXR pathways in cell differentiation. Using U937 cells as a model, we delineated the relative contributions of RAR and RXR by assessing the effects of receptor-selective synthetic retinoids. The synergy between VD and all-trans RA or RAR-specific agonists (TTNPB and Ro 40-6055) was abrogated by a RARα-specific antagonist (Ro 41-5253), confirming an involvement of RARα. However, the cooperation between VD and 9-cis RA, although reduced, was not suppressed by the antagonist, suggesting also an involvement of the RXR pathway. The role of RXR as a ligand-activated receptor was confirmed using RXR-specific agonists (CD2608 and LGD1069), which also proved able to cooperate with VD. Finally, while each synthetic agonist alone was significantly less potent than 9-cis RA, combinations of the RAR and RXR selective agonists TTNPB and LGD1069 appeared to be as effective as the pan agonist 9-cis-RA. These results confirm that various retinoids can cooperate with VD and demonstrate that, at a whole cell level, optimal effects require the activation of both RAR and RXR receptors.

Serial analysis of gene expression (SAGE) in bovine trypanotolerance: preliminary results

In Africa, trypanosomosis is a tsetse-transmitted disease which represents the most important constraint to livestock production. Several indigenous West African taurine (Bos taurus) breeds, such as the Longhorn (NDama) cattle are well known to control trypanosome infections. This genetic ability named trypanotolerance results from various biological mechanisms under multigenic control. The methodologies used so far have not succeeded in identifying the complete pool of genes involved in trypanotolerance. New post genomic biotechnologies such as transcriptome analyses are efficient in characterising the pool of genes involved in the expression of specific biological functions. We used the serial analysis of gene expression (SAGE) technique to construct, from Peripheral Blood Mononuclear Cells of an NDama cow, 2 total mRNA transcript libraries, at day 0 of a Trypanosoma congolense experimental infection and at day 10 post-infection, corresponding to the peak of parasitaemia. Bioinformatic comparisons in the bovine genomic databases allowed the identification of 187 up- and down- regulated genes, EST and unknown functional genes. Identification of the genes involved in trypanotolerance will allow to set up specific microarray sets for further metabolic and pharmacological studies and to design field marker-assisted selection by introgression programmes.

Thermal stress as an inducer of differentiation of U937 cells

The individual and combined effects of heat shock, all-trans retinoic acid and 1,25-dihydroxyvitamin D3 on inhibition of cell growth and initiation of differentiation were investigated on U937 human leukemia cells. Incubation of U937 cells at 43 degrees C for 1 h did not affect cell viability but induced a reduction of cell growth and the emergence of a differentiated phenotype, characterized by the acquisition of chemiluminescent responses to various oxidative burst inducers and by the capacity to produce IL-6 in response to bacterial lipopolysaccharide. Heat shock alone, therefore, appears to be an efficient inducer of cell differentiation. In addition, heat shock primed the cells to respond more efficiently to the action of retinoic acid and vitamin D, and amplified the phenotypic changes initiated by pretreatment of U937 cells with these agents.

NH2-terminal sequence of Calf Fetuin

Abstract Calf fetuin, one of the 3 major known fetal proteins has been isolated by a two-step purification procedure and characterized by aminoacid composition. The purified glycoprotein, which consisted of a single chain, was submitted to 47 steps of automatic aminoacid sequencing, allowing to determine 44 positions. This section of the molecule was devoid of carbohydrates. Comparison of this sequence with a variety of detectable potentially related protein did not allow to point to any detectable homology.

Bovine alpha-fetoprotein. Isolation and characterization.

Bovine AFP was purified by ion exchange chromatograph on C.M. cellulose and DEAE Sephadex A-50, gel filtration and immunosorbent technique. AFP was homogeneous when studied by gel electrophoresis under non denaturing and denaturing conditions, by ultracentrifugation and by immunological methods. The following molecular data were obtained: 1. Sedimentation equilibrium indicated a molecular weight of 66,500 and sedimentation velocity gave s degrees 20, w = 4.71 S. A partial specific volume v = 0.737 ml g-1 was derived from density measurements. 2. From these data, a Stokes radius of 3.26 nm, a diffusion coefficient D20 w = 6.61 10(-7) cm2 sec-1 and a frictional ratio f/fo = 1.21 were calculated. 2. Sodium dodecylsulphate disc electrophoresis suggests a molecular weight of 67,000. 3. Gel filtration pointed to a molecular weight of 75,000. 4. Microheterogeneity of AFP was demonstrated by isoelectric focusing. The isoelectric point of the major component is 4.6. 5. The chemical composition was determined. AFP is a glycoprotein containing 7 per cent carbohydrate including 1.67 per cent hexoses, 2.38 per cent N-acetyl glucosamine and 1.8 per cent N-acetyl neuraminic acid.

Use of the Serial Analysis of Gene Expression (SAGE) Method in Veterinary Research: A Concrete Application in the Study of the Bovine Trypanotolerance Genetic Control

Abstract: New postgenomic biotechnologies, such as transcriptome analyses, are now able to characterize the full complement of genes involved in the expression of specific biological functions. One of these is the Serial Analysis of Gene Expression (SAGE) technique, which consists of the construction of transcripts libraries for a quantitative analysis of the entire gene(s) expressed or inactivated at a particular step of cellular activation. Bioinformatic comparisons in the bovine genomic databases allow the identification of several up‐ and downregulated genes, expressed sequence tags, and unknown functional genes directly involved in the genetic control of the studied biological mechanism. We present and discuss the preliminary results in comparing the expressed genes in two total mRNA transcripts libraries obtained during an experimental Trypanosoma congolense infection in one trypanotolerant NDama animal cow. Knowing all the functional genes involved in the trypanotolerance control will permit validation of some results obtained with the quantitative trait locus approach, to set up specific microarrays sets for further metabolic and pharmacological studies, and to design field marker‐assisted selection by introgression programs.

Purification and structure of sheep haptoglobin

Haptoglobin (Hp) is a glycoprotein which is found in the serum of several animals. Its characteristic property is the ability to form with hemoglobin (Hb) a stable equimolecular complex Hp-Hb which Possess a peroxidase activity; this property allows a quantitative estimation of Hp [ 1 ] . The normal level of Hp varies according to the species: from 0.05 g/l in Bovidae and Cervidae, to 1.2 g/l in human. It always rises in any inflammatory state [2]. In the sheep, Hp is composed of a series of polymeric proteins, whose electrophoretic pattern is very similar to that of human Hp 2-2 [3]. Their mol. wts. range from lo5 to 10” [3]. They are formed in the human by combination in different numbers of two polypeptidic chains called (Y and 0 [4]. We have undertaken the purification of sheep Hp in order to study its structure. We have controlled the purification by using the Hp estimation after each stage. But, as we shall establish later, sheep Hp is easily altered: below pH 4 or above pH 6.5, it looses its ability to combine with Hb. For these reasons, the preparation of this protein in the native state is very difficult. Therefore, after two successive chromatographic purifications, we have used an immunological technique as a last step of purification. The method is based on the following fact: since the Hp level in normal sheep is very low, a rabbit immune-serum anti-normal-sheep serum contains a very small quantity of y-globulins anti-haptoglobin. After insolubilisation of these rabbit antibodies, affinity chromatography retains selectively proteins