Biomedical subjects
Publications and source records attributed to D Portetelle.
This communication reports the sequence of the beta-subunit of the insulin-like growth factor-1 receptor. It shares 91.1% homology at the nucleotide level and 97.7% at the amino acid level with the equivalent human receptor subunit.
This communication reports the complete amino acid sequence (263 amino acids) for the insulin-like growth factor binding protein 1 in the bovine (bIGFBP-1). It is 70% homologous with the equivalent IGFBP-1 in human and rat.
The bovine cyclic AMP responsive binding protein cDNA (CREB2) was isolated from a lambda-gt11 cDNA expression library using a 32P labelled oligonucleotide corresponding to the 21 bp enhancer sequence present in the BLV LTR. The deduced amino acid sequence revealed that CREB2 contains a leucine zipper structure (residue 295 to 316), a basic amino acid domain (residue 268 to 291) and several potential phosphorylation sites.
Human T-lymphotropic viruses (HTLV-I and -II) and bovine leukaemia virus (BLV) express transactivator proteins able to increase long terminal repeat (LTR) directed viral expression. These transacting factors are though to be involved in the induction of leukaemia by these viruses. Transfection of BLV transactivator p34tax together with Ha-ras immortalizes and transforms rat embryo fibroblasts, in vitro. The transformed cell induce tumours in nude mice. These data emphasize the causal role exerted by p34tax in in vivo tumorigenesis.
Study of the antigenic structure of the Bovine Leukemia Virus (BLV) envelope glycoprotein gp51 with a panel of mouse monoclonal antibodies (MAbs) has allowed the identification of biologically important determinants directly involved in the infectivity of BLV. Considering the various facts reported in this paper, it follows that diagnostic and vaccination procedures that make use of gp51 in a native configuration constitute a prerequisite for the design of an efficient BLV eradication program. To improve the efficacy of a serological detection test, MAbs have been selected as reagents of choice to develop a competition enzyme-linked immunosorbent assay (cELISA). Recombinant vaccinia virus expressing gp51 and gp30 was indicated as a very promising protective vaccine against BLV infection.
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To study endocrinal control of growth development in cattle during puberty, major hepatic vessels were cannulated in 8-13 week old Holstein male calves. In our experimental conditions, chronic hepatic cannulation with "grey" polyurethane catheters was performed for a long period (more than 65 d) without any rejection problems. A physiological washing solution containing at least 5 x 10(5) IU/l heparin-lithium was required to prevent cannula occlusion. Moreover, echography was utilized for rapid and accurate introduction of catheters into the vessels. With chronic hepatic cannulation, we have observed that plasma testosterone and insulin-like growth factor I (IGF-I) levels in cattle were lower in portal and in portal and cava veins respectively than in the other vessels. Our experiment has also shown that average plasma testosterone and IGF-I concentrations sharply increased at puberty to reach adult values. However, no relation was observed between these two hormones during the pubertal period. In conclusion, both testosterone and IGF-I plasma levels can be used as predictive factor of age at puberty but more investigations are needed to establish standard values for each breed.
Bovine leukemia virus (BLV) p24 gene was expressed in Saccharomyces cerevisiae under the control of the PHO5 (encoding repressible acid phosphatase, rAPase) promoter. Yeast cells were transformed by a yeast-E. coli shuttle vector carrying the PHO5 promoter, the p24 gene and the CYC1 transcription terminator. After low inorganic phosphate (Pi) induction of the PHO5 promoter, p24 accumulated in the producing cells up to a concentration representing 10% of total soluble proteins. The expression level of p24 gene was not increased by insertion of the positive regulatory gene PHO4 on the p24 expression vector. The p24 produced in this system and incubated in crude yeast extract showed a remarkably high resistance to proteolytic degradation, a feature that presumably correlates with the compact globular conformation of the protein combined to the stabilizing effect of the N-terminal residue.
The nucleotide sequence coding for bovine leukemia virus (BLV) envelope glycoprotein gp51 was inserted into a yeast-Escherichia coli shuttle vector carrying the promoter and secretion signal sequence of PHO5 (the yeast gene coding for repressible acid phosphatase) and the CYC1 transcriptional terminator. Yeast cells transformed by this construction synthesized gp51 after PHO5 induction by inorganic phosphate deprivation. The yeast-expressed gp51 was partially glycosylated into heterodisperse protein molecules ranging from 40 to 48 kDa. No gp51 was excreted in the culture medium. The amount of protein accumulated in yeast cells was estimated to reach 0.06% of soluble proteins. This modest level of expression seemed to be due to the toxicity of gp51 to the yeast cell. The yeast-expressed gp51 products were used in enzyme-linked immunosorbent assays for the detection of antibodies in sera from BLV-infected animals; they were also screened for the presence of well-defined biological epitopes. In both studies poor reactivity was observed. Rabbits immunized with the recombinant gp51 showed high antibody titers to native BLV gp51. However, these antibodies did not neutralize BLV in vitro.
In the course of generating monoclonal antibodies to bovine thymus-dependent differentiation antigens, we were able to characterize an antibody, termed 8C11, that detects an antigen shared by a majority of thymocytes and peripheral T cells (in blood and thymus-dependent area of spleen and lymph-nodes), but undetectable on normal B cells. However, this antibody was reactive with B cells from cows infected with bovine leukemia virus (BLV). These BLV-infected B cells were found to express simultaneously high concentrations of both surface IgM and 8C11-detected antigen. The antigen recognized by this antibody was shown to be a 67.5 kDa molecule. Because similar findings have been made on mouse myelomas and on human chronic leukemia cells, where this antigen was considered to be the equivalent of mouse Ly-1 antigen and human Leu-1 or CD5 antigen, the T cell antigen detected on BLV-infected cells could be the bovine counterpart of the CD5 antigen. By another way, it has been found that the CD5 T cell antigen is also present on a minor subpopulation of B cells in the spleen but not in the blood. We suggest that in the bovine a similar B cell subpopulation should be the BLV target and expand as a consequence of viral insertion, leading to the persistent lymphocytosis observed on BLV-infected animals.
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Previous studies with monoclonal antibodies of the antigenic structure of bovine leukemia virus (BLV) envelope glycoprotein (gp51) have identified three epitopes (F, G, H) directly involved in the infectivity of BLV, F, G, and H lost their reactivity with the respective monoclonal antibodies after treatment with a reducing agent, indicating that these epitopes were conformational. Sequence comparisons between BLV mutants and differential reactivities of urokinase or proteinase K gp51 fragments with monoclonal antibodies indicated that the NH2 moiety of the env protein harbored the three architectural determinants F, G, and H. ELISA tests demonstrated that anti-F, -G, and -H monoclonal antibodies were maximally reactive toward intact virions whereas they showed much poorer affinities for their respective epitopes when presented on a purified protein. Accordingly, an efficient vaccine against BLV infection will include at least the identified gp51 region presented in its native architectural configuration.
Peptides corresponding to residues 21-28, 39-48, 57-67, 59-69, 78-92, 144-155, 144-157, 195-205, 255-268, and 260-268 of envelope glycoprotein gp51 of bovine leukemia virus (BLV) were chemically synthesized and coupled to keyhole limpet hemocyanin or tetanus toxoid. All peptides were immunogenic in rabbits and induced production of antipeptide antibodies. Enzyme-linked immunosorbent assays using Tween 80-purified gp51 or BLV particles showed that antibodies against peptides 78-92, 255-268, and to a lesser extent 39-48 and 144-157 were able to react with the parent glycoprotein, purified or as an integer part of BLV particles. Antisera against peptides 39-48, 78-92, and 144-157 neutralized VSV (BLV) pseudotypes in vitro, the highest neutralization titer being obtained with the 78-92 cyclized peptide. These observations confirm that the NH2 moiety of gp51 carries epitopes involved in virus infectivity.
The bovine leukemia virus (BLV) p34tax (also called tat, p34, XLOR gene product) is a 34-kDa polypeptide encoded in the 3'-terminal region of the virus. This protein is responsible for positive transcriptional trans-activation of promoter elements located within the BLV long-terminal repeat. We introduced the protein-coding region of BLV p34tax into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus. After infection of the insect Spodoptera frugiperda (SF9) cell line, this recombinant strain of baculovirus produced approximately 100 to 150 mg of p34tax per 2 X 10(9) cells. This protein, when introduced into mammalian fibroblasts by using a cell-to-cell fusion technique, functionally trans-activated the BLV long-terminal repeat. Analysis of 32P-labeled proteins of SF9 cells expressing BLV tax by two-dimensional gel electrophoresis indicated that the BLV p34tax was phosphorylated.
Mutants of the bovine leukemia virus (BLV) transactivator protein (tat, tax, p34, the XLOR gene product) were constructed by site-directed deletions, in-phase linker insertions, or fragment replacements (swapping) among BLV variants. The mutant constructs were transfected into cos cells and transiently expressed. Western blot analysis using a mixture of monoclonal antibodies to wild-type p34 revealed the presence of mutated XLOR gene products in all the mutants tested. The transactivating activity of 11 tax mutants containing site-directed deletions and in-phase linker insertions was completely abolished. Only the swapping mutant tested, a hybrid between two BLV variants, transactivated LTR-directed gene expression at wild-type levels. These data illustrate the narrow range of structural variations that allow full activity of the BLV tax product and suggest that the present molecular structure of the transactivator protein results from heavy evolutionary constraints.
Expression of bovine leukemia virus (BLV) antigens in vivo has not been shown. After BLV infection, however, production of antibodies directed towards BLV proteins (e.g. gp51) can be easily demonstrated. Thus, production of BLV proteins has to take place somewhere in infected cattle. Tissues and organs of experimentally infected cattle were fixed in acetone and embedded in paraffin. Monoclonal antibodies directed to gp51 were used to demonstrate BLV expression immunohistologically by the peroxidase-antiperoxidase (PAP) method. The same samples were also used to demonstrate a tumor associated antigen (TAA) employing a monoclonal antibody. Our results indicate that very few cells, found in the intestinal mucosa, produce gp51 in vivo. The expression of TAA, however, increases significantly shortly after infection with BLV and remains high throughout life.
A competition ELISA technique involving two monoclonal anti-gp51 antibodies has been developed for the detection of bovine leukaemia virus (BLV) antibodies. Precoated gp51 antigen-microtitre plates were obtained by incubation of plastic adsorbed monoclonal antibody with a non-purified BLV preparation. Samples to be tested were incubated in the wells of the gp51-coated plates; the presence of anti-gp51 antibodies was indicated by competition for antigen binding with an enzyme linked monoclonal antibody directed to an important epitope on gp51. This test is as sensitive as a routinely used indirect ELISA test; it is highly specific, reliable and easy to perform.
The glycoprotein gp51 of bovine leukaemia virus (BLV) has been included in an immunostimulating complex (ISCOM). The ISCOM was characterized biochemically in SDS-polyacrylamide gel electrophoresis showing the presence of proteins of estimated molecular weights of 50 and 30 kDa. Immunoblotting showed that gp51 was present in the ISCOM. The BLV-ISCOM had a S-value of 19 S and the electronmicrograph showed the cage-like structure as previously reported for other ISCOMs. About 17% of the total amount of gp51 in the cell culture fluid was recovered in the ISCOMs. The largest loss of gp51 was encountered during the sedimentation of the virus. An ELISA, utilizing monoclonal antibodies to defined epitopes for capture was developed to control the antigenicity of epitopes, e.g. those known to induce neutralizing antibodies. Using this device as a quality control for epitopes the following could be stated. First, ISCOMs prepared from virus solubilized with the non-ionic detergents Triton X-100 or MEGA did not react with neutralizing monoclonal antibodies. In contrast, ISCOMs prepared from virus solubilized with the non-ionic detergents Tween-20, Tween-80 or octyl glucoside did react with the neutralizing antibodies. Second, the neutralizing epitopes were better exposed in ISCOMs than the other epitopes of gp51. In a preliminary experiment it was shown that gp51 in ISCOMs was highly immunogenic.