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Biomedical subjects

A Burny

Publications and source records attributed to A Burny.

At least 163 records · Page 9Linked to original sources
PubMed1991

The amino acid (157-197) peptide segment of bovine leukemia virus p34tax encompass a leucine-rich globally neutral activation domain.

The specific DNA-binding Gal4 amino-terminal portion (amino acids 1 to 147) was fused to protein segments of BLV transactivator p34tax and tested for its capacity to activate CAT-gene expression in mammalian cells. The p34tax peptide segment 157 to 197 encompasses an activating region. The segment is approximately located in the middle of p34tax and is globally neutral (net charge zero). The tax (157-197) domain contains 24% of leucine residues, possibly involved in heterologous protein interactions.

Amino Acid Sequence↗
PubMed1990

Sequence analysis of the pig phosphoglucose isomerase gene promoter region.

A cDNA for pig phosphoglucose isomerase (PGI) was used to isolate genomic clones representing the 5' portion of the corresponding gene. A total of 656 bases of the pig PGI gene were obtained that include 5'-flanking information and part of exon 1. A major transcription start was localized at 74 nucleotides from the translation start. The sequence organization of the pig PGI promoter is similar to that of other housekeeping genes. This GC-rich region includes a TATA-like box, two putative Sp1 recognition sites but no CCAAT box.

Amino Acid Sequence↗
PubMed1990

Orientation into the lipid bilayer of an asymmetric amphipathic helical peptide located at the N-terminus of viral fusion proteins.

The complete amino-acid sequence of viral fusion proteins has been analyzed by the Eisenberg procedure. The region surrounding the cleavage site contains a highly hydrophilic region immediately followed by a membrane-like region. Since the effective cleavage between these two domains seems required to expose the fusogenic domain (located at the N-terminal sequence of the transmembrane like region) which is assumed to interact with the lipid membrane of the host cell, we have focused our analysis on the conformation and mode of insertion of this membrane-like domain in a lipid monolayer. It was inserted as an alpha-helical structure into a dipalmitoylphosphatidylcholine (DPPC) monolayer and its orientation at the lipid/water interface was determined using a theoretical analysis procedure allowing the assembly of membrane components. For each viral protein sequence these N-terminal helical segments oriented obliquely with respect to the lipid/water interface. This rather unusual orientation is envisaged as a prerequisite to membrane destabilization and fusogenic activity.

Amino Acid Sequence↗
PubMed1990

Production of human monoclonal IgG antibodies reacting with cytomegalovirus (CMV).

A human monoclonal antibody (MoAb) reacting with cytomegalovirus (CMV) has been produced using somatic cell hybridization between Epstein-Barr virus (EBV) infected B lymphocytes and a human-mouse heteromyeloma cell line (SHM-D33). The hybrids were selected in HAT medium containing 5 x 10(-7) ouabain. The median level of Ig production was 5 (0.1-20) micrograms/10(6) cells/day. One selected hybridoma (IB-8E9H5) has been maintained in continuous culture for more than 30 months with a stable IgG2, lambda production. Molecular hybridization using EBV-specific probes demonstrate that our hybrids have lost the IR-1 EBV sequence during fusion. Unexpectedly, these blotting experiments revealed the presence of multiple EBNA-1 sequences dispersed among the genomic DNA of the SHM-D33 cell line. Screening for anti-CMV specificity was performed by ELISA and confirmed by immunofluorescence staining. Thus far, three CMV reference strains and 14 local strains are stained by the MoAb as early as 3 h after CMV infection of human fibroblasts, apparently through the recognition of a nuclear viral antigen of 67 kDa. In conclusion, this technique permits (a) the removal of the EBV genome contained in the lymphoblastoid parental cell line and (b) the production of human anti-CMV MoAb with potential applications in the prevention of life threatening CMV infections.

Antibodies, Monoclonal↗
PubMed1990

Cooperation between bovine leukaemia virus transactivator protein and Ha-ras oncogene product in cellular transformation.

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.

Animals↗
PubMed1990

Identification and characterization of an enhancer in the coding region of the genome of human immunodeficiency virus type 1.

Transcription of human immunodeficiency virus type 1 (HIV-1) is regulated by cis-acting DNA elements located in the viral long terminal repeats, by viral transregulatory proteins, and by cellular transcription factors acting in concert to modulate the degree of viral expression. We demonstrate that a DNA fragment corresponding to the central portion of the HIV-1 genome exhibits enhancer activity when cloned upstream of the thymidine kinase promoter of herpes simplex virus. This enhancer is inducible by phorbol 12-myristate 13-acetate in HeLa cells and is independent of its position and orientation with respect to the promoter. We have mapped the activity of the enhancer to two independent domains encompassing nucleotides 4079-4342 (end of the pol gene) and nucleotides 4781-6026 (vif gene and first coding exon of tat). This intragenic enhancer and its subdomains demonstrate cellular specificity because they are only active in specific cell lines. The presence of similar intragenic enhancer elements in other retroviruses suggests that they might be a conserved feature of this family of viruses.

Base Sequence↗
PubMed1990

Sequence comparison between the fusion protein of human and bovine respiratory syncytial viruses.

The nucleotide sequence was determined for the fusion (F) protein-coding mRNA of the bovine respiratory syncytial virus (strain RB 94) and the amino acid sequence of the F protein was deduced for comparison with the sequence of human respiratory syncytial virus subtypes A and B (RSS-2 and 18537 strains). The human and bovine RS virus F proteins (excluding the cleaved signal peptide) share 83 to 84% homology. The greatest divergence occurred within the F2 subunit in the region preceding the cleavage activation site.

Amino Acid Sequence↗
PubMed1990

B cells from bovine leukemia virus- (BLV) infected sheep with hematological disorders express the CD5 T cell marker.

Though peripheral blood B cells from healthy sheep were known to be devoid of the CD5 T cell marker, it appears from our study that most B cells from bovine leukemia virus- (BLV) infected sheep with hematological disorders express both the CD5 marker and surface IgM. The possible meaning of this T cell marker expression on B cells from BLV-infected sheep is briefly discussed.

Animals↗
PubMed1990

Development of a specific serological test and an efficient subunit vaccine to control bovine leukemia virus infection.

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.

Amino Acid Sequence↗
PubMed1990

[Evolution of plasma concentrations of testosterone and insulin-like growth factor I during the peripubertal period of cattle catheterized at the hepatic level].

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.

Animals↗
PubMed1989

High yield synthesis of the bovine leukemia virus (BLV) p24 major internal protein in Saccharomyces cerevisiae.

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.

Acid Phosphatase↗
PubMed1989

Biochemical and immunological characterization of the bovine leukemia virus (BLV) envelope glycoprotein (gp51) produced in Saccharomyces cerevisiae.

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.

Acid Phosphatase↗
PubMed1989

Bovine leukemia virus (BLV)-infected B-cells express a marker similar to the CD5 T cell marker.

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.

Animals↗
PubMed1989

The GAG precursor of simian immunodeficiency virus assembles into virus-like particles.

To examine the potential role of the GAG precursor polyprotein in morphogenesis and assembly of the simian immunodeficiency virus (SIV), we have expressed the gag gene of SIVMac using a baculovirus expression vector. Infection of insect cells with recombinant virus containing the entire gag gene results in high expression of the GAG precursor protein, Pr57gag. The recombinant protein is myristylated and is released in the culture supernatant in an insoluble particulate form. A point mutation in the N-terminal glycine codon (Gly----Ala) inhibits myristylation. This mutated product is highly expressed but is not found in the culture supernatant. Electron microscopy and immunogold labelling of infected cells show that the native Pr57gag protein assembles into 100-120 nm virus-like particles that bud from the cell plasma membrane and are released in the culture supernatant. The unmyristylated protein also assembles into particulate structures which only accumulate inside the cells. These results demonstrate that the unprocessed GAG precursor of SIV can spontaneously assemble into particles in the absence of other viral proteins. Myristylation of the Pr57gag precursor is necessary for its association with the cell plasma membrane, for budding and for extracellular release.

Animals↗
PubMed1989

Antigenic variants of bovine leukemia virus (BLV) are defined by amino acid substitutions in the NH2 part of the envelope glycoprotein gp51.

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.

Amino Acid Sequence↗