Biomedical subjects

G Antoine

Publications and source records attributed to G Antoine.

At least 19 recordsLinked to original sources

PubMed2005

Cloning, expression and functional characterization of the full-length murine ADAMTS13.

Functional deficiency or absence of the human von Willebrand factor (VWF)-cleaving protease (VWF-cp), recently termed ADAMTS13, has been shown to cause acquired and congenital thrombotic thrombocytopenic purpura (TTP), respectively. As a first step towards developing a small animal model of TTP, we have cloned the complete (non-truncated) murine Adamts13 gene from BALB/c mice liver poly A+ mRNA. Murine ADAMTS13 is a 1426-amino-acid protein with a high homology and similar structural organization to the human ortholog. Transient expression of the murine Adamts13 cDNA in HEK 293 cells yielded a protein with a molecular weight of approximately 180 kDa which degraded recombinant murine VWF (rVWF) in a dose-dependent manner. The cleavage products of murine rVWF had the expected size of 140 and 170 kDa. Murine ADAMTS13 was inhibited by EDTA and the plasma from a TTP patient.

ADAM Proteins↗

PubMed2000

Recombinant human factor X: high yield expression and the role of furin in proteolytic maturation in vivo and in vitro.

Factor X/Xa plays a pivotal role in the coagulation cascade and exhibits a therapeutic potential for the treatment of factor X-deficient as well as FVIII and FIX inhibitor patients. This report describes the establishment of Chinese hamster ovary cell clones expressing recombinant human factor X up to 120 microg/mL x day and 78 microg/10(6) cells x day, that is to 100-fold higher levels than reported previously. Although propeptide removal and single chain precursor to light and heavy chain processing as well as vitamin K-dependent gamma-carboxylation became impaired at these expression levels, up to 25% of the recombinant human factor X produced was active. This represents the highest functional activity ever reported for a vitamin K-dependent protein at such an expression level. Expression of recombinant human factor X in Chinese hamster ovary cells lacking the endoprotease Furin revealed that propeptide removal still occurred, whereas single chain precursor to light/heavy chain processing was abolished. This suggests that a protease different from Furin mediates propeptide removal, a unique finding compared with the other vitamin K-dependent coagulation factors. In contrast, exposure of incompletely processed rFX molecules to soluble recombinant Furin in vitro mediated both of these cleavage reactions despite the absence of a typical argP4-xP3-lys/argP2-argP1 Furin cleavage site in the propeptide, indicating relaxed specificity in vitro. Concomitantly with the degree of processing, the functional activity of recombinant human factor X increased. Interestingly, Furin was shown to even perform correct N-terminal proteolytic trimming of FX molecules truncated amino-terminal to the P3 residue in vitro. Depending on the absence or presence of warfarin in the culture media, as well as on the processing state, four distinct recombinant human factor X light chain isoforms were observed and their structure characterized. One of these light chain forms correlated with the functional activity. Finally, the distribution of the individual light chain isoforms suggests that gamma-carboxylation may be a prerequisite for propeptide removal.

Animals↗

PubMed1999

Highly efficient induction of protective immunity by a vaccinia virus vector defective in late gene expression.

Vaccinia viruses defective in the essential gene coding for the enzyme uracil DNA glycosylase (UDG) do not undergo DNA replication and do not express late genes in wild-type cells. A UDG-deficient vaccinia virus vector carrying the tick-borne encephalitis (TBE) virus prM/E gene, termed vD4-prME, was constructed, and its potential as a vaccine vector was evaluated. High-level expression of the prM/E antigens could be demonstrated in infected complementing cells, and moderate levels were found under noncomplementing conditions. The vD4-prME vector was used to vaccinate mice; animals receiving single vaccination doses as low as 10(4) PFU were fully protected against challenge with high doses of virulent TBE virus. Single vaccination doses of 10(3) PFU were sufficient to induce significant neutralizing antibody titers. With the corresponding replicating virus, doses at least 10-fold higher were needed to achieve protection. The data indicate that late gene expression of the vaccine vector is not required for successful vaccination; early vaccinia virus gene expression induces a potent protective immune response. The new vaccinia virus-based defective vectors are therefore promising live vaccines for prophylaxis and cancer immunotherapy.

Animals↗

PubMed1998

The complete genomic sequence of the modified vaccinia Ankara strain: comparison with other orthopoxviruses.

The complete genomic DNA sequence of the highly attenuated vaccinia strain modified vaccinia Ankara (MVA) was determined. The genome of MVA is 178 kb in length, significantly smaller than that of the vaccinia Copenhagen genome, which is 192 kb. The 193 open reading frames (ORFs) mapped in the MVA genome probably correspond to 177 genes, 25 of which are split and/or have suffered mutations resulting in truncated proteins. The left terminal genomic region of MVA contains four large deletions and one large insertion relative to the Copenhagen strain. In addition, many ORFs in this region are fragmented, leaving only eight genes structurally intact and therefore presumably functional. The inserted DNA codes for a cluster of genes that is also found in the vaccinia WR strain and in cowpox virus and includes a highly fragmented gene homologous to the cowpox virus host range gene, providing further evidence that a cowpox-like virus was the ancestor of vaccinia. Surprisingly, the central conserved region of the genome also contains some fragmented genes, including ORF F5L, encoding a major membrane protein, and ORFs F11L and O1L, encoding proteins of 39.7 and 77.6 kDa, respectively. The right terminal genomic region carries three large deletions all classical poxviral immune evasion genes and all ankyrin-like genes located in this region are fragmented except for those encoding the interleukin-1 beta receptor and the 68-kDa ankyrin-like protein B18R. Thus, the attenuated phenotype of MVA is the result of numerous mutations, particularly affecting the host interactive proteins, including the ankyrin-like genes, but also involving some structural proteins.

Ankyrins↗

PubMed1996

Specific quantitation of genomic DNA in the femtogram range by amplification of repetitive sequences.

An assay was developed to specifically quantitate concentrations as low as 50 fg/ml genomic DNA based on the amplification of repetitive sequences. Reliable results were obtained by using internal standard molecules which were coextracted, coamplified, and coanalyzed with the nucleic acids of interest. Amplification was performed by the polymerase chain reaction in the presence of fluorescent dye-labeled primers followed by quantitation of fluorescence derived from the reaction products after separation by PAGE. Based on the known amount of added internal standard molecules and the intensities of the fluorescence of the reaction products, the primary results of the assay were obtained as copies per milliliter of sample. These were converted into mass units of DNA by applying an experimentally determined conversion factor. Chicken DNA has been used as an example for genomic DNA, and the sequences amplified were CR1 repetitive elements. This type of assay may be applied in cases where a sensitive and precise quantitation of genomic DNA is required, such as in the quality control of biological products.

Animals↗

PubMed1996

Characterization of the vaccinia MVA hemagglutinin gene locus and its evaluation as an insertion site for foreign genes.

The 'Modified Vaccinia Ankara' (MVA) strain is a potential live vaccine vector. The use of the hemagglutinin (ha) gene of the MVA strain as an insertion site for foreign genes was evaluated. To identify the molecular basis of the hemagglutinin-negative (HA-) phenotype of MVA, the ha gene and the region around this gene were sequenced. Amino acid (aa) sequence comparisons with functional hemagglutinins of other vaccinia strains predicted a functional polypeptide. The late part of the promoter region of the ha gene, however, was deleted, causing the apparent loss of the ha gene function. Nevertheless, insertion of foreign DNA into the ha gene allowed generation of functional recombinant viruses, indicating that the ha-gene region is a suitable insertion site.

Base Sequence↗

PubMed1996

Production of highly homogeneous and structurally intact recombinant von Willebrand factor multimers by furin-mediated propeptide removal in vitro.

Recombinant human von Willebrand Factor (rvWF), a multimeric glycoprotein essential to haemostasis, has been developed as a potential therapeutic agent for treatment of von Willebrand disease (vWD). Permanent Chinese-hamster ovary (CHO)-rvWF cell clones co-expressing recombinant furin (rfurin) were established in order to ensure complete rvWF propeptide removal [Fischer, Schlokat, Mitterer, Reiter, Mundt, Turecek, Schwarz and Dorner (1995) FEBS Lett. 375, 259-262]. Large quantities of material are required for in vivo tests and clinical studies. This demand is commonly met by achieving high-yield expression of the desired protein via amplification. Co-amplification of rfurin, necessary to completely process increasing amounts of rvWF precursor, could not be accomplished, presumably due to lethal effects of overexpressed rfurin for the host cells [Creemers (1994) Ph.D. Thesis, University of Leuven]. Recent reports have inferred that rfurin can only mediate rvWF processing intracellularly [Rehemtulla and Kaufman (1992) Blood 79, 2349-2355; Rehemtulla, Dorner and Kaufman (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8235-8239]. We report here that rvWF-precursor processing, however, occurs predominantly extracellularly upon rfurin co-expression. Mixing experiments employing rfurin- as well as rvWF-precursor-containing conditioned media demonstrate that rvWF precursors are accessible and cleavable by rfurin in vitro. Exposure to rfurin in vitro converts the heterogeneous multimer pattern typical of incompletely processed rvWF multimers into highly homogeneous and structurally intact multimers superior to the ones exhibited by plasma-derived vWF. These findings thus demonstrate the feasibility of large-scale production of a completely processed, intact and homogeneous rvWF preparation, based on individual rvWF-precursor high-yield expression and subsequent propeptide removal by rfurin in vitro.

Animals↗

PubMed1992

The complete DNA sequence of yeast chromosome III.

The entire DNA sequence of chromosome III of the yeast Saccharomyces cerevisiae has been determined. This is the first complete sequence analysis of an entire chromosome from any organism. The 315-kilobase sequence reveals 182 open reading frames for proteins longer than 100 amino acids, of which 37 correspond to known genes and 29 more show some similarity to sequences in databases. Of 55 new open reading frames analysed by gene disruption, three are essential genes; of 42 non-essential genes that were tested, 14 show some discernible effect on phenotype and the remaining 28 have no overt function.

Base Sequence↗

PubMed1990

A case of facial diplegia associated with acute bilateral otitis media.

A 32-month-old toddler developed facial diplegia in association with a bout of acute bilateral otitis media. After 28 days of antibiotic therapy and placement of tympanostomy tubes, the patient demonstrated 100% symmetrical recovery. A brief discussion of the pathophysiology is included.

Acute Disease↗

PubMed1984

A size analysis of the adenovirus replicon.

The linear double-stranded genome of adenovirus DNA replicates semiconservatively from two origins of replication at either of the two molecular ends. Using an in vitro replication system which is able to initiate de novo DNA synthesis we have mapped the origin of DNA replication within the terminal 19 bp of the viral genome. Our conclusions are based on the use of different natural DNA templates, i.e., adenovirus type 2 and mouse adenovirus Fl DNA. In addition, we have employed linearized plasmid DNA templates which contain cloned terminal restriction enzyme fragments as well as chemically synthesized adenovirus termini of different length.

Adenoviridae↗

PubMed1982

Defective expression of mouse adenovirus Fl in human cells.

HeLa cells infected with mouse adenovirus strain Fl ( AdFl ) produce at least 2000 times less virus than permissive mouse 3T3 cells. Viral DNA synthesis, however, proceeds unimpaired. The defect in virion production was linked to a dramatic reduction in the synthesis of AdFl structural proteins, in particular the hexon. The identity of the AdFl hexon gene product was recognized through its immunogenic reactivity towards an antiserum raised against the human adenovirus type 2 (Ad2) hexon gene product. This cross-reactivity is reflected in an extensive DNA sequence homology between AdFl and Ad2 DNA at position 53-60, the locus of the hexon gene, on the Ad2 physical map. Through hybridization at different formamide concentrations, the present study identifies one additional, highly conserved region at map positions 14-15 on the Ad2 genome.

Adenoviridae↗

PubMed1981