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R Biemans

Publications and source records attributed to R Biemans.

12 recordsLinked to original sources
PubMed2002

DNA immunisation. New histochemical and morphometric data.

Splenic germinal center reactions were measured during primary response to a plasmidic DNA intramuscular injection. Cardiotoxin-pretreated Balb/c mice were immunized with DNA plasmids encodmg or not the SAG1 protein, a membrane antigen of Toxoplasma gondii. Specific anti-SAG1 antibodies were detected on days 16 and 36 after injection of coding plasmids. The results of ELISAs showed that the SAG1-specific antibodies are of the IgG2a class. Morphometric analyses were done on serial immunostained cryosections of spleen and draining or non-draining lymph nodes. This new approach made it possible to evaluate the chronological changes induced by DNA immunisation in the germinal centres (in number and in size). Significant increases in the number of germinal centres were measured in the spleen and only in draining lymph nodes after plasmid injection, the measured changes of the germinal centers appeared to result from the adjuvant stimulatory effect of the plasmidic DNA since both the coding and the noncoding plasmid DNA induced them. No measurable changes were recorded in the T-dependent zone of lymph organs.

Animals↗
PubMed2001

The surface antigen SAG3 mediates the attachment of Toxoplasma gondii to cell-surface proteoglycans.

The attachment of Toxoplasma gondii to target cells is mediated by recognition of cellular heparan sulfate proteoglycans (HSPGs). The present study was performed to determine whether SAG1 and SAG3, two of the parasite surface antigens anchored to the membrane via glycosylphosphatidylinositol groups (GPIs), are involved in the tachyzoite binding to proteoglycans. The use of recombinant soluble forms of these proteins allowed us to demonstrate that SAG3, but not SAG1, interacts specifically with cellular HSPGs. Indeed, soluble recombinant SAG3 protein (recSAG3) was found to bind to immobilized heparin, whereas recSAG1 did not interact with this glycoaminoglycan. The specific adherence of recSAG3 to CHO cells was inhibited by soluble glycoconjugates, of which heparin, fucoidan and dextran sulfate were the most effective. Moreover, binding of recSAG3 to two HSPGs-deficient cell mutants was reduced by up to 80%. Proteoglycan sulfation was critical for SAG3 adherence to HSPGs as incubation of cells in the presence of sodium chlorate drastically reduced the recSAG3 binding. Finally, preincubation of CHO cells with recSAG3 blocked the adsorption of radiolabelled Toxoplasma tachyzoites. Taken together, these results indicate that SAG3 is a first glycoaminoglycan-binding protein associated with Toxoplasma, and SAG3-HSPGs interactions are involved in the parasite attachment to target cells.

Animals↗
PubMed2000

Crystallization and preliminary x-ray diffraction analysis of beta-cinnamomin, an elicitin secreted by the phytopathogenic fungus Phytophthora cinnamomin.

Cinnamomin (CIN) belongs to a family of 10 kDa proteins designated as elicitins. Some of these proteins induce a hypersensitive response in diverse plant species, leading to resistance against fungal and bacterial plant pathogens. CIN was crystallized by the vapour-diffusion method using either ammonium sulfate or polyethyleneglycol (PEG) as precipitants in solutions buffered at around pH 7. These crystals are isomorphous and belong to the triclinic space group, with unit-cell parameters a = 31.69, b = 36. 99, c = 44.09 A, alpha = 76.86, beta = 84.41, gamma = 80.26 degrees. A frozen crystal diffracted X-rays beyond 1.45 A resolution on a synchrotron-radiation source.

Algal Proteins↗
PubMed2000

Protective immunity against congenital toxoplasmosis with recombinant SAG1 protein in a guinea pig model.

Primary infection with Toxoplasma gondii during pregnancy can induce fetal pathology and abortion in both humans and animals. The present study describes the development of an experimental model of congenital toxoplasmosis in the guinea pig. In this animal model, we evaluated the protective effect of vaccination with a recombinant form of SAG1 against maternofetal transmission of tachyzoites. The presence of parasites in fetuses was determined by nested PCRs and by an in vivo readout after fetal brain homogenate injections in mice. The absence of parasites was demonstrated in 66 to 86% of fetuses derived from adult guinea pigs immunized with SAG1 and challenged with the mildly virulent T. gondii strain C56. In contrast, more than 80% of fetuses from mock-immunized guinea pigs were infected. The protection was not correlated with titers of antibody to SAG1. Our results indicated that this experimental model constitutes a relevant model for evaluation of vaccine candidates against congenital toxoplasmosis and that SAG1 elicits significant protection against maternofetal transmission.

Animals↗
PubMed1999

Expression of a recombinant Toxoplasma gondii ROP2 fragment as a fusion protein in bacteria circumvents insolubility and proteolytic degradation.

A 268-amino-acid-residue carboxy-terminal antigenic fragment of the Toxoplasma gondii rhoptry protein ROP2 (recROP2(t), residues 196-464) was expressed in Escherichia coli. This recombinant fragment was produced at low concentration and in a highly insoluble form. By contrast, the level of recROP2(t) production was drastically greater when the same coding sequence was fused to the C-terminus of thioredoxin (TRX) or to the maltose-binding protein (MBP) gene. While both fusion proteins were found to be mainly insoluble, solubilization could be achieved without significant degradation. MBP was more efficient than TRX in increasing the recovery of soluble protein with more than 10% of total MBP-recROP2(t) being readily expressed in a soluble form. Moreover, the insoluble form of MBP-recROP2(t) could be correctly refolded with a recovery of more than 80%. Both forms of MBP-recROP2(t) were purified to homogeneity by amylose chromatography. In contrast, the refolding of TRX-recROP2(t) promoted aggregation of the protein, which was prevented by the use of zwitterionic detergent during the one-step purification by gel filtration. Subsequent proteolytic cleavages of purified TRX-recROP2(t) and of MBP-recROP2(t) led respectively to the complete degradation or to the truncation of the recROP2(t) moiety. However, recROP2(t), despite the presence of the fusion partners, adopted a suitable conformation recognized by human serum-derived antibodies from T. gondii-seropositive individuals. Finally, both fusion proteins were able to induce specific humoral and cell-mediated immune response to the ROP2 fragment. Such fusions could represent an alternative to study the immunogenicity of T. gondii proteins which are difficult to produce because of insolubility and degradation.

ATP-Binding Cassette Transporters↗
PubMed1998

The conformation of purified Toxoplasma gondii SAG1 antigen, secreted from engineered Pichia pastoris, is adequate for serorecognition and cell proliferation.

A truncated form of SAG1, the immunodominant surface antigen of Toxoplasma gondii, has been produced in the methylotrophic yeast, Pichia pastoris. By construction, the recombinant protein lacks C-terminal residues 308-336 which, in native SAG1, encompass the glycosylphosphatidylinositol anchorage site. Secretion of anchor-less SAG1 proceeded via the yeast prepro alpha-mating factor signal peptide and yielded two immunoreactive protein species having apparent molecular masses of 31.5 and 34.5 kDa, respectively, and differing only by N-glycosylation of the single Asn-X-Ser site present in the molecule. Purification of the anchor-less SAG1 was achieved by a combination of ion-exchange and size-exclusion chromatographies. N-terminal amino acid sequencing of the products indicated the presence of additional residues glutamic acid--alanine at the N-terminal end of the products. Despite incomplete processing and unnatural glycosylation, anchor-less SAG1 proteins apparently adopted a suitable conformation recognized by monoclonal and human serum-derived antibodies, specific for the native SAG1. In addition, the recombinant anchor-less SAG1 proved competent for inducing proliferation, in vitro, of mononuclear cells from seropositive individuals. Finally, properly adjuvanted anchor-less SAG1 was able to induce protection of mice against a lethal challenge with T. gondii tachyzoites.

Amino Acid Sequence↗
PubMed1992

Immunoelectron microscopic detection of the hepatitis B virus major surface protein in dilated perinuclear membranes of yeast cells.

The major surface protein of hepatitis B virus produced in Saccharomyces cerevisiae can be recovered from cell lysates in the form of 22-mm lipoprotein particles. Immunoelectron microscopy was applied to investigate site and time of particle assembly. Thin sections of yeast cells revealed that production of the S protein provoked a dilation of the endoplasmic reticulum. Dilated areas were specifically labeled with a polyclonal antibody raised against glutaraldehyde-treated yeast-derived HBsAg particles. In contrast to previous postulates of particle formation during cell lysis and extract preparation, these results suggest that particle formation in yeast occurs in the endoplasmic reticulum and that transport of particles along the secretion pathway is blocked.

Cell Transformation, Viral↗
PubMed1992

Subcellular localization of recombinant truncated Gag precursor proteins of HIV expressed in Saccharomyces cerevisiae.

OBJECTIVE: To determine signals contained in the HIV-1 Gag precursor implicated in protein transport. DESIGN: To study the localization of truncated Gag proteins expressed in Saccharomyces cerevisiae. METHODS: Thin-section immunoelectron microscopy studies were performed on S. cerevisiae cells producing myristoylated or non-myristoylated Pr55gag, the core protein (p24) and several truncated Gag proteins. RESULTS: Pr55gag and the carboxy-terminal truncated Gag proteins were myristoylated and localized at the plasma membrane. p24 was localized in the nucleus or perinuclear membrane. However, addition of a myristoyl group to p24 targeted this molecule to the plasma membrane. CONCLUSIONS: The myristoylated amino-terminal 214 amino acids are sufficient to target Pr55gag to the plasma membrane. Subcellular signals implicated in protein transport are present in the core p24 polypeptide which may become dominant or accessible in the absence of the amino-myristoyl group.

Base Sequence↗
PubMed1991

The large surface protein of hepatitis B virus is retained in the yeast endoplasmic reticulum and provokes its unique enlargement.

The coding sequences for each of the three envelope proteins of hepatitis B virus (HBV), the major (S), middle (M), and large (L) surface proteins, were expressed in Saccharomyces cerevisiae. Analysis by immunoelectron microscopy of thin sections of yeast cells showed that production of L protein but not of M or S protein provoked morphological changes in the yeast endoplasmic reticulum. A large accumulation of membranous structures connected with the perinuclear cysternae and specifically labeled by a monoclonal antibody directed against the amino-terminal (preS1) sequence of the L protein, was observed. The L protein was post-translationally modified by N- and O-linked glycosylation, indicative of its entry into the yeast secretory pathway and by N-myristoylation of its amino-terminal glycine residue. Deletion of this glycine residue resulted in the synthesis of a nonmyristoylated L protein. Proliferation of the endoplasmic reticulum was comparable in cells producing either the myristoylated or nonmyristoylated L protein, indicating that myristoylation alone is not responsible for the induction of the abnormal membrane morphology.

Blotting, Western↗
PubMed1987

Porcine D-amino acid oxidase: determination of the mRNA nucleotide sequence by the characterization of genomic and cDNA clones.

Oligodeoxynucleotide probes derived from the published amino acid (aa) sequence for D-amino acid oxidase (DAO) [Ronchi et al. J. Biol. Chem. 259 (1982) 8824-8834] were used to screen cDNA libraries made from porcine kidney cortex and liver. Whereas no clones were obtained from kidney mRNAs, 20 independent ones were isolated from the liver library. Surprisingly, all of them carried only partial cDNAs for DAO starting around aa 100 in the coding sequence and extending for up to 250 bp in the 3'-noncoding sequence. One of these clones, pULB9103, was used to screen a porcine genomic library and allowed the isolation of DAO gene clone phULB001. Four exons encoding aa 1-151 were identified and sequenced, as well as the relevant exon-intron junctions. The mRNA sequence coding for DAO has been reconstituted from the genomic and cDNA sequences; its analysis by computer did not reveal any significant secondary structure, or particular feature, which could explain the failure to obtain full-length cDNAs.

Amino Acid Sequence↗