Biomedical subjects

G Devauchelle

Publications and source records attributed to G Devauchelle.

At least 19 recordsLinked to original sources

PubMed2006

Members of the Hyposoter didymator Ichnovirus repeat element gene family are differentially expressed in Spodoptera frugiperda.

BACKGROUND: The abundance and the conservation of the repeated element (rep) genes in Ichnoviruses genomes suggest that this gene family plays an important role in viral cycles. In the Ichnovirus associated with the wasp Hyposoter didymator, named HdIV, 10 rep genes were identified to date. In this work, we report a relative quantitative transcription study of these HdIV rep genes in several tissues of the lepidopteran host Spodoptera frugiperda as well as in the H. didymator wasps. RESULTS: The data obtained in this work indicate that, in the early phases of infection (24 hours), HdIV rep genes each display different levels of transcripts in parasitized 2nd instar or HdIV-injected last instar S. frugiperda larvae. Only one, rep1, is significantly transcribed in female wasps. Transcript levels of the HdIV rep genes were found as not correlated to their copy number in HdIV genome. Our results also show that HdIV rep genes display different tissue specificity, and that they are primarily transcribed in S. frugiperda fat body and cuticular epithelium. CONCLUSION: This work is the first quantitative analysis of transcription of the ichnovirus rep gene family, and the first investigation on a correlation between transcript levels and gene copy numbers in Ichnoviruses. Our data indicate that, despite similar gene copy numbers, not all the members of this gene family are significantly transcribed 24 hours after infection in lepidopteran larvae. Additionally, our data show that, as opposed to other described HdIV genes, rep genes are little transcribed in hemocytes, thus suggesting that they are not directly associated with the disruption of the immune response but rather involved in other physiological alterations of the infected lepidopteran larva.

Amino Acid Sequence↗

PubMed2003

Annotation pattern of ESTs from Spodoptera frugiperda Sf9 cells and analysis of the ribosomal protein genes reveal insect-specific features and unexpectedly low codon usage bias.

MOTIVATION: A whole set of Expressed Sequence Tags (ESTs) from the Sf9 cell line of Spodoptera frugiperda is presented here for the first time. By this way we want to identify both conserved and specific genes of this pest species. We also expect from this analysis to find a class of protein sequences providing a tool to explore genomic features and phylogeny of Lepidoptera. RESULTS: The ESTs display both housekeeping as well as developmentally regulated genes, and a high percentage of sequences with unknown function. Among the identified ORFs, almost all ribosomal proteins (RPs) were found with high EST redundancy and hence sequence accuracy. The codon usage found among RP genes is in average surprisingly much less biased in Lepidoptera than in other organisms. Other Spodoptera genes also displayed a low bias, suggesting a general genome expression feature in this Lepidoptera. We also found that the L35A and L36 RP sequences, respectively, display 40 and 10 amino-acid insertions, both being present only in insects. Sequence analysis suggests that they are probably not subjected to a strong selective pressure and may be good phylogenetic markers for Lepidoptera. Most interestingly, the Lepidoptera sequences of 9 RP genes displayed a specific signature different from the canonical one. We conclude that the RP family allows valuable comparative genomics and phylogeny of Lepidoptera. AVAILABILITY: All EST sequence data are available from the private 'Spodo-Base' upon request.

Abstracting and Indexing↗

PubMed2001

Efficient CD4 binding and immunosuppressive properties of the 13B8.2 monoclonal antibody are displayed by its CDR-H1-derived peptide CB1.

A systematic exploration of the V(H)2/V(kappa)12-13 variable domains of the anti-CD4 monoclonal antibody (mAb) 13B8.2 was performed by the Spot method to screen for paratope-derived peptides (PDPs) demonstrating CD4 binding ability. Nine peptides, named CB1 to CB9, were identified, synthesized in a cyclic and soluble form and tested for binding to recombinant soluble CD4. Among them, CB1, CB2 and CB8 showed high anti-CD4 activity. Competition studies for CD4 binding indicated that PDPs CB1, CB8, and the parental mAb 13B8.2 recognized the same complementarity determining region (CDR)3-like loop region. PDP CB1 was shown to mimic the biological properties of 13B8.2 mAb in two independent cellular assays, demonstrating inhibitory activities in the micromolar range on antigen presentation and human immunodeficiency virus promoter activation. Our results indicate that the bioactive CDR-H1 PDP CB1 has retained a significant part of the parental 13B8.2 mAb properties and might be a lead for the design of anti-CD4 peptidomimetics of clinical interest.

Animals↗

PubMed2001

Characterization of the cDNA encoding the 90 kDa heat-shock protein in the Lepidoptera Bombyx mori and Spodoptera frugiperda.

This report presents the first hsp90 complete cDNA sequences from two Lepidoptera. The Bombyx mori full sequence was reconstituted from 15 partial cDNA clones belonging to expressed sequence tag libraries obtained from different tissues or cultured cells, thus showing the ubiquitous expression of the hsp90 gene. The Spodoptera frugiperda cDNA was isolated as a full-length clone from a cDNA library established from the Sf9 cell line. Both cDNAs are highly homologous and display the classical amino acid (aa) stretches representing the HSP90 signature. They potentially encode a 716 aa (B. mori) and a 717 aa (S. frugiperda) protein, with a calculated molecular mass of 83 kDa similar to the Drosophila homologous protein. We show that, unlike the vertebrates, hsp90 is a unique gene in both S. frupiperda and B. mori genomes. Sequencing of the corresponding genomic region shows that, contrary to the dipteran homologous gene, the lepidopteran hsp90 gene does not display any intron. Phylogenetic analysis based on the two lepidopteran and 23 other HSP90 aa sequences displays a high consistency with known phylogeny at both high and low taxonomic levels. Transcriptional analysis performed in S. frugiperda shows that the induction of the hsp90 gene only occurs 14 degrees C above physiological growth conditions (42 degrees C).

Amino Acid Sequence↗

PubMed2001

Expression of a membrane-bound form of Trypanosoma cruzi trans-sialidase in baculovirus-infected insect cells: a potential tool for sialylation of glycoproteins produced in the baculovirus-insect cells system.

A chimeric protein containing the catalytic domain of Trypanosoma cruzi trans-sialidase, the transmembrane domain of the major envelope glycoprotein of the baculovirus (gp67), and the signal peptide of ecdysteroid glucosyltransferase of the baculovirus was expressed under the control of the very late promoter p10 in baculovirus-infected lepidopteran cells. The recombinant protein was found to be enzymatically active. Three days after infection, equal amounts of activity were found associated to the plasma membrane and in the infection medium, both forms having the same apparent molecular weight and being N-glycosylated. When exogenous galactosylated acceptors (lactose or asialo-alpha1-acid glycoprotein) were added in the culture medium of cells infected with the recombinant baculovirus in the presence of a sialylated donor, a sialylation could be observed. Therefore, we propose the use of trans-sialidase as a potential tool for sialylation of glycoconjugates in the baculovirus-insect cells system.

Animals↗

PubMed2001

The chimeric mouse-human anti-CD4 Fab 13B8.2 expressed in baculovirus inhibits both antigen presentation and HIV-1 promoter activation.

The anti-CD4 mAb 13B8.2, directed against the CDR3-like loop of the D1 domain of CD4, inhibits signal transduction pathways leading to both T cell activation and HIV replication. VH9/DSP2/JH2 and Vkappa12-13/Jkappa2 rearrangements, corresponding to genes encoding the heavy and light chain variable regions of the 13B8.2 mAb, were inserted into baculovirus cassettes upstream from pre-installed human Fdgamma1 and Ckappa genes, respectively. After expression in insect cells, a complete correctly-processed Fab was secreted into the culture medium; it was protein-G immunopurified with a yield of 5 mg/L. The chimeric Fab 13B8.2 showed anti-CD4 binding activity with an affinity value of 3.3 nM and recognized the same region on the CDR3-like loop as the parental mAb. The mouse-human Fab inhibited IL2 secretion following antigen presentation and displayed a strong capacity to prevent HIV-1 promoter activation. Taken together, these results indicate that the chimeric Fab retained a major part of the parental 13B8.2 mAb properties and suggest that it might be a valuable therapeutic tool.

Amino Acid Sequence↗

PubMed2001

Pertinence of kappa and lambda recombinant antibodies directed against thyroid peroxidase in thyroid autoimmune disease.

Forty-one single-chain variable region fragments (scFvs) directed against thyroid peroxidase (TPO) were obtained by phage display libraries constructed from thyroid-infiltrating B cells of Graves' disease patients. Among these scFvs, 24.4% used a Vkappa light chain whereas 75.6% shows a light chain of Vlamda origin. Study of light chain gene usage in the TPO antibody repertoire demonstrated a dominance of the Vkappa 1-39 and Vlambda 1-51 genes. Thyroid peroxidase probing of overlapping peptides covering the amino acid sequences of anti-TPO T2/kappa and T13/lambda variable regions demonstrated a more restricted antigen recognition on T13/lambda than on T2/kappa. These two recombinant antibodies, expressed as whole IgG1 in the baculovirus/insect cell system, inhibited the binding to TPO of serum TPO autoantibodies whatever the light chain. Our study indicates that lambda as well as kappa light chain usage are found in the TPO antibody repertoire of thyroid-infiltrating B cells and are pertinent in the pathogenesis of autoimmune thyroid disease.

Amino Acid Sequence↗

PubMed2000

Adaptation of an insect cell line of Spodoptera frugiperda to grow at 37 degrees C: characterization of an endodiploid clone.

Sf21 and Sf9 cell lines established from the lepidoptera Spodoptera frugiperda do not display major induction of heat shock proteins when exposed to a temperature of 37 degrees C. After some months of adaptation at 37 degrees C we obtained two new cell lines, Sf21-HT and Sf9-HT, which have now been established for several years in our laboratory. The Sf9-HT line displays a slightly shorter doubling time at 37 degrees C than the wild type at 28 degrees C, but cell lethality gives rise to an earlier growth arrest. The composition of total lipid extract from heat-adapted cells reveals a higher sphingomyelin to phosphatidylcholine ratio and a higher percentage of saturated fatty acids, which are expected for the lower membrane fluidity, required for thermotolerance. The cell volume of Sf9-HT is doubled, and by flow cytometry we showed that the DNA content is twice that in the parental cell line. Karyotypic examination of metaphasic cells achieved under epifluorescence microscopy revealed a doubled chromosome number in Sf9-HT.

Animals↗

PubMed1999

Related RNAs in lepidopteran cells after in vitro infection with Hyposoter didymator virus define a new polydnavirus gene family.

In the present study, we describe the isolation and the characterization of three different Hyposoter didymator virus (HdV) lepidopteran host-expressed genes, the products of which might interfere with the host physiology during parasitism. In this report, we study the expression of HdV genes in Sf9 cells infected with HdV since results indicate that the Sf9 model mimics to some extent the in vivo model and may be utilized to study expression of HdV genes in lepidopteran host cells. This system allowed us to isolate three HdV-specific cDNAs, termed M24, M27, and M40. cDNA nucleotide sequence analysis demonstrated significant regions of homology. The three cDNAs displayed repeated sequences arranged in tandem array that might have evolved through domain duplication. Similar to other previously described polydnavirus host-expressed genes, two intron positions have been found in the M24 leader region. The cDNAs corresponded to RNAs of 1.5, 1.6, and 2.3 kb that are also detected in parasitized Spodoptera littoralis larvae. They are encoded by different genes likely located on different HdV DNA molecules. Corresponding RNAs are detected early postinfection and remain detectable for at least 10 days postinfection. They encode secreted glycine- and proline-rich proteins. An antiserum raised against a baculovirus recombinant M24-encoded protein detected similar proteins in the culture medium of infected lepidopteran cells and in parasitized host hemolymph. We propose that the three cloned genes belong to an HdV gene family specifically expressed in parasitized lepidopteran hosts.

Amino Acid Sequence↗

PubMed1999

Efficient amplification and direct sequencing of mouse variable regions from any immunoglobulin gene family.

We have designed two original sets of oligonucleotide primers hybridizing the relatively conserved motifs within the immunoglobulin signal sequences of each of the 15 heavy chain and 18 kappa light chain gene families. Comparison of these 5' primers with the immunoglobulin signal sequences referenced in the Kabat database suggests that these oligonucleotide primers should hybridize with 89.4% of the 428 mouse heavy chain signal sequences and with 91.8% of the 320 kappa light chain signal sequences with no mismatch. Following PCR amplification using the designed primers and direct sequencing of the amplified products, we obtained full-length variable sequences belonging to major (V(H)1, V(H)2, V(H)3, Vkappa1 and Vkappa21) but also small-sized (V(H)9, V(H)14, Vkappa2, Vkappa9A/9B, Vkappa12/13, Vkappa23 and Vkappa33/34) gene families, from nine murine monoclonal antibodies. This strategy could be a powerful tool for antibody sequence assessment whatever the V gene family before humanization of mouse monoclonal antibody or identification of paratope-derived peptides.

Animals↗

PubMed1999

A CD4-independent interaction of human immunodeficiency virus-1 gp120 with CXCR4 induces their cointernalization, cell signaling, and T-cell chemotaxis.

The gp120 envelope glycoprotein of human immunodeficiency virus-1 (HIV-1) interacts with the CXCR4 chemokine receptor, but it is not known whether gp120 activates CXCR4-mediated signaling cascades in the same manner as its natural ligand, SDF1alpha. We assessed the effects of wild-type gp120 and a mutant gp120 that interacts with CXCR4 but not CD4 on CD4(-)/CXCR4(+) cells and CD4(+)/CXCR4(+) cells, respectively. Under both experimental conditions, the interaction of CXCR4 and gp120 resulted in their CD4-independent cointernalization. Both molecules were translocated into early endosomes, whereas neither protein could be detected in late endosomes. Binding of gp120 to CXCR4 resulted in a CD4-independent phosphorylation of Pyk2 and an induction of chemotactic activity, demonstrating that this interaction has functional consequences. Interestingly, however, whereas SDF1alpha activated the ERK/MAP kinase pathway, this cascade was not induced by gp120. Together, these results suggest that the pathology of HIV-1 infection may be modulated by the distinct signal transduction pathway mediated by gp120 upon its interaction with CXCR4.

Antigens, CD↗

PubMed1999

Cystic fibrosis transmembrane conductance regulator (CFTR) confers glibenclamide sensitivity to outwardly rectifying chloride channel (ORCC) in Hi-5 insect cells.

Increasing evidence is now accumulating for the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) in the control of the outwardly rectifying chloride channel (ORCC). We have examined the sensitivity of ORCC to the sulfonylurea drug glibenclamide in Hi-5 (Trichoplusia ni) insect cells infected with recombinant baculovirus expressing either wild-type CFTR, DeltaF508-CFTR or E. coli beta galactosidase cDNA and in control cells either infected with virus alone or uninfected. Iodide efflux and single channel patch-clamp experiments confirmed that forskolin and 1-methyl-3-isobutyl xanthine (IBMX) or 7-methyl-1,3 dipropyl xanthine (DPMX) activate CFTR channels (unitary conductance: 9.1 +/- 1.6 pS) only in cells expressing CFTR. In contrast, we identified 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS)-sensitive ORCC in excised membrane patches in any of the cells studied, with similar conductance (22 +/- 2.5 pS at -80 mV; 55 +/- 4.1 pS at +80 mV) and properties. In the presence of 500 microm SITS, channel open probability (Po) of ORCC was reversibly reduced to 0.05 +/- 0.01 in CFTR-cells, to 0.07 +/- 0.02 in non-CFTR expressing cells and to 0.05 +/- 0.02 in DeltaF508-cells. In Hi-5 cells that did not express CFTR, glibenclamide failed to inhibit ORCC activity even at high concentrations (100 microm), whereas 500 microm SITS reversibly inhibited ORCC. In contrast in cells expressing CFTR or DeltaF508, glibenclamide dose dependently (IC50 = 17 microm, Hill coefficient 1.2) and reversibly inhibited ORCC. Cytoplasmic application of 100 microm glibenclamide reversibly reduced Po from 0.88 +/- 0.03 to 0.09 +/- 0.02 (wash: Po = 0.85 +/- 0.1) in CFTR cells and from 0.89 +/- 0.05 to 0.08 +/- 0.05 (wash: Po = 0.87 +/- 0.1) in DeltaF508 cells. In non-CFTR expressing cells, glibenclamide (100 microm) was without effect on Po (control: Po = 0. 89 +/- 0.09, glib.: Po = 0.86 +/- 0.02; wash: Po = 0.87 +/- 0.05). These data strongly suggest that the expression of CFTR confers glibenclamide sensitivity to the ORCC in Hi-5 cells.

1-Methyl-3-isobutylxanthine↗

PubMed1999

Synthetic peptides derived from the variable regions of an anti-CD4 monoclonal antibody bind to CD4 and inhibit HIV-1 promoter activation in virus-infected cells.

The monoclonal antibody (mAb) ST40, specific for the immunoglobulin complementarity-determining region (CDR) 3-like loop in domain 1 of the CD4 molecule, inhibits human immunodeficiency virus type 1 (HIV-1) promoter activity and viral transcription in HIV-infected cells. To design synthetic peptides from the ST40 paratope that could mimic these biological properties, a set of 220 overlapping 12-mer peptides frameshifted by one residue, corresponding to the deduced ST40 amino acid sequence, was synthesized by the Spot method and tested for binding to recombinant soluble CD4 antigen. Several peptides that included in their sequences amino acids from the CDRs of the antibody and framework residues flanking the CDRs were found to bind soluble CD4. Eleven paratope-derived peptides (termed CM1-CM11) were synthesized in a cyclic and soluble form. All the synthetic peptides showed CD4 binding capacity with affinities ranging from 1.6 to 86.4 nM. Moreover, peptides CM2, CM6, CM7, CM9, and CM11 were able to bind a cyclic peptide corresponding to the CDR3-like loop in domain 1 of CD4 (amino acids 81-92 of CD4). Peptide CM9 from the light chain variable region of mAb ST40 and, to a lesser extent, peptides CM2 and CM11 were able to inhibit HIV-1 promoter long terminal repeat-driven beta-galactosidase gene expression in the HeLa P4 HIV-1 long terminal repeat beta-galactosidase indicator cell line infected with HIV-1. The binding of mAb ST40 to CD4 was also efficiently displaced by peptides CM2, CM9, and CM11. Our results indicate that the information gained from a systematic exploration of the antigen binding capacity of synthetic peptides from immunoglobulin variable sequences can lead to the identification of bioactive paratope-derived peptides of potential pharmacological interest.

Amino Acid Sequence↗

PubMed1999

[Baculovirus: an example of an insect virus of use to humans].

Baculovirus is a viral pathogen of insects in general and lepidoptera in particular. The genome of this large virus consists of a circular, infectious bicatenary DNA molecule. At the end of its replication cycle in insects, baculovirus produces a large quantity of at least two proteins, i.e., polyedrine and polypeptide P10. These proteins are essential for transmission of the virus in nature, but are not necessary in cell cultures. Using molecular recombinant techniques, one or both of the genes coding for these proteins can be replaced by heterologous genes. In this way, baculovirus raised in vitro can be used to produce large quantities of the alien proteins at the end of the multiplication cycle. So far more than 3,000 different proteins have been expressed including several presenting interest as diagnostic tools (Puumala virus, Herpes simplex virus) or therapeutic treatment in man and animals (vaccinations against dengue, flu, malaria and production of anti-Rhesus immunoglobulins). Since it is based on the use of lepidoptera virus, this system would appear to be particularly safe. No vertebrate virus is able to replicate in the cell system used. Use of this genetic engineering tool will undoubtedly expand and holds great promise for the future.

Animals↗

PubMed1998

Anti-digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties.

A gene encoding a single-chain antibody fragment directed against digoxin (named 1C10 scFv) was cloned in two expression systems. For this purpose, a new baculovirus transfer cassette fully compatible with the procaryotic pHEN vector was constructed. Baculovirus production led to higher yield than did Escherichia coli expression. The procaryotic fragment showed variations in the fine specificity profile but an affinity constant nearly identical to that of the 1C10 Fab, whereas the eucaryotic scFv fragment had a lower affinity with a specificity profile identical to original mAb. The half-lives of the digoxin:scFv complexes and the global specificity are compatible with therapeutic use of this antibody fragment.

Amino Acid Sequence↗

PubMed1998

Dissociation of the CD4 and CXCR4 binding properties of human immunodeficiency virus type 1 gp120 by deletion of the first putative alpha-helical conserved structure.

To evaluate conserved structures of the surface gp120 subunit (SU) of the human immunodeficiency virus type 1 (HIV-1) envelope in gp120-cell interactions, we designed and produced an HIV-1 IIIB (HXB2R) gp120 carrying a deletion of amino acids E61 to S85. This sequence corresponds to a highly conserved predicted amphipathic alpha-helical structure located in the gp120 C1 region. The resultant soluble mutant with a deleted alpha helix 1 (gp120 DeltaalphaHX1) exhibited a strong interaction with CXCR4, although CD4 binding was undetectable. The former interaction was specific since it inhibited the binding of the anti-CXCR4 monoclonal antibody (12G5), as well as SDF1alpha, the natural ligand of CXCR4. Additionally, the mutant gp120 was able to bind to CXCR4(+)/CD4(-) cells but not to CXCR4(-)/CD4(-) cells. Although efficiently expressed on cell surface, HIV envelope harboring the deleted gp120 DeltaalphaHX1 associated with wild-type transmembrane gp41 was unable to induce cell-to-cell fusion with HeLa CD4(+) cells. Nevertheless, the soluble gp120 DeltaalphaHX1 efficiently inhibited a single round of HIV-1 LAI infection in HeLa P4 cells, with a 50% inhibitory concentration of 100 nM. Our data demonstrate that interaction with the CXCR4 coreceptor was maintained in a SUgp120 HIV envelope lacking alphaHX1. Moreover, in the absence of CD4 binding, the interaction of gp120 DeltaalphaHX1 with CXCR4 was sufficient to inhibit HIV-1 infection.

Amino Acid Sequence↗

PubMed1997

Human growth hormone receptor: cloning and expression of the full-length complementary DNA after site-directed inactivation of a cryptic bacterial promoter.

Growth hormone receptor is a cytokine-type receptor which is required for normal somatic growth and for numerous metabolic processes. Its complementary DNA (cDNA) has been isolated in various species leading to intensive studies to elucidate the mechanism of action of the growth hormone. However, serious difficulties have been reported in cloning in Escherichia coli, an intact full-length human cDNA. In this study, the cDNA is shown to contain a cryptic bacterial promoter driving inappropriate expression of a part of human growth hormone (hGH) receptor which is toxic for E. coli growth. Identification of this promoter and its inactivation by changing only one nucleotide led us to obtain stable bacterial clones containing a high copy number of full-length coding sequences. This molecular clone was used in a baculovirus/insect cell system to produce large amounts of glycosylated recombinant receptor. Binding studies with 125I-labelled hGH revealed an affinity constant of 2.8 x 10(9) M(-1), similar to that reported for the native liver receptor. This report described a general method of cloning which could be applied to similar unclonable cDNA fragments.

Animals↗

PubMed1996

The baculovirus/insect cell system as an alternative to Xenopus oocytes. First characterization of the AKT1 K+ channel from Arabidopsis thaliana.

Two plant (Arabidopsis thaliana) K+ transport systems, KAT1 and AKT1, have been expressed in insect cells (Sf9 cell line) using recombinant baculoviruses. Microscopic observation after immunogold staining revealed that the expressed AKT1 and KAT1 polypeptides were mainly associated with internal membranes, but that a minute fraction was targeted to the cell membrane. KAT1 was known, from earlier electrophysiological characterization in Xenopus oocytes, to be an inwardly rectifying voltage-gated channel highly selective for K+, while similar experiments had failed to characterize AKT1. Insect cells expressing KAT1 displayed an exogenous inwardly rectifying K+ conductance reminiscent of that described previously in Xenopus oocytes expressing KAT1. Under similar conditions, cells expressing AKT1 showed a disturbed cell membrane electrical stability that precluded electrophysiological analysis. Use of a baculovirus transfer vector designed so as to decrease the expression level allowed the first electrophysiological characterization of AKT1. The baculovirus system can thus be used as an alternative method when expression in Xenopus oocytes is unsuccessful for electrophysiological characterization of the ion channel of interest. The plant AKT1 protein has been shown in this way to be an inwardly rectifying voltage-gated channel highly selective for K+ ions and sensitive to cGMP.

Adenosine Triphosphate↗