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Biomedical subjects

J Dubuisson

Publications and source records attributed to J Dubuisson.

At least 19 recordsLinked to original sources
PubMed2006

[Spontaneous rupture of the uterine pedicle during pregnancy: Three case reports].

We report a retrospective review of three cases presenting with spontaneous uterine artery rupture during pregnancy. Clinical presentations were intra-peritoneal haemorrhage for two of them and in utero fetal death for the last one. Fetal outcome was poor in all three cases. Uterine vessels spontaneous rupture during pregnancy is an exceptional event. Considering our experience and the literature review, we propose a guideline for vascular rupture during pregnancy.

Adult↗
PubMed2005

[Decision-to-deliver interval for forceps delivery and cesarean section: 137 extractions for abnormal fetal heart rhythm during labor].

AIM: Comparison of the decision to delivery interval in cases of forceps delivery and in cases of cesarean sections. MATERIAL AND METHOD: A retrospective analysis was performed on 137 cases of forceps deliver (n = 63) and cesarean section (n = 74) indicated for abnormal fetal heart rhythm. All cases were observed in a level 3 maternity unit between October 2003 and August 2004. RESULTS: The mean decision-to-delivery interval was significantly shorter in the forceps group (14.84 min +/- 6.54 versus 29.31 min +/- 11.79 p < 0.0001). Maternal and neonatal morbidity were comparable. CONCLUSION: This study suggest that once the fetal head is engaged, forceps delivery can significantly reduced the decision-to-delivery interval.

Adult↗
PubMed2004

Positive effect of the hepatitis C virus nonstructural 5A protein on viral multiplication.

Hepatitis C virus infection (HCV), is a major cause of liver disease worldwide, and are frequently resistant to the interferon alpha treatment. The nonstructural (NS) 5A protein of HCV has been proposed to be involved in this resistance. Additional studies have pointed out a role for NS5A in several other cellular interactions as well as an important role of its adaptative mutations in HCV genome replication. However, no infectious system is available to assess the role of NS5A in the HCV life cycle. Thus, we have constructed a recombinant system directly demonstrating for the first time that the expression of NS5A confers a multiplicative advantage to Sindbis virus, a virus close to HCV. This advantage seemed to be related to an anti-apoptotic effect of the NS5A protein. At a later stage, a possible nuclear localization of NS5A was observed, likely due to apoptotic cleavages of this protein. The NS5A protein was also shown to induce the interleukin-8 (IL-8) mRNA and to activate the NF-kappaB pathway independently of the Sindbis virus. Together, our data suggest that the activation of NF-kappaB could lead to the anti-apoptotic activity of NS5A and explain the viral multiplicative advantage conferred by the expression of the NS5A protein.

Apoptosis↗
PubMed2001

Mimotopes of the hepatitis C virus hypervariable region 1, but not the natural sequences, induce cross-reactive antibody response by genetic immunization.

The hypervariable region 1 (HVR1) of the putative envelope protein E2 of hepatitis C virus (HCV) contains a principal neutralization epitope, and anti-HVR1 antibodies have been shown to possess protective activity in ex vivo neutralization experiments. However, the high rate of variability of this antigenic fragment may play a major role in the mechanism of escape from host immune response and might represent a major obstacle to developing an HCV vaccine. Thus, even if direct experimental evidence of the neutralizing potential of anti-HVR1 antibodies by active immunization is still missing, the generation of a vaccine candidate with a cross-reactive potential would be highly desirable. To overcome the problem of HVR1 variability, we have engineered cross-reactive HVR1 peptide mimics (mimotopes) at the N terminus of the E2 ectodomain in plasmid vectors suitable for genetic immunization. High levels of secreted and biologically active mimotope/E2 chimeras were obtained by transient transfection of these plasmids in cultured cells. All plasmids elicited anti-HVR1 antibodies in mice and rabbits with some of them leading to a cross-reacting response against many HVR1 variants from natural isolates. Epitope mapping revealed a pattern of reactivity similar to that induced by HCV infection. In contrast, plasmids encoding naturally occurring HVR1 sequences displayed either on full-length E2 in the context of the whole HCV structural region, or on a soluble, secreted E2 ectodomain, did not induce a cross-reacting anti-HVR1 response.

Amino Acid Sequence↗
PubMed2001

Nonenveloped nucleocapsids of hepatitis C virus in the serum of infected patients.

One of the characteristics of hepatitis C virus (HCV) is the high incidence of persistent infection. HCV core protein, in addition to forming the viral nucleocapsid, has multiple regulatory functions in host-cell transcription, apoptosis, cell transformation, and lipid metabolism and may play a role in suppressing host immune response. This protein is thought to be present in the bloodstream of the infected host as the nucleocapsid of infectious, enveloped virions. This study provides evidence that viral particles with the physicochemical, morphological, and antigenic properties of nonenveloped HCV nucleocapsids are present in the plasma of HCV-infected individuals. These particles have a buoyant density of 1.32 to 1.34 g/ml in CsCl, are heterogeneous in size (with predominance of particles 38 to 43 or 54 to 62 nm in diameter on electron microscopy), and express on their surface epitopes located in amino acids 24 to 68 of the core protein. Similar nucleocapsid-like particles are also produced in insect cells infected with recombinant baculovirus bearing cDNA for structural HCV proteins. HCV core particles isolated from plasma were used to generate anti-core monoclonal antibodies (MAbs). These MAbs stained HCV core in the cytoplasm of hepatocytes from experimentally infected chimpanzees in the acute phase of the infection. These chimpanzees had concomitantly HCV core antigen in serum. These findings suggest that overproduction of nonenveloped nucleocapsids and their release into the bloodstream are properties of HCV morphogenesis. The presence of circulating cores in serum and accumulation of the core protein in liver cells during the early phase of infection may contribute to the persistence of HCV and its many immunopathological effects in the infected host.

Amino Acid Sequence↗
PubMed2000

The transmembrane domains of hepatitis C virus envelope glycoproteins E1 and E2 play a major role in heterodimerization.

Oligomerization of viral envelope proteins is essential to control virus assembly and fusion. The transmembrane domains (TMDs) of hepatitis C virus envelope glycoproteins E1 and E2 have been shown to play multiple functions during the biogenesis of E1E2 heterodimer. This makes them very unique among known transmembrane sequences. In this report, we used alanine scanning insertion mutagenesis in the TMDs of E1 and E2 to examine their role in the assembly of E1E2 heterodimer. Alanine insertion within the center of the TMDs of E1 or E2 or in the N-terminal part of the TMD of E1 dramatically reduced heterodimerization, demonstrating the essential role played by these domains in the assembly of hepatitis C virus envelope glycoproteins. To better understand the alanine scanning data obtained for the TMD of E1 which contains GXXXG motifs, we analyzed by circular dichroism and nuclear magnetic resonance the three-dimensional structure of the E1-(350-370) peptide encompassing the N-terminal sequence of the TMD of E1 involved in heterodimerization. Alanine scanning results and the three-dimensional molecular model we obtained provide the first framework for a molecular level understanding of the mechanism of hepatitis C virus envelope glycoprotein heterodimerization.

Alanine↗
PubMed2000

Glycosylation of the hepatitis C virus envelope protein E1 is dependent on the presence of a downstream sequence on the viral polyprotein.

The addition of N-linked oligosaccharides to Asn-X-(Ser/Thr) sites is catalyzed by the oligosaccharyltransferase, an enzyme closely associated with the translocon and generally thought to have access only to nascent chains as they emerge from the ribosome. However, the presence of the sequon does not automatically ensure core glycosylation because many proteins contain sequons that remain either nonglycosylated or glycosylated to a variable extent. In this study, hepatitis C virus (HCV) envelope protein E1 was used as a model to study the efficiency of N-glycosylation. HCV envelope proteins, E1 and E2, were released from a polyprotein precursor after cleavage by host signal peptidase(s). When expressed alone, E1 was not efficiently glycosylated. However, E1 glycosylation was improved when expressed as a polyprotein including full-length or truncated forms of E2. These data indicate that glycosylation of E1 is dependent on the presence of polypeptide sequences located downstream of E1 on HCV polyprotein.

Glycoproteins↗
PubMed2000

Reciprocal relationship between alpha1,2 mannosidase processing and reglucosylation in the rough endoplasmic reticulum of Man-P-Dol deficient cells.

The study of the glycosylation pathway of a mannosylphosphoryldolichol-deficient CHO mutant cell line (B3F7) reveals that truncated Glc(0-3)Man5GlcNAc2 oligosaccharides are transferred onto nascent proteins. Pulse-chase experiments indicate that these newly synthesized glycoproteins are retained in intracellular compartments and converted to Man4GlcNAc2 species. In this paper, we demonstrate that the alpha1,2 mannosidase, which is involved in the processing of Man5GlcNAc2 into Man4GlcNAc2, is located in the rough endoplasmic reticulum. The enzyme was shown to be inhibited by kifunensine and deoxymannojirimycin, indicating that it is a class I mannosidase. In addition, Man4GlcNAc2 species were produced at the expense of Glc1Man5GlcNAc2 species. Thus, the trimming of Man5GlcNAc2 to Man4GlcNAc2, which is catalyzed by this mannosidase, could be involved in the control of the glucose-dependent folding pathway.

1-Deoxynojirimycin↗
PubMed2000

Functional characterization of intracellular and secreted forms of a truncated hepatitis C virus E2 glycoprotein.

The E2 protein of hepatitis C virus (HCV) is believed to be a virion surface glycoprotein that is a candidate for inclusion in an antiviral vaccine. A truncated soluble version of E2 has recently been shown to interact with CD81, suggesting that this protein may be a component of the receptor for HCV. When expressed in eukaryotic cells, a significant proportion of E2 forms misfolded aggregates. To analyze the specificity of interaction between E2 and CD81, the aggregated and monomeric forms of a truncated E2 glycoprotein (E2(661)) were separated by high-pressure liquid chromatography and analyzed for CD81 binding. Nonaggregated forms of E2 preferentially bound CD81 and a number of conformation-dependent monoclonal antibodies (MAbs). Furthermore, intracellular forms of E2(661) were found to bind CD81 with greater affinity than the extracellular forms. Intracellular and secreted forms of E2(661) were also found to differ in reactivity with MAbs and human sera, consistent with differences in antigenicity. Together, these data indicate that proper folding of E2 is important for its interaction with CD81 and that modifications of glycans can modulate this interaction. Identification of the biologically active forms of E2 will assist in the future design of vaccines to protect against HCV infection.

Amino Acid Sequence↗
PubMed2000

Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a major role in the processing, subcellular localization, and assembly of these envelope proteins.

For most membrane proteins, the transmembrane domain (TMD) is more than just an anchor to the membrane. The TMDs of hepatitis C virus (HCV) envelope proteins E1 and E2 are extreme examples of the multifunctionality of such membrane-spanning sequences. Indeed, they possess a signal sequence function in their C-terminal half, play a major role in endoplasmic reticulum localization of E1 and E2, and are potentially involved in the assembly of these envelope proteins. These multiple functions are supposed to be essential for the formation of the viral envelope. As for the other viruses of the family Flaviviridae, these anchor domains are composed of two stretches of hydrophobic residues separated by a short segment containing at least one fully conserved charged residue. Replacement of these charged residues by an alanine in HCV envelope proteins led to an alteration of all of the functions performed by their TMDs, indicating that these functions are tightly linked together. These data suggest that the charged residues of the TMDs of HCV glycoproteins play a key role in the formation of the viral envelope.

Amino Acid Sequence↗
PubMed1999

Retention and degradation of N-glycoproteins in the rough endoplasmic reticulum.

Recent studies have shown that newly synthesized proteins and glycoproteins are submitted to a quality control mechanism in the rough endoplasmic reticulum (ER). In this report we present two models: One model will illustrate a transient retention in rough ER leading to a further degradation of glycoproteins in the cytosol, (soluble alkaline phosphatase expressed in Man-P-Dol deficient CHO cells lines). The second model will illustrate a strict retention of glycoproteins in rough ER without degradation nor recycling through the Golgi (E1, E2 glycoproteins of Hepatitis C virus in stably transfected UHCV-11.4 cells and in infected Hep G2 cells). In both cases, oligomannoside structures are markers of these phenomena, either as free soluble released oligomannosides in the case of degradation, or as N-linked oligomannosides for strict retention in rough ER.

Alkaline Phosphatase↗
PubMed1999

Analysis of the glycosylation sites of hepatitis C virus (HCV) glycoprotein E1 and the influence of E1 glycans on the formation of the HCV glycoprotein complex.

The hepatitis C virus (HCV) genome encodes two membrane-associated envelope glycoproteins (E1 and E2), which are released from the viral polyprotein precursor by host signal peptidase cleavages. These glycoproteins interact to form a noncovalent heterodimeric complex, which is retained in the endoplasmic reticulum. HCV glycoproteins, E1 and E2, are heavily modified by N-linked glycosylation. A recent study has revealed that upon partial deglycosylation with endoglycosidase H only four of the five potential glycosylation sites of HCV glycoprotein E1 are utilized. In this work, the unused glycosylation site on the E1 glycoprotein was identified and the influence of N-linked glycosylation on the formation of the HCV glycoprotein complex was studied by expressing a panel of E1 glycosylation mutants in HepG2 cells. Each of the five potential N-linked glycosylation sites, located at amino acid positions 196, 209, 234, 305 and 325, respectively, on the HCV polyprotein, was mutated separately as well as in combination with the other sites. Expression of the mutated E1 proteins in HepG2 cells indicated that the fifth glycosylation site is not used for the addition of N-linked oligosaccharides and the Pro immediately following the sequon (Asn-Trp-Ser) precludes core glycosylation. The effect of each mutation on the formation of noncovalent E1E2 complexes was also analysed. As determined with the use of a conformation-sensitive monoclonal antibody, mutations at positions N2 and N3 had no, or only minor, effects on the assembly of the E1E2 complex, whereas a mutation at position N1 and predominantly at position N4 dramatically reduced the efficiency of the formation of noncovalent E1E2 complexes.

Animals↗
PubMed1999

A single nucleotide change in the 5' noncoding region of Sindbis virus confers neurovirulence in rats.

Two pairs of Sindbis virus (SV) variants that differ in their neuroinvasive and neurovirulent traits in mice have been isolated. Recently, we mapped the genetic determinants responsible for neuroinvasiveness in weanling mice. Here, we extend this study to newborn and adult rats and to rat neuronal cultures. Remarkably, certain aspects of the pathogenesis of these strains in rats were found to be quite distinct from the mouse model. Suckling rats were susceptible to all four isolates, and replication in the brain was observed after both intraperitoneal and intracranial (i.c.) inoculation. None of the isolates was neuroinvasive in adult rats, although all replicated after i.c. inoculation. For the isolate pair that was highly neurovirulent in mice, SVN and SVNI, only SVNI caused death after i.c. inoculation of adult rats. Similarly, only SVNI was cytotoxic for primary cultures of mature neurons. The genetic determinants responsible for the pathogenic properties of SVNI were mapped to the E2 glycoprotein and the 5' noncoding region (5'NCR). Substitution of two amino acids in SVN E2 with the corresponding residues of SVNI (Met-190 and Lys-260) led to paralysis in 3- and 5-week-old rats. More dramatically, a single substitution in the 5'NCR of SVN (G at position 8) transformed the virus into a lethal pathogen for 3-week-old rats like SVNI. In 5-week-old rats, however, this recombinant was attenuated relative to SVNI by 2 orders of magnitude. Combination of the E2 and 5'NCR determinants resulted in a recombinant with virulence properties indistinguishable from those of SVNI. These data indicate that the 5'NCR and E2 play an instrumental role in determining the age-dependent pathogenic properties of SV in rats.

5' Untranslated Regions↗
PubMed1999

The transmembrane domain of hepatitis C virus glycoprotein E1 is a signal for static retention in the endoplasmic reticulum.

Hepatitis C virus (HCV) glycoproteins E1 and E2 assemble to form a noncovalent heterodimer which, in the cell, accumulates in the endoplasmic reticulum (ER). Contrary to what is observed for proteins with a KDEL or a KKXX ER-targeting signal, the ER localization of the HCV glycoprotein complex is due to a static retention in this compartment rather than to its retrieval from the cis-Golgi region. A static retention in the ER is also observed when E2 is expressed in the absence of E1 or for a chimeric protein containing the ectodomain of CD4 in fusion with the transmembrane domain (TMD) of E2. Although they do not exclude the presence of an intracellular localization signal in E1, these data do suggest that the TMD of E2 is an ER retention signal for HCV glycoprotein complex. In this study chimeric proteins containing the ectodomain of CD4 or CD8 fused to the C-terminal hydrophobic sequence of E1 were shown to be localized in the ER, indicating that the TMD of E1 is also a signal for ER localization. In addition, these chimeric proteins were not processed by Golgi enzymes, indicating that the TMD of E1 is responsible for true retention in the ER, without recycling through the Golgi apparatus. Together, these data suggest that at least two signals (TMDs of E1 and E2) are involved in ER retention of the HCV glycoprotein complex.

Amino Acid Sequence↗
PubMed1999

Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81.

A truncated soluble form of the hepatitis C virus E2 glycoprotein, E2661, binds specifically to the surface of cells expressing human CD81 (hCD81) but not other members of the tetraspanin family (CD9, CD63, and CD151). No differences were noted between the level of E2661 binding to hCD81 expressed on the surface of rat RBL or KM3 cells compared to Daudi and Molt-4 cells, suggesting that additional human-cell-specific factors are not required for the primary interaction of E2 with the cell surface. E2 did not interact with African green monkey (AGM) CD81 on the surface of COS cells, which differs from the hCD81 sequence at four residues within the second extracellular region (EC2) (amino acids [aa] 163, 186, 188, and 196), suggesting that one or more of these residues defines the site of interaction with E2. Various recombinant forms of CD81 EC2 show differences in the ability to bind E2, suggesting that CD81 conformation is important for E2 recognition. Regions of E2 involved in the CD81 interaction were analyzed, and our data suggest that the binding site is of a conformational nature involving aa 480 to 493 and 544 to 551 within the E2 glycoprotein. Finally, we demonstrate that ligation of CD81 by E2661 induced aggregation of lymphoid cells and inhibited B-cell proliferation, demonstrating that E2 interaction with CD81 can modulate cell function.

Animals↗
PubMed1998

Hepatitis C virus glycoprotein complex localization in the endoplasmic reticulum involves a determinant for retention and not retrieval.

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2). These glycoproteins interact to form a noncovalent heterodimeric complex which in the cell accumulates in endoplasmic reticulum (ER)-like structures. The transmembrane domain of E2, at least, is involved in HCV glycoprotein complex localization in this compartment. In principle, ER localization of a protein can be the consequence of actual retention in this organelle or of retrieval from the Golgi. To determine which of these two mechanisms is responsible for HCV glycoprotein complex accumulation in the ER, the precise localization of these proteins was studied by immunofluorescence, and the processing of their glycans was analyzed. Immunolocalization of HCV glycoproteins after nocodazole treatment suggested an ER retention. In addition, HCV glycoprotein glycans were not modified by Golgi enzymes, indicating that the ER localization of these proteins is not because of their retrieval from the cis Golgi. Retention of HCV glycoprotein complexes in the ER without retrieval suggests that this compartment plays an important role for the acquisition of the envelope of HCV particles. A true retention in the ER was also observed for E2 expressed in the absence of E1 or for a chimeric protein containing the ectodomain of CD4 in fusion with the transmembrane domain of E2. These data indicate that, in HCV glycoprotein complex, the transmembrane domain of E2, at least, is responsible for true retention in the ER, without recycling through the Golgi.

Animals↗
PubMed1998

Characterization of human monoclonal antibodies specific to the hepatitis C virus glycoprotein E2 with in vitro binding neutralization properties.

Both linear and conformational determinants of hepatitis C virus (HCV) are believed to be involved in viral neutralization. After immortalization of B cells from HCV chronically infected patients with Epstein-Barr virus, we obtained two polyclonal lymphoblastoid cell lines (LCL) secreting human monoclonal antibodies (HMabs). One clone was derived from a patient infected with a genotype 4 isolate while the second was isolated from a genotype 1b-infected patient. Immunoprecipitation studies, Western blot, and immunofluorescence analysis, peptide scanning, and ELISA studies indicated that the HMabs (1) recognized conformation-dependent determinant(s), (2) were capable of recognizing genotype 1a and 1b derived antigens, and (3) were able to precipitate noncovalently associated E1E2 complexes believed to exist on the surface of virion particles. The HMab derived from the genotype 4-infected patient was in addition shown to neutralize the in vitro binding of recombinant E2 protein onto susceptible cells suggesting a potential for in vivo neutralization. These data indicate that anti-E2 antibodies directed at conserved conformational-dependent determinant(s) exist in chronic HCV infection.

Antibodies, Monoclonal↗