Osmolyte trimethylamine N-oxide converts recombinant alpha-helical prion protein to its soluble beta-structured form at high temperature.

The thermal unfolding of full-length human recombinant alpha-helical prion protein (alpha-PrP) in neutral pH is reversible, whereas, in the presence of the osmolyte N-trimethylamine oxide (TMAO), the protein acquires a beta-sheet structure at higher temperatures and the thermal unfolding of the protein is irreversible. Lysozyme, an amyloidogenic protein similar to prion protein, regains alpha-helical structure on cooling from its thermally unfolded form in buffer and in TMAO solutions. The thermal stability of alpha-PrP decreases, whereas that of lysozyme increases in TMAO solution. Light-scattering and turbidity values indicate that beta-sheet prion protein exists as soluble oligomers that increase thioflavin T fluorescence and bind to 1-anilino 8-naphthalene sulfonic acid (ANS). The oligomers are resistant to proteinase K digestion and during incubation for long periods they form linear amyloids>5 microm long. The comparable fluorescence polarization of the tryptophan groups and their accessibility to acrylamide in alpha-PrP and oligomers indicate that the unstructured N-terminal segments of the protein, which contain the tryptophan groups, do not associate among themselves during oligomerization. Partial unfolding of alpha-helical prion protein in TMAO solution leads to its structural conversion to misfolded beta-sheet form. The formation of the misfolded prion protein oligomers and their polymerization to amyloids in TMAO are unusual, since the osmolyte generally induces denatured protein to fold to a native-like state and protects proteins from thermal denaturation and aggregation.

Design and evaluation of a diabody to improve protection against a potent scorpion neurotoxin.

Diabodies are recombinant, dimeric, antibody-based molecules composed of two non-covalently associated single-chain antibody fragments that bind to an antigen in a divalent manner. In an attempt to develop more effective therapeutic molecules against scorpion venoms, we designed a diabody derived from monoclonal antibody 9C2, which neutralizes the toxicity of scorpion neurotoxin AahI in mammals. The recombinant diabody produced in the periplasm of Escherichia coli was purified to homogeneity in a single step by protein L-agarose affinity chromatography. It was functional, and possessed a high binding affinity to AahI (8 x 10(-11) M). The bivalence of the diabody was confirmed by size-exclusion chromatography, isoelectrofocussing and electron microscopic observations. Finally, the diabody showed high thermal stability in serum and demonstrated protective activity when injected intraperitoneally in mice experimentally envenomed with toxin AahI. In conclusion, the diabody format gives the 9C2 molecule advantageous properties that are particularly important for potential clinical applications in the treatment of envenomations.

[Scintigraphic imaging of macrophages involved in lung vasoreflex: rat model]. / Scintigraphie des macrophages impliqués dans la vasoréactivité pulmonaire: modélisation chez le rat.

At time of pathological situations, a pulmonary fixation of labelled substances injected by intravenous way is observed. This fixation would result from a phagocytosis of these substances by abnormal cells whose presence was induced in the endothelium: Pulmonary Intravascular Macrophages (PIM's). After activation by phagocytosis, these cells are able to secrete powerful vasoactive mediators capable of inducing cardiopulmonary accidents. Hepatic cholestase was induced in Wistar rats by ligation and section of common bile duct. The recruitment of PIM's was followed in vivo by phagocytosis scintigraphic imaging after labelled colloid injection. During the 35 days of evolution of the pathology, we observe a pulmonary fixation of the colloid agents which progresses up to 70% as well as a concomitant decease in the hepatic activity. Histologic examination showed numerous cells related to pulmonary capillaries' endothelium belonging to mononuclear phagocytes line and expressing an activated phenotype of monocytes. The scintigraphic and histological tests carried out enabled us to validate the model of induction of PIM's in rat by ligation of the choledoque one. The study of the vasoactive response via certain mediators can from now be approached, a Doppler technique on the pig aorta is being in the course of evaluation.

Gene transfer using human papillomavirus pseudovirions varies according to virus genotype and requires cell surface heparan sulfate.

Artificial viruses consisting of DNA plasmid packaged in vitro into virus-like particles (VLPs) are new vehicles for gene transfer. We therefore investigated the ability of nine human papillomavirus (HPV) VLPs to interact with heterologous DNA and transfer genes. HPV 16, 18, 31, 33, 39, 45, 58, 59, and 68 VLPs were able to bind heterologous DNA and to transfer genes into Cos-7 cells. Inhibition of gene transfer by preincubation of the pseudovirions with heparin confirmed that heparan sulfate on the cell surface plays a role as cell receptor for HPVs. As HPV neutralizing antibodies are mainly type-specific, gene transfer with different HPV pseudovirions offers the possibility of their sequential use in vivo for a greater efficacy.

Gene therapy for cystic fibrosis with aerosolized adenovirus-CFTR: characterization of the aerosol and scintigraphic determination of lung deposition in baboons.

For cystic fibrosis (CF) gene therapy using an aerosolized adenovirus expressing the CFTR gene, optimization of the inhalation conditions is a prerequisite to obtain sufficient amount of CFTR protein expression in the target areas of the respiratory tract. For such a purpose, in vivo radioisotopic imaging of the radiolabeled virus is a unique strategy for a quantitative assessment of the actual deposition. In the present study, an adenovirus CFTR (AdCFTR) was labeled with 99m Technetium gamma emitting isotope in such conditions that its bioactivity was preserved. The 99mTc-AdCFTR aerosol was characterized using both laser diffraction and cascade impaction for sizing with further determination of nebulized and inhalable fractions. After administration to baboons, scintigraphic quantitation of the regional lung distribution was performed and the actual dose deposited in the target area was estimated and expressed as an equivalent viral titer. Since a virus scintigraphy is not realistic in a hospital setting, we have developed an approach using 99mTc-DTPA (diethylene triamino pentaacetic acid) that could be used to predict the virus deposition. Indeed, similarities observed between 99mTc-DTPA and 99mTc-adenovirus aerosol imaging patterns validates the use of the 99mTc-DTPA scintigraphy that we propose as a pretherapeutic test for each patient prior to gene transfer.

Murine recombinant prion protein induces ordered aggregation of linear nucleic acids to condensed globular structures.

Interaction between nucleic acid and recombinant murine prion protein, MoPrPC resulted in a time-dependent change in the nucleic acid morphology revealed by electron microscopy. After the addition of the protein to DNA, association of small number of nucleic acid molecules (nucleo-protein complex) was followed by aggregation of large number of them still retaining their initial linear morphology. With increase in the incubation time, ordered aggregation resulted in small condensed spherical globules. Subsequently, the formation of large condensed particles took place either by fusion of the already formed small globules or by accumulation of more nucleic acid molecules on them. The condensed nucleic acid structures observed here were different from other known morphologically altered nucleic acid structures induced by different cellular proteins. The condensed nucleic acid structures dissociated spontaneously. The formation of the prion protein-induced condensed nucleic acid structures resembled the human immunodeficiency virus 1 nucleocapsid protein NCp7-induced condensed ordered aggregates of nucleic acids. In the latter system, both the processes of condensation and dissociation of the nucleoprotein complex are believed to be responsible for the functional properties of the HIV-1 virus. Demonstration of functional activity of the prion protein-nucleic acid complex would be relevant for a role of nucleic acid in prion diseases.

Gene transfer using recombinant rabbit hemorrhagic disease virus capsids with genetically modified DNA encapsidation capacity by addition of packaging sequences from the L1 or L2 protein of human papillomavirus type 16.

The aim of this study was to produce gene transfer vectors consisting of plasmid DNA packaged into virus-like particles (VLPs) with different cell tropisms. For this purpose, we have fused the N-terminally truncated VP60 capsid protein of the rabbit hemorrhagic disease virus (RHDV) with sequences which are expected to be sufficient to confer DNA packaging and gene transfer properties to the chimeric VLPs. Each of the two putative DNA-binding sequences of major L1 and minor L2 capsid proteins of human papillomavirus type 16 (HPV-16) were fused at the N terminus of the truncated VP60 protein. The two recombinant chimeric proteins expressed in insect cells self-assembled into VLPs similar in size and appearance to authentic RHDV virions. The chimeric proteins had acquired the ability to bind DNA. The two chimeric VLPs were therefore able to package plasmid DNA. However, only the chimeric VLPs containing the DNA packaging signal of the L1 protein were able efficiently to transfer genes into Cos-7 cells at a rate similar to that observed with papillomavirus L1 VLPs. It was possible to transfect only a very limited number of RK13 rabbit cells with the chimeric RHDV capsids containing the L2-binding sequence. The chimeric RHDV capsids containing the L1-binding sequence transfer genes into rabbit and hare cells at a higher rate than do HPV-16 L1 VLPs. However, no gene transfer was observed in human cell lines. The findings of this study demonstrate that the insertion of a DNA packaging sequence into a VLP which is not able to encapsidate DNA transforms this capsid into an artificial virus that could be used as a gene transfer vector. This possibility opens the way to designing new vectors with different cell tropisms by inserting such DNA packaging sequences into the major capsid proteins of other viruses.

Identification of the glycoprotein 41(TM) cytoplasmic tail domains of human immunodeficiency virus type 1 that interact with Pr55Gag particles.

We investigated the protein/protein interactions that occur during human immunodeficiency virus (HIV-1) budding. We evaluated the binding to Pr55Gag particles of peptides mapping to the cytoplasmic tail of gp41TM and of host-cell proteins, in a cell-free, in vitro assay. Host-cell proteins and irrelevant viral envelope peptides did not bind. Peptides corresponding to a large central domain of the gp41TM cytoplasmic tail (93 residues) bound to Pr55Gag particles. This demonstrates that a Gag/Env interaction is responsible for the specific incorporation of the Env glycoprotein into nascent HIV-1 virions, and defines more accurately the gp41TM domain involved in this interaction.

The nine C-terminal amino acids of the major capsid protein of the human papillomavirus type 16 are essential for DNA binding and gene transfer capacity.

Four C-terminal deletion mutants of the human papillomavirus 16 L1 protein were expressed in the baculovirus expression system. They consist of the deletion of amino acids 497-505, 477-505, 403-505 and 302-505 (delta C9, delta C31, delta C103 and delta C204 respectively). Only two of the C-terminally deleted proteins, delta C9 and delta C31, retained the ability to form virus-like particles (VLPs) resembling those obtained with the full length L1 protein. Analysis of deleted L1 proteins and corresponding VLPs indicated that the C-terminus was necessary both for DNA binding and DNA packaging. These results were corroborated by the loss of the gene transfer capacities of C-terminal deleted VLPs.

Different role of platelet glycoprotein GP Ia/IIa in platelet contact and activation induced by type I and type III collagens.

The role of glycoprotein Ia/IIa was studied during platelet contact and aggregation induced by type I and type III collagen. The anti-glycoprotein Ia/IIa (6F1) antibody inhibited type I collagen-induced aggregation but did not inhibit the first contact between platelets and collagen. In contrast, it was without effect either on type III collagen-induced contact or platelet interaction with the subendothelium in a static assay. Platelet aggregation induced by type III collagen was only slightly slowed down by 6F1 but pp72 spleen tyrosine kinase phosphorylation was not modified even at concentrations of 6F1 that completely blocked platelet activation induced by type I collagen. Our results indicate that glycoprotein Ia/IIa is not a primary binding site for type I or type III collagen on the platelet membrane. This receptor is more specifically involved in type I collagen-induced platelet spreading and aggregation.

Nucleotide sequences of genes coding for fimbrial proteins in a cryptic genospecies of Haemophilus spp. isolated from neonatal and genital tract infections.

Nineteen isolates belonging to a cryptic genospecies of Haemophilus (referred to here as genital strains) isolated from genital tract infections (6 strains) and from neonatal infections (13 strains) were studied for fimbrial genes. Sixteen strains exhibit peritrichous fimbriae observed by electron microscopy. By PCR with primers corresponding to the extreme ends of the Haemophilus influenzae type b (Hib) hifA and hifD genes and Southern blotting, a hifA-like gene (named ghfA) and a hifD-like gene (named ghfD) were identified in 6 of the 19 strains. Five of these six strains were from the genital tracts of adults, and one was from a neonate. For each gene, the nucleotide sequence was identical for the six strains. A hifE-like gene (named ghfE) was amplified from only one of the 19 genital strains of Haemophilus, but the ghfE probe gave a signal in Southern hybridization with the five other strains positive for ghfA and ghfD. Therefore, these strains may carry a ghfE-like gene. The Hib fimbrial gene cluster is located between the purE and pepN genes as previously described. For the 13 genital Haemophilus strains that lack fimbrial genes, this region corresponds to a noncoding sequence. Another major fimbrial gene designated the fimbrin gene was previously identified in a nontypeable H. influenzae strain. A fimbrin-like gene was identified for all of our 19 genital strains. This gene is similar to the ompP5 gene of many Haemophilus strains. Therefore, other, unidentified genes may explain the piliation observed in electron microscopy on genital Haemophilus strains which do not possess LKP-like fimbrial genes. Fimbrial genes were significantly associated with strains isolated from the genital tract. They may confer on the strain the ability to survive in the genital tract.

The L1 major capsid protein of human papillomavirus type 16 variants affects yield of virus-like particles produced in an insect cell expression system.

The L1 major capsid proteins of six human papillomavirus type 16 (HPV-16) strains were expressed in insect cells by using recombinant baculoviruses. Virus-like particles (VLPs) which appeared similar to empty virions were identified by electron microscopy for all HPV strains investigated. However, the yield of VLPs produced varied in a range from 1 to 79 depending on the HPV-16 strain. The L1 proteins of these strains differed by up to 15 amino acids from the L1 protein of the prototype HPV-16 strain. Mutations in the amino acid region from residues 83 to 97 seemed to affect the level of expression of the L1 protein. These results are important when considering the development of HPV vaccines and serological tests. They indicate that strains inducing high levels of VLP production must be selected for the development of vaccines. Moreover, the L1 proteins of all strains investigated were able to bind with DNA. We also investigated the seroreactivities of VLPs derived from three different HPV-16 strains from Algeria, Senegal, and the Philippines by testing sera from women from 11 countries in immunoglobulin G-specific enzyme-linked immunosorbent assays. We observed a strong correlation between the reactivities of the three different VLP variants, independent of the geographical origin of the sera investigated. These results indicate that the three strains investigated are serologically cross-reactive despite the fact that their L1 proteins differ in 14 amino acids and suggest that VLPs derived from only one HPV-16 strain could be sufficient for the development of an HPV-16 vaccine and anti-HPV-16 tests.

Production of recombinant virus-like particles from human papillomavirus types 6 and 11, and study of serological reactivities between HPV 6, 11, 16 and 45 by ELISA: implications for papillomavirus prevention and detection.

The L1 major capsid proteins of human papillomaviruses types 6 and 11 were expressed in insect cells using recombinant baculoviruses. These L1 proteins were shown to self-assemble into virus-like particles resembling papillomavirus virions as previously observed for HPV 16 and 45. However, we observed variations in the yield of virus-like particles among the four genotypes investigated. This suggests that more than one strain of each genotype has to be investigated to obtain the high level of virus-like particle production necessary to develop HPV vaccines or serological tests. Cross-reactivities between HPV 6, 11, 16 and 45 were studied using polyclonal and monoclonal antibodies to virus-like particles, L1 proteins and synthetic peptides. Although antisera react strongly against homologous virus-like particles, there is evidence of some cross-reactivity. This could be one of the explanations for the fact that antibodies to one genotype are detected in individuals infected with another genotype. This study also identified a linear epitope recognized by anti-HPV 16 virus-like particle sera.

Particle and genomic characteristics of a new member of the Ascoviridae: Diadromus pulchellus ascovirus.

A new member of the family Ascoviridae, Diadromus pulchellus ascovirus (DpAV), has been found in the lepidopteran nymphs of Acrolepiopsis assectella parasitized by the hymenopteran wasp Diadromus pulchellus. Virions have the standard features of the ascovirus group; each particle is about 220 nm long and 150 nm wide. They are multilayered, with two clear 7-nm-thick outer layers and one 15-nm-thick inner layer surrounding an electron-dense core (155 x 110 nm). However, the flattened rice-grain shape and fragility of the DpAV particles are unlike that of known ascoviruses infecting Noctuidae species. They form large vesicles containing virions in infected cells. The DpAV genome is about 116 kb long and has a circular and relaxed structure. It contains 6-8 repeated and interspersed sequences of 494 bp. The structural and genomic features of DpAV suggest that this virus belongs to an ascovirus sub-family different from that containing the ascoviruses previously found to infect species of Noctuidae (Federici et al., 1991).

Biological and molecular features of the relationships between Diadromus pulchellus ascovirus, a parasitoid hymenopteran wasp (Diadromus pulchellus) and its lepidopteran host, Acrolepiopsis assectella.

The Diadromus pulchellus ascovirus (DpAV) has been isolated from laboratory strains of Diadromus pulchellus and in natural wild populations collected from the Antibes locality (southern France). The DpAV genome was found in the cells of the head, thorax and abdomen of this hymenopteran wasp. DpAV virions are present in the female genitalia and are transmitted to the nymphal lepidopteran host, Acrolepiopsis assectella, at each oviposition of the female wasp. The presence of the DpAV genome in all Diadromus somatic cells suggests that it is inherited by vertical transmission. DpAV is amplified in the host tissues during the larval development of D. pulchellus in A. assectella. Cell lysis due to amplification of the virus does not prevent the development of the hymenopteran larva. Virus amplification appears to be slower in nymphs parasitized by D. pulchellus than in nymphs artificially infected with DpAV alone. Lysis of the nymphal cells due to viral replication seems to be synchronous with egg hatching and the development of the hymenopteran larva. The features of DpAV and its relationship with the parasitoid wasp D. pulchellus during its development are compared to those of the ichnoviruses.

The genome segments of DpRV, a commensal reovirus of the wasp Diadromus pulchellus (Hymenoptera).

The complete nucleotide sequences of the five double-stranded RNA genome segments of the Diadromus pulchellus reovirus (DpRV) have been determined. They consist of 985, 1240, 1318, 1652, and 4230 bp. Each segment contains at least one putative open reading frame encoding 33-, 40-, 45-, 49-, and 148-kDa proteins, respectively. The proteins have no significant similarities with sequences in data banks. Analysis of these segments and of two other previously published segments revealed the presence of degenerate consensus inverted repeats at both ends (5'-rCAAUUUUnnACU...AGUAAAAAAAUnrG-3'). The biological, structural, and genomic features of DpRV suggest that this virus is related to members of the Orthoreovirus genus.

A member of the reoviridae (DpRV) has a ploidy-specific genomic segment in the wasp Diadromus pulchellus (Hymenoptera).

Wild and laboratory populations of the parasitoid wasp Diadromus pulchellus are infected with a new member of the reoviridae (DpRV) described in this paper. The particles of this virus possess two capsid shells (diameters: 35 and 70 nm) made up of 11 proteins. The virus is present mainly in the gut of the wasp, with smaller quantities in its venom gland. The genome of virus particles purified from haploid insects (functional males) contains 10 segments, whereas virus from diploid insects (females and sterile diploid males) contains a supernumerary 3.33-kb segment. The sequence of this dsRNA segment revealed that it is a triplicated 1050-bp motif which is 97.5% similar to the 5' region of one of the 10 basic segments, the 3.80-kb segment.