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1.
J Gen Virol ; 89(Pt 1): 341-347, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18089759

ABSTRACT

It has been shown previously that ovine prion protein (PrP(C)) renders rabbit epithelial RK13 cells permissive to the multiplication of ovine prions, thus providing evidence that species barriers can be crossed in cultured cells through the expression of a relevant PrP(C). The present study significantly extended this observation by showing that mouse and bank vole prions can be propagated in RK13 cells that express the corresponding PrP(C). Importantly, the respective molecular patterns of abnormal PrP (PrP(res)) and, where examined, the neuropathological features of the infecting strains appeared to be maintained during the propagation in cell culture. These findings indicate that RK13 cells can be genetically engineered to replicate prion strains faithfully from different species. Such an approach may facilitate investigations of the molecular basis of strain identity and prion diversity.


Subject(s)
Prions/pathogenicity , Animals , Arvicolinae , Cell Line , Kidney/pathology , Mice , Prions/genetics , Prions/isolation & purification , Rabbits
2.
Biochem Biophys Res Commun ; 305(3): 548-51, 2003 Jun 06.
Article in English | MEDLINE | ID: mdl-12763028

ABSTRACT

Prion diseases are fatal neurodegenerative disorders associated with an abnormal isoform of the PrPc host-encoded protein. Invalidation of the Prnp gene, that encodes PrPc, led to transgenic mice that are viable, apparently healthy, and resistant to challenge by the infectious agent. These results indicated that a down-regulation of the Prnp gene expression is a potential therapeutic approach. In the present report, we demonstrate that RNAi targeted towards the Prnp mRNA can efficiently and highly specifically reduce the level of PrPc in transfected cells. It, thus, indicates that RNAi is an attractive therapeutic approach to fight against prion diseases.


Subject(s)
PrPC Proteins/genetics , RNA Interference , Animals , Base Sequence , Cell Line , Down-Regulation , PrPC Proteins/metabolism , Promoter Regions, Genetic , RNA, Messenger/metabolism , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , Rabbits , Transfection
3.
J Neurochem ; 83(5): 1208-14, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12437592

ABSTRACT

We examined the influence of cellular prion protein (PrPc) in the control of cell death in stably transfected HEK293 cell line and in the PrPc-inducible Rov9 cells. PrPc expression in stably transfected HEK293 human cells did not modify basal apoptotic tonus but drastically potentiated staurosporine-stimulated cellular toxicity and DNA fragmentation as well as caspase 3-like activity and immunoreactivity. An identical staurosporine-induced caspase 3 activation was observed after doxycycline in the PrPc-inducible Rov9 cell line. Interestingly, proteasome inhibitors increase PrPc-like immunoreactivity and unmasked a basal caspase 3 activation. Conversely, we show that anti-PrPc antibodies sequestrate PrPc at the cell surface and drastically lower PrPc-dependent caspase activation. We suggest that intracellular PrPc could sensitize human cells to pro-apoptotic phenotype and that blockade of PrPc internalization could be a track to prevent intracellular toxicity associated with PrPc overexpression.


Subject(s)
Antibodies/pharmacology , Caspases/metabolism , Enzyme Inhibitors/pharmacology , Multienzyme Complexes/antagonists & inhibitors , PrPC Proteins/biosynthesis , Animals , Apoptosis/drug effects , Caspase 3 , Cell Line , Cell Membrane/metabolism , Cysteine Endopeptidases , DNA Fragmentation , Doxycycline/pharmacology , Enzyme Activation/drug effects , Gene Expression Regulation/drug effects , Humans , Kidney/cytology , Kidney/metabolism , Mice , PrPC Proteins/antagonists & inhibitors , PrPC Proteins/genetics , Proteasome Endopeptidase Complex , Sheep , Staurosporine/pharmacology , Transfection
4.
J Virol ; 75(13): 5977-84, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11390599

ABSTRACT

The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrP(VRQ)-encoding transgenes under an endogenous PrP-deficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrP(VRQ) provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie.


Subject(s)
Disease Models, Animal , PrPSc Proteins/toxicity , Scrapie/etiology , Animals , Disease Susceptibility , Mice , Mice, Transgenic , PrPSc Proteins/genetics , Scrapie/pathology , Sheep , Transgenes
5.
Proc Natl Acad Sci U S A ; 98(7): 4055-9, 2001 Mar 27.
Article in English | MEDLINE | ID: mdl-11259656

ABSTRACT

Transmissible spongiform encephalopathies, or prion diseases, are fatal degenerative disorders of the central nervous system that affect humans and animals. Prions are nonconventional infectious agents whose replication depends on the host prion protein (PrP). Transmission of prions to cultured cells has proved to be a particularly difficult task, and with a few exceptions, their experimental propagation relies on inoculation to laboratory animals. Here, we report on the development of a permanent cell line supporting propagation of natural sheep scrapie. This model was obtained by stable expression of a tetracycline-regulatable ovine PrP gene in a rabbit epithelial cell line. After exposure to scrapie agent, cultures were repeatedly found to accumulate high levels of abnormal PrP (PrPres). Cell extracts induced a scrapie-like disease in transgenic mice overexpressing ovine PrP. These cultures remained healthy and stably infected upon subpassaging. Such data show that (i) cultivated cells from a nonneuronal origin can efficiently replicate prions; and (ii) species barrier can be crossed ex vivo through the expression of a relevant PrP gene. This approach led to the ex vivo propagation of a natural transmissible spongiform encephalopathy agent (i.e., without previous experimental adaptation to rodents) and might be applied to human or bovine prions.


Subject(s)
Epithelial Cells/metabolism , PrPSc Proteins/biosynthesis , Prion Diseases/veterinary , Prions/biosynthesis , Sheep Diseases/metabolism , Animals , Cells, Cultured , Genetic Variation , Mice , Prion Diseases/metabolism , Prions/genetics , Rabbits , Sheep
6.
In Vitro Cell Dev Biol Anim ; 36(1): 45-9, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10691040

ABSTRACT

Primary cultures of the brain from sheep embryos were used to establish cell lines after transfection by the simian virus 40 (SV40) large T gene. Two of the lines (A15 and 4A6) displayed astroglial properties. They expressed the glial fibrillary acidic protein (GFAP), intermediate filament protein vimentin, and S-100 (beta-subunit) protein. While numerous rodent and human glial cell lines are available, this is to our knowledge the first description of ovine cell lines with astrocyte features. In addition, these cell lines were derived from sheep embryos chosen for their genetic susceptibility to scrapie (PrP genotype: VV136, QQ171). Therefore, they could be attractive tissue culture models for the study of propagation and pathogenesis of the scrapie agent ex vivo.


Subject(s)
Astrocytes/cytology , Genetic Predisposition to Disease , Scrapie/genetics , Animals , Cell Line , Sheep/embryology
7.
J Virol ; 74(1): 320-5, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10590120

ABSTRACT

Propagation of the agents responsible for transmissible spongiform encephalopathies (TSEs) in cultured cells has been achieved for only a few cell lines. To establish efficient and versatile models for transmission, we developed neuroblastoma cell lines overexpressing type A mouse prion protein, MoPrP(C)-A, and then tested the susceptibility of the cells to several different mouse-adapted scrapie strains. The transfected cell clones expressed up to sixfold-higher levels of PrP(C) than the untransfected cells. Even after 30 passages, we were able to detect an abnormal proteinase K-resistant form of prion protein, PrP(Sc), in the agent-inoculated PrP-overexpressing cells, while no PrP(Sc) was detectable in the untransfected cells after 3 passages. Production of PrP(Sc) in these cells was also higher and more stable than that seen in scrapie-infected neuroblastoma cells (ScN2a). The transfected cells were susceptible to PrP(Sc)-A strains Chandler, 139A, and 22L but not to PrP(Sc)-B strains 87V and 22A. We further demonstrate the successful transmission of PrP(Sc) from infected cells to other uninfected cells. Our results corroborate the hypothesis that the successful transmission of agents ex vivo depends on both expression levels of host PrP(C) and the sequence of PrP(Sc). This new ex vivo transmission model will facilitate research into the mechanism of host-agent interactions, such as the species barrier and strain diversity, and provides a basis for the development of highly susceptible cell lines that could be used in diagnostic and therapeutic approaches to the TSEs.


Subject(s)
Neuroblastoma/virology , PrPSc Proteins/pathogenicity , Prions/genetics , Scrapie/transmission , Animals , Mice , PrPSc Proteins/genetics , Species Specificity , Tumor Cells, Cultured
8.
Mamm Genome ; 10(6): 585-7, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10341090

ABSTRACT

A sheep BAC library of over three genome equivalents was constructed and arrayed in superpools and row, column, and plate pools. The library contains 90,000 clones distributed in 39 superpools. The average insert size was estimated at 123 kb. The library was screened by PCR with 77 primer pairs corresponding to ovine microsatellites distributed throughout the genome. The probability of finding a random sequence in the library could be estimated at 0.96.


Subject(s)
Chromosomes, Bacterial , Gene Library , Sheep/genetics , Animals , Deoxyribonuclease HindIII/genetics , Genome , In Situ Hybridization, Fluorescence , Male , Microsatellite Repeats , Prions/genetics , X Chromosome
9.
Mol Microbiol ; 24(6): 1225-34, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9218771

ABSTRACT

The mechanism of recombination of tandem repeats in the chromosome of Escherichia coli was investigated by genetic means. Tandem repeats 624 bp long were introduced into the lacZ gene of E. coli and the efficiency of deletion of one repeat was compared in different recombination mutants. No effects of the recA, recBC, recF, ruvA or ruvA recG mutations were detected. Hence, tandem repeat deletion appears to not proceed via the RecBCD or RecF homologous recombination pathways. A new mutant in which RecA-independent recombination is increased 15-fold was isolated. The mutation lies in the dnaE gene coding for the alpha subunit of polymerase III: it is a Gly to Asp change at codon 133. Another dnaE mutation, dnaE486, was tested and also shown to stimulate RecA-independent recombination. It is proposed that tandem-repeat recombination occurs by a replication slippage mechanism. RecA-independent recombination is also enhanced in a rep mutant, in which chromosomal replication is slowed down by the absence of the Rep helicase, suggesting that replication pausing may facilitate slippage.


Subject(s)
DNA Polymerase III/genetics , Escherichia coli/genetics , Rec A Recombinases/metabolism , Recombination, Genetic , Chromosomes, Bacterial , DNA Replication , Escherichia coli/metabolism , Mutation , Rec A Recombinases/genetics , Repetitive Sequences, Nucleic Acid , Sequence Analysis, DNA , Sequence Deletion
10.
Mol Gen Genet ; 252(4): 398-403, 1996 Sep 25.
Article in English | MEDLINE | ID: mdl-8879240

ABSTRACT

We have previously shown that concurrent progression of pBR322 replication and pTac-directed transcription in opposite orientations induces illegitimate recombination events. We tested here the effects of M13 rolling circle replication on the incidence of plasmid deletions. The progression of the M13 replication fork leads to an increase of more than 300-fold in the frequency of transcription-dependent deletion events. pBR322 derivatives carrying the M13 replication origin and a 511 bp transcribed region under the control of the pTac promoter were used. Up to 12% of the plasmid population has sustained deletions within 4 h following the induction of pTac-directed transcription and M13 DNA replication, provided that the two proceed in opposite orientations. We observed that induction of transcription of the whole Escherichia coli lacZ gene (3244 bp) in the direction opposite to M13 replication leads to a fivefold decrease in plasmid copy number within 2 h, which is consistent with the proposal that deletions arise because replication fork progression is impeded. This decrease in parental plasmid copy number leads in turn to an enrichment in deleted plasmid forms. Our data confirm and extend the notion that simultaneous transcription and replication in opposite directions can efficiently promote deletion formation. In addition, this instability may be amplified when the rearranged molecules acquire a replicative advantage.


Subject(s)
Bacteriophage M13/genetics , DNA Helicases , DNA Replication , Plasmids/genetics , Sequence Deletion , Transcription, Genetic , Adenosine Triphosphatases/genetics , Escherichia coli/genetics , Lac Operon/genetics , Mutation , Nuclear Proteins/genetics , Replication Origin , Transformation, Genetic
11.
Mol Microbiol ; 17(3): 493-504, 1995 Aug.
Article in English | MEDLINE | ID: mdl-8559068

ABSTRACT

Characterization of functions that render DNA susceptible to rearrangement is important for a better understanding of genome instability. In a previous work, we showed that sequences located downstream of a strong promoter are particularly prone to deletion. In this paper, the parameters that influence transcription-induced deletions were studied. pBR322 derivatives carrying the M13 (+) replication origin and a PTac-dependent transcription region were used. Deletion formation was analysed in the presence of the replication protein of M13, which introduces a nick at the phage replication origin, and in a rep- strain to avoid M13-driven replication. Our study showed that: (i) 4 h after induction of transcription, a few per cent of the plasmids have experienced a deletion; (ii) these deletions result in joining of the M13 replication origin to a nucleotide located in or downstream of the transcribed region; (iii) deletion formation strongly depends on the orientation of transcription, on promoter strength and transcript length, but is independent of translation; (iv) formation of transcription-induced supercoiling domains does not induce deletion formation. We propose that deletions in the transcribed region result from collisions between converging replication and transcription machineries.


Subject(s)
Escherichia coli/genetics , Plasmids/genetics , Sequence Deletion , Base Sequence , DNA Replication/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Superhelical/chemistry , DNA, Superhelical/genetics , Lac Operon , Molecular Sequence Data , Plasmids/chemistry , Transcription, Genetic
12.
Gene ; 135(1-2): 161-6, 1993 Dec 15.
Article in English | MEDLINE | ID: mdl-8276254

ABSTRACT

Illegitimate recombination, which is one of the major causes of genome rearrangements, can occur in a number of ways. These might involve enzymes which cut and join DNA or enzymes which replicate DNA, as illustrated by two examples: (i) formation of deletions at the replication origin (ori) of an Escherichia coli bacteriophage, M13; and (ii) excision of E. coli transposon Tn10. It is proposed that a common theme to various ways by which illegitimate recombination can occur might be the capacity to create ends in the DNA molecule and to make the ends meet.


Subject(s)
Recombination, Genetic , Bacteriophage M13/genetics , DNA Transposable Elements , Escherichia coli/genetics
13.
Thromb Haemost ; 69(5): 485-9, 1993 May 03.
Article in English | MEDLINE | ID: mdl-7686694

ABSTRACT

The human platelet antigen (HPA) 3 system is expressed on GPIIb, one subunit of GPIIb-IIIa, the platelet fibrinogen receptor. It was recently shown that HPA-3 was associated with an Ile843/Ser polymorphism. To investigate further HPA-3 determinant structure, we localized an HPA-3a determinant, recognized by the alloantiserum Lek(a), within the last 29 amino acids of GPIIb alpha. This region encompasses the polymorphic Ile843, which, as expected, is substituted into Ser in Lek(a)-negative individuals, as shown by DNA sequence after polymerase chain reaction on platelet RNA. In addition, contribution of glycosylation to the determinant structure was demonstrated since the Lek(a) antigenicity was strongly decreased after specifically removing non-terminal O-linked sugars, but not terminal sialic acids. We have thus refined the localization of an HPA-3a determinant within the last 29 amino acids, including Ile843, of GPIIb heavy chain, and shown that the Lek(a) HPA-3a determinant is dependent, in part, upon the serine-linked carbohydrates adjacent to Ile/Ser843.


Subject(s)
Antigens, Human Platelet/immunology , Carbohydrates/immunology , Epitopes/immunology , Isoantibodies/immunology , Platelet Membrane Glycoproteins/immunology , Antibodies, Monoclonal/immunology , Antigens, Human Platelet/genetics , Base Sequence , Glycosylation , Humans , Molecular Sequence Data , Platelet Membrane Glycoproteins/genetics , Polymerase Chain Reaction , Protein Processing, Post-Translational , RNA, Messenger/genetics
14.
EMBO J ; 11(10): 3629-34, 1992 Oct.
Article in English | MEDLINE | ID: mdl-1396563

ABSTRACT

Chimeric plasmids containing phage M13 and plasmid pBR322 sequences undergo deletions in Escherichia coli with a high frequency. In all plasmids one deletion endpoint is the M13 replication origin nick site. We examined the effects of transcription on the position of the other deletion end-point, by inserting in the plasmids an inducible promoter followed by a transcription terminator. Transcription dramatically affected deletions in an orientation-dependent way, such that greater than 95% of end-points were localized downstream from the inserted promoter when it faced the major plasmid transcripts. The end-points were not constrained to the transcribed region and were not affected by the orientation of pBR322 DNA replication. We propose that deletion events occur preferentially in a plasmid domain which is rendered positively supercoiled by convergent transcription. We also show that interaction of LacI repressor with the cognate operator generates a localized deletion hot spot. This hot spot is dependent on pBR322 replication, and therefore probably acts by arresting progression of DNA replication.


Subject(s)
Bacteriophage M13/genetics , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA, Viral/genetics , DNA, Viral/metabolism , Escherichia coli/genetics , Gene Deletion , Plasmids , Repressor Proteins/metabolism , Transcription, Genetic , Base Sequence , DNA Replication , Escherichia coli/metabolism , Molecular Sequence Data , Restriction Mapping , Sequence Homology, Nucleic Acid
15.
Thromb Res ; 62(3): 127-35, 1991 May 01.
Article in English | MEDLINE | ID: mdl-1716379

ABSTRACT

We have adapted the acid-guanidinium-phenol-chloroform extraction procedure of Chomczinsky and Sacchi to achieve efficient rapid recovery of total RNA from human platelets. Sufficient platelet RNA (20 micrograms of total RNA per 30 ml of whole blood) can be recovered from relatively small individual samples to perform Northern blot analysis on individual donors and detect the mRNAs for glycoproteins IIb1(GP IIb) and IIIa1(GP IIIa), 3.4 kb and 6.2 kb, respectively. Platelet GP IIb and GP IIIa mRNAs could also be reverse transcribed, and amplified in vitro by the polymerase chain reaction (PCR). Thus, our technique allows simultaneous Northern blotting and PCR, and therefore should be of great help to the characterization of inherited platelet disorders such as Glanzmann's thrombasthenia.


Subject(s)
Blood Platelets/physiology , Platelet Membrane Glycoproteins/genetics , RNA, Messenger/blood , RNA/blood , Blotting, Northern , DNA Probes , Electrophoresis, Agar Gel , Humans , Lymphocytes/physiology , Polymerase Chain Reaction/methods , RNA/genetics , RNA/isolation & purification , RNA, Messenger/genetics , RNA, Messenger/isolation & purification
16.
Exp Cell Res ; 188(2): 219-25, 1990 Jun.
Article in English | MEDLINE | ID: mdl-2335188

ABSTRACT

The control of endothelial cell proliferation is important in a variety of processes including wound healing and tumor-induced angiogenesis. We have observed that normal unstimulated human monocytes isolated from the blood can inhibit human endothelial cell proliferation. Monocyte-conditioned medium was fractionated by gel filtration chromatography, yielding a 175-fold enrichment of a growth inhibitory activity, designated monocyte-derived endothelial cell inhibitory factor (MECIF). MECIF was found to be protease sensitive, resistant to acid treatment, and heat labile. When conditioned medium was subjected to HPLC gel filtration, the inhibitory activity was eluted as a single peak with a molecular weight of 50-70 kDa. Several characteristics distinguish MECIF from previously described monocyte/macrophage-derived inhibitory factors. Unlike TGF-beta, MECIF is heat labile and does not induce a mitogenic response in growth-arrested normal rat kidney cells. In addition, polyclonal antibodies specific for TGF-beta or INF-gamma do not inhibit MECIF activity. MECIF preparations show low levels of TNF-alpha, insufficient to promote the observed growth inhibitory effect. MECIF activity on human endothelial cells was found to be dose dependent and reversible. MECIF also appeared to be target cell selective in that it did not significantly alter the growth of human smooth muscle cells or skin fibroblasts. These data suggest that monocyte-derived factors may play a key role in inhibiting endothelial cell proliferation.


Subject(s)
Endothelium, Vascular/cytology , Growth Inhibitors/biosynthesis , Monocytes/metabolism , Cell Division/drug effects , Cells, Cultured , Culture Media , Fibroblasts/cytology , Growth Inhibitors/pharmacology , Hot Temperature , Humans , Molecular Weight , Muscle, Smooth, Vascular/cytology , Transforming Growth Factors/pharmacology , Tumor Necrosis Factor-alpha/pharmacology
17.
Biorheology ; 27(3-4): 425-32, 1990.
Article in English | MEDLINE | ID: mdl-2261508

ABSTRACT

The adhesion of leukocytes to endothelium is a physiological phenomenon which is the first step for leukocyte emigration. The adhesion can be dramatically increased in pathological situations such as inflammation and vascular diseases. The molecular basis of leukocyte-endothelium interaction has been largely investigated in the last ten years. Using monoclonal antibodies it is possible to characterize the leukocyte adhesion molecule (LeuCAM) also named CD11/CD18 complex. These molecules responsible for leukocyte adhesion are heterodimers consisting of a common beta subunit and different subunit CD11a/CD18 corresponding to LFA-1; CD11b/CD18 to Mac1/Mol; CD11c/CD18 to GP150-95. Beside these receptors, other leukocyte structures such as the fibronectin receptors are involved in the adhesive process. On the endothelial cell side specialized structures implicated in leukocyte adhesion have been identified. Structures like Intercellular Adhesion Molecule (ICAM) are expressed on endothelial cells in the absence of stimulation, while other receptors Endothelial Leukocyte Adhesion Molecule (ELAM) are only detectable on activated endothelial cells. Cytokines such as IL-1 induced the expression of ELAM, increased the number of ICAM and Human Leukocyte Antigens (HLA) DR, DP, DQ. In various pathological circumstances, namely extracorporeal circulation, Acute Respiratory Distress Syndrome (ARDS), hypercholesterolemia and diabetes mellitus increased leukocyte adhesion has been reported and is potentially responsible for vascular damage. Therefore, the modulation of leukocyte-endothelial cell interactions is a possible target for antithrombotic and antiatherosclerotic therapy.


Subject(s)
Leukocytes/physiology , Cell Adhesion/physiology , Endothelium/cytology , Endothelium/physiology , Humans , Receptors, Leukocyte-Adhesion/physiology
18.
Int J Cancer ; 43(2): 327-33, 1989 Feb 15.
Article in English | MEDLINE | ID: mdl-2563721

ABSTRACT

Lymphoid cell lines derived from the peripheral blood of French West Indian patients with HTLV-I sero-positive Tropical Spastic Paraparesis and HTLV-I isolates were characterized. While patients' peripheral blood lymphocytes did not express detectable HTLV-I antigens when uncultured, they did so after short-term culture. Established cell lines were of T-cell lineage: CD2+, CD3+, CD4+, CD7+, WT31+ with activated T-cell markers CD25+, DR+ and a clonal rearrangement of the beta and gamma genes of the T-cell receptor. HTLV-I antigens were detected in cell lines by indirect immunofluorescence, Western blot and radio-immunoprecipitation assays. After 4 months in culture, low levels of Mg2+ dependent reverse transcriptase activity were detected and electron microscopy revealed numerous type-C retroviral particles similar to HTLV-I virions. Western blot and radio-immunoprecipitation analysis of purified viruses revealed gp46, p24, p19 and Pr53gag proteins similar to those detected in HUT 102 and MT2 cell lines. Deep analysis of env-coded precursor of one TSP versus ATL isolates revealed minor differences in their molecular weights. Southern blot analysis using 32P HTLV-I env gene as a probe showed the presence of HTLV-I proviral fragments clonally integrated into the genome of the cell lines. Our data suggest that HTLV-I isolated from Tropical Spastic Paraparesis does not differ significantly from the leukemogenic prototypes. Does HTLV-I induce either acute lymphoproliferative diseases or chronic neuromyelopathies depending upon as yet unknown co-factors? This question remains to be determined.


Subject(s)
Deltaretrovirus Antigens/analysis , Human T-lymphotropic virus 1/isolation & purification , Paraparesis, Tropical Spastic/microbiology , T-Lymphocytes/microbiology , Blotting, Southern , Cell Line , Female , Fluorescent Antibody Technique , France , Human T-lymphotropic virus 1/immunology , Humans , Male , Martinique/ethnology , Microscopy, Electron , Middle Aged , Paraparesis, Tropical Spastic/pathology , Phenotype , Viral Proteins/analysis
19.
Biol Cell ; 65(3): 215-9, 1989.
Article in English | MEDLINE | ID: mdl-2665885

ABSTRACT

Ras proto-oncogenes are thought to be involved in both proliferation and malignant processes. We examined some growth properties of a murine embryonal carcinoma cell line (ECC), PCC4, that have been shown previously to be amplified for the c-KI-ras gene. Our results show that doubling time and plating efficiency are not significantly affected by such amplification. To examine the possible link between malignant behavior and c-Ki-ras alteration, we investigated activating mutations in this PCC4 cell line as well as in other ECC. Analysis of the in vitro amplified Ki-ras gene by PCR technology has not revealed point mutations in any of the ECC examined.


Subject(s)
Carcinoma , Gene Amplification , Genes, ras , Mutation , Proto-Oncogenes , Tumor Cells, Cultured/cytology , Animals , Cell Division , Cell Line , Mice
20.
C R Acad Sci III ; 307(2): 53-8, 1988.
Article in French | MEDLINE | ID: mdl-2843260

ABSTRACT

HTLV-I, the causative agent of Adult T cell Leukemia, has recently been found to be associated with chronic neuromyelopathies common in tropical areas and in Japan. We report here the establishment of a lymphoid T cell line from peripheral blood lymphocytes of a patient with Tropical Spastic Paraparesis. This cell line produces a retrovirus whose morphologic, antigenic and genetic characteristics show no detectable difference from the leukemogenic prototype of HTLV-I.


Subject(s)
Deltaretrovirus/growth & development , Paralysis/blood , T-Lymphocytes/microbiology , Antigens, Viral, Tumor/analysis , Cell Line , DNA, Viral/analysis , Deltaretrovirus/genetics , Deltaretrovirus/immunology , Female , Humans , Microscopy, Electron , Muscle Spasticity/blood , T-Lymphocytes/pathology , T-Lymphocytes/ultrastructure
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