Diagnostic par PCR des rétroviroses félines

Le virus FELV (Feline Leukemia Virus) et FIV (Feline Immunodediciency Virus) constituent, de part leurs conséquences cliniques, des pathogènes majeurs du chat. En l'absence de traitement et de vaccin parfaitement efficaces, il est impératif de dépister les animaux infectés afin d'assurer une prohylaxie sanitaire des rétroviroses félines. Après une présentation générale des virus, l'auteur expose les différentes méthodes disponibles pour leur mise en évidence chez l'animal, en soulignant leurs avantages et inconvénients . La PCR (Polymerase Chain Reaction) apparaît comme une technique de choix, permettant de dépister les deux rétrovirus à des stades cliniques où les tests utilisant la recherche d'anticorps ou d'antigènes échouent. L'auteur présente ensuite la mise au point de tests de diagnostic utilisant le principe de la PCR. Deux tests sensibles et spécifiques, l'un permettant le dépistage du FIV, l'autre du FELV, sont élaborés. En ayant recours à la PCR multiplex, l'auteur envisage alors de réaliser un test permettant le dépistage simultané des deux virus. Malgré plusieurs tentatives d'optimisation de ce test, il s'avère moins sensible que les tests qui dépistent séparément les deux virus. Cependant, les diagnostics réalisés avec les deux types de tests sur plusieurs prélèvements de sang de chats donnent les mêmes résultats. Le test multiplex apparaît donc assez sensible pour être utilisé en routine pour le diagnostic

Transcriptome analysis of Sinorhizobium meliloti during symbiosis

BACKGROUND: Rhizobia induce the formation on specific legumes of new organs, the root nodules, as a result of an elaborated developmental program involving the two partners. In order to contribute to a more global view of the genetics underlying this plant-microbe symbiosis, we have mined the recently determined Sinorhizobium meliloti genome sequence for genes potentially relevant to symbiosis. We describe here the construction and use of dedicated nylon macroarrays to study simultaneously the expression of 200 of these genes in a variety of environmental conditions, pertinent to symbiosis. RESULTS: The expression of 214 S. meliloti genes was monitored under ten environmental conditions, including free-living aerobic and microaerobic conditions, addition of the plant symbiotic elicitor luteolin, and a variety of symbiotic conditions. Five new genes induced by luteolin have been identified as well as nine new genes induced in mature nitrogen-fixing bacteroids. A bacterial and a plant symbiotic mutant affected in nodule development have been found of particular interest to decipher gene expression at the intermediate stage of the symbiotic interaction. S. meliloti gene expression in the cultivated legume Medicago sativa (alfalfa) and the model plant M. truncatula were compared and a small number of differences was found. CONCLUSIONS: In addition to exploring conditions for a genome-wide transcriptome analysis of the model rhizobium S. meliloti, the present work has highlighted the differential expression of several classes of genes during symbiosis. These genes are related to invasion, oxidative stress protection, iron mobilization, and signaling, thus emphasizing possible common mechanisms between symbiosis and pathogenesis

A MANBA mutation resulting in residual beta-mannosidase activity associated with severe leukoencephalopathy: a possible pseudodeficiency variant

<p>Abstract</p> <p>Background</p> <p>β-Mannosidosis (OMIM 248510) is a rare inborn lysosomal storage disorder caused by the deficient activity of β-mannosidase, an enzyme encoded by a single gene (<it>MANBA</it>) located on chromosome 4q22-25. To date, only 20 cases of this autosomal recessive disorder have been described and 14 different <it>MANBA </it>mutations were incriminated in the disease. These are all null mutations or missense mutations that abolish β-mannosidase activity. In this study, we characterized the molecular defect of a new case of β-mannosidosis, presenting with a severe neurological disorder.</p> <p>Methods</p> <p>Genomic DNA was isolated from peripheral blood leukocytes of the patient to allow <it>MANBA </it>sequencing. The identified mutation was engineered by site-directed mutagenesis and the mutant protein was expressed through transient transfection in HEK293T cells. The β-mannosidase expression and activity were respectively assessed by Western blot and fluorometric assay in both leukocytes and HEK293T cells.</p> <p>Results</p> <p>A missense disease-associated mutation, c.1922G>A (p.Arg641His), was identified for which the patient was homozygous. In contrast to previously described missense mutations, this substitution does not totally abrogate the enzyme activity but led to a residual activity of about 7% in the patient's leukocytes, 11% in lymphoblasts and 14% in plasma. Expression studies in transfected cells also resulted in 7% residual activity.</p> <p>Conclusion</p> <p>Correlations between MANBA mutations, residual activity of β-mannosidase and the severity of the ensuing neurological disorder are discussed. Whether the c.1922G>A mutation is responsible for a yet undescribed pseudodeficiency of β-mannosidase is also discussed.</p

Poxivirus et tumeurs (influence des facteurs de croissance viraux sur la formation des fibromes de Shope)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Diagnostic par PCR des rétroviroses félines

TOULOUSE3-BU Santé-Centrale (315552105) / SudocTOULOUSE-EN Vétérinaire (315552301) / SudocSudocFranceF

Palmitoyl protein thioesterase 1 modulates tumor necrosis factor alpha-induced apoptosis.

International audienceInduction of apoptosis by TNF has recently been shown to implicate proteases from lysosomal origin, the cathepsins. Here, we investigated the role in apoptosis of palmitoyl protein thioesterase 1 (PPT1), another lysosomal enzyme that depalmitoylates proteins. We show that transformed fibroblasts derived from patients with the infantile form of neuronal ceroid lipofuscinosis (INCL), a neurodegenerative disease due to deficient activity of PPT1, are partially resistant to TNF-induced cell death (57-75% cell viability vs. 15-30% for control fibroblasts). TNF-initiated proteolytic cleavage of caspase-8, Bid and caspase-3, as well as cytochrome c release was strongly attenuated in INCL fibroblasts as compared to control cells. Noteworthy, activation of p42/p44 mitogen-activated protein kinase and of transcription factor NF-kappaB by TNF, and induction of cell death by staurosporine or chemotherapeutic drugs in INCL cells were unaffected by PPT1 deficiency. Resistance to TNF-induced apoptosis was also observed in embryonic fibroblasts derived from Ppt1/Cln1-deficient mice but not from mice with a targeted deletion of Cln3 or Cln5. Finally, reconstitution of PPT1 activity in mutant cells was accompanied by resensitization to TNF-induced caspase activation and toxicity. These observations emphasize for the first time the role of PPT1 and, likely, protein depalmitoylation in the regulation of TNF-induced apoptosis

Functions of sphingolipid metabolism in mammals--lessons from genetic defects.

International audienceMuch is known about the pathways that control the biosynthesis, transport and degradation of sphingolipids. During the last two decades, considerable progress has been made regarding the roles this complex group of lipids play in maintaining membrane integrity and modulating responses to numerous signals. Further novel insights have been provided by the analysis of newly discovered genetic diseases in humans as well as in animal models harboring mutations in the genes whose products control sphingolipid metabolism and action. Through the description of the phenotypic consequences of genetic defects resulting in the loss of activity of the many proteins that synthesize, transport, bind, or degrade sphingolipids, this review summarizes the (patho)physiological functions of these lipids

Beta-mannosidosis: a new cause of spinocerebellar ataxia.

International audienceBeta-mannosidosis (OMIM 248510) is an inborn lysosomal storage disorder caused by deficiency of beta-mannosidase activity. This enzyme is encoded by a single gene (MANBA), located on chromosome 4q22-25. This autosomal recessive disorder is characterized by a wide range of symptoms including mental retardation, behavioural problems, hearing loss, recurrent respiratory infections, angiokeratoma, facial dysmorphism, skeletal deformation, seizures, hypotonia, demyelinating polyneuropathy, and hepatosplenomegaly. The age of symptom onset is variable. We describe a 14-year clinical follow-up of a patient with beta-mannosidase deficiency with symptoms of mental retardation, progressive spasticity and cerebellar ataxia, a clinical spectrum that so far has never been reported in beta-mannosidosis. A novel mutation in the MANBA gene was found in our patient. Evoked potentials were in favour of a demyelinating pathology of the central nervous system. Serial MRI showed generalized cortical and subcortical atrophy in the absence of white matter changes suggesting an additional axonal pathophysiological component

Are glucosylceramide-related sphingolipids involved in the increased risk for cancer in Gaucher disease patients? Review and hypotheses

The roles of ceramide and its catabolites, i.e., sphingosine and sphingosine 1-phosphate, in the development of malignancies and the response to anticancer regimens have been extensively described. Moreover, an abundant literature points to the effects of glucosylceramide synthase, the mammalian enzyme that converts ceramide to β-glucosylceramide, in protecting tumor cells from chemotherapy. Much less is known about the contribution of β-glucosylceramide and its breakdown products in cancer progression. In this chapter, we first review published and personal clinical observations that report on the increased risk of developing cancers in patients affected with Gaucher disease, an inborn disorder characterized by defective lysosomal degradation of β-glucosylceramide. The previously described mechanistic links between lysosomal β-glucosylceramidase, β-glucosylceramide and/or β-glucosylphingosine, and various hallmarks of cancer are reviewed. We further show that melanoma tumor growth is facilitated in a Gaucher disease mouse model. Finally, the potential roles of the β-glucosylceramidase protein and its lipidic substrates and/or downstream products are discussed.</p

First Report of a Patient with MPS Type VII, Due to Novel Mutations in GUSB, Who Underwent Enzyme Replacement and Then Hematopoietic Stem Cell Transplantation

We report the case of a boy who was diagnosed with mucopolysaccharidosis (MPS) VII at two weeks of age. He harbored three missense &beta;-glucuronidase (GUSB) variations in exon 3: two novel, c.422A&gt;C and c.424C&gt;T, inherited from his mother, and the rather common c.526C&gt;T, inherited from his father. Expression of these variations in transfected HEK293T cells demonstrated that the double mutation c.422A&gt;C;424C&gt;T reduces &beta;-glucuronidase enzyme activity. Enzyme replacement therapy (ERT), using UX003 (vestronidase alfa), was started at four months of age, followed by a hematopoietic stem cell allograft transplantation (HSCT) at 13 months of age. ERT was well tolerated and attenuated visceromegaly and skin infiltration. After a severe skin and gut graft-versus-host disease, ERT was stopped six months after HSCT. The last follow-up examination (at the age of four years) revealed a normal psychomotor development, stabilized growth curve, no hepatosplenomegaly, and no other organ involvement. Intriguingly, enzyme activity had normalized in leukocytes but remained low in plasma. This case report illustrates: (i) The need for an early diagnosis of MPS, and (ii) the possible benefit of a very early enzymatic and/or cellular therapy in this rare form of lysosomal storage disease