Lysosomes and lysosomal proteins in cancer cell death (new players of an old struggle)

Death of cancer cells influences tumor development and progression, as well as the response to anticancer therapies. This can occur through different cell death programmes which have recently been shown to implicate components of the acidic organelles, lysosomes. The role of lysosomes and lysosomal enzymes, including cathepsins and some lipid hydrolases, in programmed cell death associated with apoptotic or autophagic phenotypes is presented, as evidenced from observations on cultured cells and living animals. The possible molecular mechanisms that underlie the action of lysosomes during cell death are also described. Finally, the contribution of lysosomal proteins and lysosomes to tumor initiation and progression is discussed. Elucidation of this role and the underlying mechanisms will shed a new light on these 'old' organelles and hopefully pave the way for the development of novel anticancer strategies.     

Development of a multiplex real-time PCR for contagious agalactia diagnosis in small ruminants

Contagious agalactia is an important disease worldwide that affects small ruminants. Clinical manifestations vary from mastitis, pneumonia, arthritis and keratoconjunctivitis to septicemia. Four mycoplasmal etiological agents have been identified: Mycoplasma (M.) agalactiae, M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens. The current procedure for direct diagnosis, recommended by the World Organization for Animal Health, involves the isolation of one or several causative agents from clinical specimens and further time-consuming identification steps. The present study reports the development of a new multiplex real-time PCR (including an internal positive control) that detects all four pathogens simultaneously and distinguishes M. agalactiae from the others. First, intra- and inter-species polymorphisms of the two target house-keeping genes, namely polC and fusA, were analyzed to design primers and probes adapted to the diversity of currently circulating strains. The specificity and the sensitivity of the assay were then challenged and the limit of detection was found to be as low as 6 to 12 copies of the target genes. The assay requires further assessment on clinical specimens but its performances (notably low intra- and inter-assay variability) are already very promising for use in large-scale diagnosis and prophylactic surveys of contagious agalactia.     

Unexpected genetic diversity of Mycoplasma agalactiae caprine isolates from an endemic geographically restricted area of Spain

ABSTRACT: BACKGROUND: The genetic diversity of Mycoplasma agalactiae (MA) isolates collected in Spain from goats in an area endemic with contagious agalactia (CA) was assessed using a set of validated and new molecular typing methods. Validated methods included pulsed field gel electrophoresis (PFGE), variable number of tandem repeats (VNTR) typing, and Southern blot hybridization using a set of MA DNA probes, including those for typing the vpma genes repertoire. New approaches were based on PCR and targeted genomic regions that diverged between strains as defined by in silico genomic comparisons of sequenced MA genomes. RESULTS: Overall, the data showed that all typing tools yielded consistent results, with the VNTR analyses being the most rapid method to differentiate the MA isolates with a discriminatory ability comparable to that of PFGE and of a set of new PCR assays. All molecular typing approaches indicated that the Spanish isolates from the endemic area in Murcia were very diverse, with different clonal isolates probably restricted to separate, but geographically close, local areas. CONCLUSIONS: The important genetic diversity of MA observed in infected goats from Spain contrasts with the overall homogeneity of the genomic background encountered in MA from sheep with CA in Southern France or Italy, suggesting that assessment of the disease status in endemic areas may require different approaches in sheep and in goats. A number of congruent sub-typing tools are now available for the differentiation of caprine isolates with comparable discriminatory powers.     

Effects of trans MUFA from dairy and industrial sources on muscle mitochondrial function and insulin sensitivity

Epidemiological studies suggest that chronic consumption of trans MUFA may alter muscle insulin sensitivity. The major sources of dietary trans MUFA (dairy fat vs. industrially hydrogenated oils) have different isomeric profiles and thus probably different metabolic consequences. These effects may involve alterations in muscle mitochondrial oxidative capacity, which may in turn promote insulin resistance if fatty acid oxidation is reduced. We report that in Wistar rats, an 8 week diet enriched (4% of energy intake) in either dairy, industrial, or control MUFA did not alter insulin and glucose responses to an intraperitoneal glucose tolerance test (1g/kg). In C2C12 myotubes, vaccenic and elaidic acids did not modify insulin sensitivity compared with oleic acid. Furthermore, the ex vivo total, mitochondrial and peroxisomal oxidation rates of [1-(14)C]oleic, vaccenic, and elaidic acids were similar in soleus and tibialis anterior rat muscle. Finally, an 8 week diet enriched in either dairy or industrial trans MUFA did not alter mitochondrial oxidative capacity in these two muscles compared with control MUFA but did induce a specific reduction in soleus mitochondrial ATP and superoxide anion production (P<0.01 vs. control). In conclusion, dietary trans MUFA of dairy or industrial origin have similar effects and do not impair muscle mitochondrial capacity and insulin sensitivity.     

Immunological diagnosis of cytomegalovirus infections. Application to blood transfusion centers

The Blood Transfusion Centers (B.T.C.) are mainly concerned with the selection of CMV infection free blood donors, the screening of the anti CMV antibody high titre plasma donors and the evaluation of specific anti CMV Immunoglobulin preparations. Various serological methods could be used but they are of different value depending the purposes of the B.T.C. The neutralization test (Nt), with the addition of complement is specific and detects the protecting AB against the glycoproteins of the viral envelope. The complement fixation test (CF) using extracts of CMV infected cells as antigen largely varies in its sensitivity according to the quality of the antigen. In any case, the CF test is not sensitive enough to detect a latent CMV infection in a certain percentage of the non immunosuppressed adults, but could be used for the selection of anti CMV antibody high titres carriers. Three sensitive methods: passive haemagglutination, indirect immunofluorescence and indirect ELISA tests, might be used for the detection of latent CMV infections. They detect various AB against various internal and external components of the CMV. They are submitted to various sources of errors. The sensitivity of the indirect IF test is mainly restricted by the quality of the antigen preparation, its specificity by the presence of anti cells antibodies in the sera, the Fc receptors in the antigens and the specificity of the conjugates. The indirect ELISA which is submitted to the same causes of errors is a highly sensitive test, easy to perform, reagents are available, and automatic processors have been developed. When compared with the previous techniques, the ELISA test is suitable for the screening of CMV free donors, when it is performed with an highly sensitive antigen. It could be also used for the screening of high antibody titre carriers: its correlation with the CF test is quite good (r = 0,82). When comparatively applied to the titration of Immunoglobulins preparations, made from plasmas or placentas, for either IM or IV administration, the Elisa test gives AB titres different from those obtained with Nt and indirect IF. The calibration of a standard Immunoglobulin reagent is urgently needed and double blind clinical assays of the protective effect of various preparations of specific anti CMV Immunoglobulins have to be promptly designed.     

Ontogenic and nutritional modifications in the intestinal fucosylation process at the weaning period. Influence of dietary fibers

In the rat small intestine, the glycosylation changes which normally take place at the weaning period are characterized by a shift from sialylation of fucosylation. The introduction of dietary fibers at weaning is one of the more striking nutritional modification so that some authors have suggested that the presence of fibers and the development of colonic fermentation might be important for the development of the small intestine, as for the colon. In order to define the respective contribution of ontogenic and nutritional factors to the intestinal glycosylation changes at this period, some aspects of the intestinal glycosylation were studied in five groups of rats (16-day-old suckling rats, prolonged nursing 23-day-old rats, 23-day-old rats weaned at day 19 with either a fiber-free, a cellulose or a pectin diet). Intestinal glycoproteins of suckling rats are characterized by a low fucose content and a high proportion of mannose. The amounts of the neutral sugars (fucose, mannose and galactose), expressed either per gram of intestine or for one intestine, are alwars higher in the fiber-fed groups than in the prolonged-nursing group or the group fed the fiber-free diet. Activities which promote fucosylation process (GDP-fucose production and fucosyltransferase activities) and those which are opposed to fucosylation (endogenous inhibitor of fucosyltransferase and GDP-fucose pyrophosphatase) are strongly modified in opposite ways at day 23 as compared to day 16. These modifications depend on the age of the animal (ontogenic factors) with additional modifications induced by the dietary factors. In particular, similar sugar contents and patterns are obtained with cellulose and pectin diests though the enzymatic activities of the fucosylation pathway are very different. No correlation was found between the caecal content of short chain fatty acids and any of the parameters under study. Thus, dietary fibers induce metabolic changes in the small intestine glycosylation in short-term experiments independently of colonic fermentation. Besides, these results point out that the consideration of fucosyl-transferase activities alone are not sufficient to predict glycoprotein fucose content and that other regulatory sites are involved. Dietary manipulations at the weaning period could represent a good model for the study of glycosylation regulation.     

The flavonoid rutin induces astrocyte and microglia activation and regulates TNF-alpha and NO release in primary glial cell cultures

Astrocyte and microglia cells play an important role in the central nervous system (CNS). They react to various external aggressions by becoming reactive and releasing neurotrophic and/or neurotoxic factors. Rutin is a flavonoid found in many plants and has been shown to have some biological activities, but its direct effects on cells of the CNS have not been well studied. To investigate its potential effects on CNS glial cells, we used both astrocyte primary cultures and astrocyte/microglia mixed primary cell cultures derived from newborn rat cortical brain. The cultures were treated for 24 h with rutin (50 or 100 μmol/L) or vehicle (0.5% dimethyl sulfoxide). Mitochondrial function on glial cells was not evidenced by the MTT test. However, an increased lactate dehydrogenase activity was detected in the culture medium of both culture systems when treated with 100 μmol/L rutin, suggesting loss of cell membrane integrity. Astrocytes exposed to 50 μmol/L rutin became reactive as revealed by glial fibrillary acidic protein (GFAP) overexpression and showed a star-like phenotype revealed by Rosenfeld’s staining. The number of activated microglia expressing OX-42 increased in the presence of rutin. A significant increase of nitric oxide (NO) was observed only in mixed cultures exposed to 100 μmol/L rutin. Enhanced TNFα release was observed in astrocyte primary cultures treated with 100 μmol/L rutin and in mixed primary cultures treated with 50 and 100 μmol/L, suggesting different sensitivity of both activated cell types. These results demonstrated that rutin affects astrocytes and microglial cells in culture and has the capacity to induce NO and TNFα production in these cells. Hence, the impact of these effects on neurons in vitro and in vivo needs to be studied.     

Comparative marker analysis of the ependymocytes of the subcommissural organ in four different mammalian species

Summary The subcommissural organ (SCO), classified as one of the circumventricular organs, is composed mainly of modified ependymal cells, attributable to a glial lineage. Nevertheless, in the rat, these cells do not possess glial markers such as glial fibrillary acidic protein (GFAP), protein S100, or the enzyme glutamine synthetase (GS). They receive a synaptic 5-HT input and show pharmacological properties for uptake of GABA resembling the uptake mechanism of neurons. In this study, we examine the phenotype of several mammalian SCO (cat, mouse, rabbit) and compare them with the corresponding features of the rat SCO. In all these species, the SCO ependymocytes possess vimentin as an intermediate filament, but never express GFAP or neurofilament proteins. They do not contain GS as do glial cells involved in GABA metabolism, and when they contain protein S100 (rabbit, mouse), its rate is low in comparison to classical glial or ependymal cells. Thus, these ependymocytes display characteristics that differentiate them from other types of glial cells (astrocytes, epithelial ependymocytes and tanycytes). Striking interspecies differences in the capacity of SCO-ependymocytes for uptake of GABA might be related to their innervation and suggest a species-dependent plasticity in their function.