A Strategy for Studying Epigenetic Diversity in Natural Populations: Proof of Concept in Poplar and Oak.

These last 20 years, several techniques have been developed for quantifying DNA methylation, the most studied epigenetic marks in eukaryotes, including the gold standard method, whole-genome bisulphite sequencing (WGBS). WGBS quantifies genome-wide DNA methylation but has several inconveniences rendering it less suitable for population-scale epigenetic studies. The high cost of deep sequencing and the large amounts of data generated prompted us to seek an alternative approach. Restricting studies to parts of the genome would be a satisfactory alternative had there not been a major limitation: the need to select upstream targets corresponding to differentially methylated regions (DMRs) as targets. Given the need to study large numbers of samples, we propose a strategy for investigating DNA methylation variation in natural populations, considering the structural complexity of the genomes with their size and their content in unique as coding regions versus repeated regions as transposable elements. We first identified regions of highly variable DNA methylation in a representative subset of genotypes representative of the biological diversity in the population by WGBS. We then analysed the variations of DNA methylation in these targeted regions at the population level by Sequencing Capture Bisulphite (SeqCapBis). The entire strategy was then validated by applying it to another species. Our strategy was developed as a proof of concept on natural populations of two forest species: Populus nigra and Quercus petraea.

O-methyltransferase(s)-suppressed plants produce lower amounts of phenolic vir inducers and are less susceptible to Agrobacterium tumefaciens infection.

The first step of Agrobacterium tumefaciens/plant interaction corresponds to the activation of a transduction pathway of the bacterium by plant exudate. Phenolic compounds rapidly secreted by wounded plant cells induce the expression of bacterial virulence (vir) genes; however, little is known about their biosynthesis in plant. Here we show that inoculation of an Agrobacterium tumefaciens virulent strain on orthodiphenol-O-methyltransferases-suppressed tobacco plants leads to significantly smaller tumors compared to control plants. These transgenic plants are inhibited for caffeic acid O-methyltransferase class I or II (OMT; EC 2.1.1.6) and/or caffeoyl-coenzyme A O-methyltransferase (CCoAOMT; EC 2.1.1.104) that are involved in monolignol biosynthesis. The significant decrease of tumor size could be suppressed by the pre-activation of bacterial virulence, before inoculation, using acetosyringone a known vir inducer. Total soluble phenolic amounts and cell wall composition analyzed by FT-IR analysis did not show significant differences between transgenic and control plants. The potential of phenolic extracts from control and OMT-suppressed plants to induce virulence was evaluated using an Agrobacterium tumefaciens reporter strain carrying a vir::LacZ gene fusion plasmid. Lower vir-inducing activities were recorded for plants that show inhibition to caffeic acid O-methyltransferase activity. HPLC analysis confirmed that the levels of several phenolic compounds were differently affected by wounding and/or by bacterial inoculation. Statistical correlations were established between tumor sizes, vir-inducing activities, O-methyltransferases proteins accumulations and the levels of various soluble phenolic compounds such as acetosyringone. These results demonstrate the role of the O-methyltransferases of the phenylpropanoid pathway in the early production of soluble Agrobacterium tumefaciens vir inducers.

[Cryptosporidium oocyst viability and infectivity evaluation by flow cytometry]. / Evaluation de la viabilité et de l'infectivité des oocystes de Cryptosporidium par cytométrie en flux.

Evaluating waterborne Cryptosporidium sp. oocyst infectivity is presently a major Issue for the estimation of environmental risks. The aim of this presentation, was to describe a new model suitable for determining in vivo oocyst infectivity. In this model, infection was assessed in suckling mice seven days after oocyst ingestion by measuring the number of gut oocysts using flow cytometry. Four days old mice were orally infected by serially diluted C. parvum oocyst suspensions. This model was found highly sensitive since ingestion of 1-10 oocysts resulted in infection in 70% of animals. Assays with Cryptosporidium oocysts from the environment suggest that this model may contribute to the evaluation of environmental risks due to the parasite.

H2O2 sensing through oxidation of the Yap1 transcription factor.

The yeast transcription factor Yap1 activates expression of antioxidant genes in response to oxidative stress. Yap1 regulation involves nuclear accumulation, but the mechanism sensing the oxidative stress signal remains unknown. We provide biochemical and genetic evidence that upon H2O2 treatment, Yap1 is activated by oxidation and deactivated by enzymatic reduction with Yap1-controlled thioredoxins, thus providing a mechanism for autoregulation. Two cysteines essential for Yap1 oxidation are also essential for its activation by H2O2. The data are consistent with a model in which oxidation of Yap1 leads to disulfide bond formation with the resulting change of conformation masking recognition of the nuclear export signal by Crm1/Xpo1, thereby promoting nuclear accumulation of the protein. In sharp contrast to H2O2, diamide does not lead to the same Yap1 oxidized form and still activates mutants lacking cysteines essential for H2O2 activation, providing a molecular basis for differential activation of Yap1 by these oxidants. This is the first example of an H2O2-sensing mechanism in a eukaryote that exploits the oxidation of cysteines in order to respond rapidly to stress conditions.

Quantitative flow cytometric evaluation of maximal Cryptosporidium parvum oocyst infectivity in a neonate mouse model.

The importance of waterborne transmission of Cryptosporidium parvum to humans has been highlighted by recent outbreaks of cryptosporidiosis. The first step in a survey of contaminated water currently consists of counting C. parvum oocysts. Data suggest that an accurate risk evaluation should include a determination of viability and infectivity of counted oocysts in water. In this study, oocyst infectivity was addressed by using a suckling mouse model. Four-day-old NMRI (Naval Medical Research Institute) mice were inoculated per os with 1 to 1,000 oocysts in saline. Seven days later, the number of oocysts present in the entire small intestine was counted by flow cytometry using a fluorescent, oocyst-specific monoclonal antibody. The number of intestinal oocysts was directly related to the number of inoculated oocysts. For each dose group, infectivity of oocysts, expressed as the percentage of infected animals, was 100% for challenge doses between 25 and 1,000 oocysts and about 70% for doses ranging from 1 to 10 oocysts/animal. Immunofluorescent flow cytometry was useful in enhancing the detection sensitivity in the highly susceptible NMRI suckling mouse model and so was determined to be suitable for the evaluation of maximal infectivity risk.

Efficacy of nitazoxanide, tizoxanide and tizoxanide glucuronide against Cryptosporidium parvum development in sporozoite-infected HCT-8 enterocytic cells.

The effects of nitazoxanide and its metabolites, tizoxanide and tizoxanide glucuronide, on the development of the asexual and sexual stages of Cryptosporidium parvum in differentiated human enterocytic HCT-8 cells were evaluated in a quantitative alkaline phosphatase immunoassay. Nitazoxanide, tizoxanide and tizoxanide glucuronide were inhibitory for up to 46 h when added after sporozoite invasion (MIC50 1.2, 22.6 and 2.2 mg/L, respectively). Tizoxanide had only limited activity, but nitazoxanide and tizoxanide glucuronide strongly inhibited asexual and sexual stages, respectively.

Cryptosporidium parvum infection stimulates the secretion of TGF-beta, IL-8 and RANTES by Caco-2 cell line.

Cryptosporidium parvum is a common cause of diarrhea in humans. Although mild inflammatory mucosal infiltrate is usually observed, limited information is currently available on the pathogenic mechanisms involved in this phenomenon. The aim of this work was to investigate in vitro the influence of C. parvum infection on the secretion of lymphocyte-targeted chemokines (RANTES. MIP-1alpha, MIP-1beta, IL-8), proinflammatory cytokines (TNF-alpha, GM-CSF and IL-6) and TGF-beta by human enterocytic Caco-2 cells. C. parvum infection stimulates IL-8, RANTES and TGF-beta secretion by both the basal and apical side of caco-2 cells. A slight increase in TNF-alpha production by infected cells was observed in the apical compartment. Data suggest that enterocytic chemokines and/or TGF-beta are involved in the initiation and regulation of the mucosal response to C. parvum infection.

Enzyme immunoassay detection of Cryptosporidium parvum inhibition by sinefungin in sporozoite infected HCT-8 enterocytic cells.

Complete parasite development was obtained in differentiated human enterocytic HCT-8 cells infected at confluence with Cryptosporidium parvum sporozoites, and evaluated in a quantitative enzyme immunoassay. Forty-eight hours after infection, a linear correlation was found between optical density values and the number of parasites determined in an immunofluorescent assay. Sinefungin exerted an inhibitory effect when added within 4 h after sporozoite addition to HCT-8 cultures (MIC50 = 38 mumol L-1), while the inhibitory effects of paromomycin and pentamidine dimethanesulfonate were also easily detected (MIC50 = 0.87 mumol L-1 and 0.27 mumol L-1, respectively). Except for high pentamidine dimethanesulfonate concentrations, no alteration in optical microscopy morphology or trypan blue exclusion of HCT-8 cells was observed in the presence of anticryptosporidial agents, which suggests that they were primarily active against developing parasites. Data suggest that EIA detection of C. parvum development in sporozoite-infected HCT-8 cells provides an accurate and convenient model for in vitro evaluation of parasite infectivity, growth and response to anticryptosporidial agents.

Characterization and Localization of a Phenoloxidase in Mung Bean Hypocotyl Cell Walls.

The occurrence of proteins able to oxidize polyphenols even in the absence of H2O2 was recently reported in mung bean (Vigna radiata L.) hypocotyl cell wall extracts (R. Goldberg, A. Chabanet, A.M. Catesson [1993] In K.G. Welinder, S.K. Rasmussen, C. Penel, H. Greppin, eds, Plant Peroxidases: Biochemistry and Physiology, pp. 296-300). Therefore, the possible presence of a laccase in the extracts was investigated using immunocytological and biochemical approaches. An enzyme catalyzing phenol oxidation in the presence of molecular O2 was extracted and purified from the cell walls. This 38-kD cationic protein, like o-diphenoloxidases, was unable to oxidize p-diphenols or p-diamines. However, it crossreacted with an anti-laccase antiserum and, like laccases, its activity was inhibited by N-cetyl-N,N,N-trimethylammonium bromide but not by ferulic acid salts. Immunolabeling data showed that the 38-kD oxidase was absent from all cellulosic cell walls. It was localized only in lignifying and lignified cell walls. This restricted localization suggests that this laccase-like phenoloxidase could participate in the lignification process but not in the primary wall stiffening, which develops in the epidermal and cortical tissues along the mung bean hypocotyl.

Decreased blood TcR gamma delta+ lymphocytes in AIDS and p24-antigenemic HIV-1-infected patients.

The significance of blood TcR gamma delta+ lymphocyte level was evaluated in the context of immunodeficiency and infections in 209 HIV-1-infected patients. Blood TcR gamma delta+ lymphocyte values were found higher in patients belonging to the CDC group II/III than those in the CDC groups IV C1 and IV D (P < 0.001) and P < 0.01, respectively). TcR gamma delta+ lymphocyte counts were lower in patients with oral candidiasis (P < 0.01), and in association with pneumocystosis or toxoplasmosis (P < 0.001). In 81 patients with a detectable HIV-1 p24 antigenemia, TcR gamma delta+ lymphocyte counts were lower than those in nonantigenemic patients (P < 0.001). In the CDC II/III group, p24-antigenemic patients exhibited lower TcR gamma delta+ cell counts than those in patients without antigenemia (P = 0.06). Data suggest that depletion of the TcR gamma delta+ lymphocyte subset characterizes HIV-1-infected patients with oral candidiasis, pneumocystosis, toxoplasmosis, and/or HIV-1-antigenemia.

Molecular cloning of the 5-aminolevulinic acid dehydratase gene from Rhodobacter sphaeroides.

A hemB mutant of Escherichia coli was used to clone the gene encoding 5-aminolevulinic acid dehydratase from Rhodobacter sphaeroides after physiological complementation of the mutation. A 2.9-kb DNA fragment was obtained and cloned in both orientations into the unique PstI restriction site of pUC19. This recombinant plasmid encodes a protein (Mr 39,000) that is immunoreactive with antibodies raised against the enzyme from higher plants.

Biochemistry and histology of the connective tissue of Dupuytren's disease lesions.

When compared to age-matched control aponeurosis, lesions of Dupuytren's disease contain higher contents of water, collagen and chondroitin-sulphate, as well as increased proportions of soluble collagens and of reducible cross-links; these indicate synthesis of new collagen. The lesions show also increased amounts of type III collagen and an increased hydroxylation and glycosylation of the reducible cross-links. All these parameters are characteristic of granulation and scar tissues. Type III collagen was located by means of immunofluorescence on thin argyrophilic fibres and also within the large fibre bundles which appeared to be disrupted into microbundles. The increase of type III collagen and the presence of myofibroblasts in the apparently unaffected aponeurosis show that the disease is widespread and suggest that it is initiated within the aponeurosis and propagated by the cells migrating along the collagen bundles.

[Variations in the hydrothermic contraction of the rat skin as a function of the age of the animals]. / Variations de la contraction hydrothermique de la peau du Rat en fonction d l'âge des animaux.

The tension developed in Rat skin by hydrothermal shrinkage is modified with ageing of the animals: the temperature of maximum tension decreases from birth to 1 month, then very slowly increases with age. At higher temperatures than that of maximum tension a relaxation occurs very rapidly in young Rats and in not yet senescent adult skins. These modifications appeared to be related to those of the nature of the intermolecular collagen cross-links with ageing.

Collagen and myofibroblasts of granulation tissue. A chemical, ultrastructural and immunologic study.

In granulation tissue produced in the rat by subcutaneous injection of turpentine oil or polyvynile sponge implantation, the great majority of fibroblasts (myofibroblasts) possess a contractile apparatus which makes them similar to smooth-muscle cells. Chemical analysis shows that these granulation tissues contain a high proportion of Type III collagen, a genetically distinct collagen normally associated with embryonic dermal tissue. Type III collagen may persist up to 9 months after sponge implantation and myofibroblasts are seen in granulation tissue by means of electron microscopy and immunofluorescence. When granulation tissue is resorbed 50 days after turpentine oil injection, myofibroblasts disappear and the dermis contains Type I collagen. The concurrent presence of myofibroblasts and Type III collagen suggests that myofibroblasts, in addition to their contractile activity, synthetize, at least in part, type III collagen.

Collagen in granulation tissues.

Collagen is abundantly synthesized in granulation tissues and reaches a concentration higher than in normal neighbouring tissues. Such newly formed collagen is characterized by an abnormally low solubility and an easy degradation by collagenases and collagenolytic cathepsins. The activities of these two types of enzymes are high (especially collagenases) in tissues of acute inflammations when the granuloma is resorbing. These activities are lower in sub-acute inflammation and the collagen content of the persistent granuloma remains high. The collagen synthesized in granulation tissues is cross linked by hydroxylysino-5-keto-norleucine, the stable cross-link of collagen in embryonic skin. It is progressively replaced by the two aldimine cross links of normal adult skin when the granuloma is resorbed (acute inflammations induced in rats; human normal scars). The cross link of embryonic skin, on the contrary, is permanently present in collagen of tissues of subacute, chronic inflammations (sponge implants in rats, human hypertrophic scars and keloids. Studies of the structure of alpha-chains revealed that type III collagen (embryonic collagen) is present in granulation tissues.