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International audience; Long-term cell cultures were used in coffee to study the cytological, genetic and epigenetic changes occurring during cell culture ageing. The objective was toidentify the mechanisms associated with somaclonal variation (SV). Three embryogenic cell lines were established in Coffea arabica (2n = 4x = 44) and somatic seedlings were regenerated after 4, 11 and 27 months. Phenotypingand AFLP, MSAP, SSAP molecular markers were performed on 199 and 124 plants, respectively. SV were only observed from the 11 and 27-month-old cultures, affecting30 and 94 % of regenerated plants, respectively. Chromosome counts performed on 15 plants showed that normal plants systematically displayed normal chromosome numbers and that, conversely, aneuploidy (monosomy) was systematically found in variants. The allopolyploid structure of C. arabica allowed aneuploid cells to survive and regenerate viable plants. No polymorphic fragments were observed between the AFLP and SSAP electrophoretic profiles of mother plants and those of the in vitro progeny.Methylation polymorphism was low and ranged between 0.087 and 0.149 % irrespective of the culture age. The number of methylation changes per plant—normal or variant—was limited and ranged from 0 (55–80 % of the plants) to 4. The three cell lines showed similar SV rate increases during cell culture ageing and produced plantswith similar molecular patterns indicating a non randomprocess. The results showed that cell culture ageing ishighly mutagenic in coffee and chromosomal rearrangements are directly linked to SV. Conversely, the analysis of methylation and transposable elements changes did notreveal any relation between the epigenetic patterns and SV.

Growing muscle tissue in culture from animal stem cells to produce meat theoretically eliminates the need to sacrifice animals. So-called “cultured” or “synthetic” or “in vitro” meat could in theory be constructed with different characteristics and be produced faster and more efficiently than traditional meat. The technique to generate cultured muscle tissues from stem cells was described long ago, but has not yet been developed for the commercial production of cultured meat products. The technology is at an early stage and prerequisites of implementation include a reasonably high level of consumer acceptance, and the development of commercially-viable means of large scale production. Recent advancements in tissue culture techniques suggest that production may be economically feasible, provided it has physical properties in terms of colour, flavour, aroma, texture and palatability that are comparable to conventional meat. Although considerable progress has been made during recent years, important issues remain to be resolved, including the characterization of social and ethical constraints, the fine-tuning of culture conditions, and the development of culture media that are cost-effective and free of animal products. Consumer acceptance and confidence in in vitro produced cultured meat might be a significant impediment that hinders the marketing process.

Description of the subject. Variability observed in sensory characteristics of “attiéké” results from an uncontrolled fermentation process due to the use of artisanal starters. Objectives. This study aims to screen microbial strains for their use during fermentation of cassava dough into attieké. Method. Technological properties of three lactic acid bacteria (LAB) isolated from artisanal starters were highlighted in vitro, and these LAB were identified as either Weissella cibaria or Weissella confusa by Matrix Assisted Laser Desorption Ionisation-Mass Spectrometry (MALDI-TOF MS).Results. The three Weissella isolates Wc 69, Wc 21 and Wc 114 induced in less than 42 h a decrease below 4.2 (main food safety factor) of the initial pH of MRS (de Man, Rogosa and Sharpe) broth. These strains are osmotolerant, present alpha-amylase activity and ferment the indigestible sugar raffinose. Moreover isolates Wc 114 and Wc 69 are thermotolerant, while Wc 114 presents a pectinase activity necessary for cassava dough softening.Conclusions. Considering their technological properties, the three isolated Weissella strains could be suitable in optimizing and standardizing the quality of attieké., Isolement et sélection de souches de Weissella comme de potentiels ferments pour la production d’attiékéDescription du sujet. La fermentation non contrôlée de l’attieké, effectuée à partir d’inocula traditionnels, est responsable de la grande variabilité sensorielle observée pour cette denrée alimentaire.Objectifs. La présente étude vise à sélectionner des micro-organismes aptes à être utilisés comme des ferments microbiens pour une fermentation contrôlée de la pâte de manioc en attieké.Méthode. Trois bactéries lactiques isolées de ferments traditionnels sont identifiées comme étant Weissella cibaria ou Weissella confusa par la technique de désorption-ionisation laser assistée par matrice couplée au spectromètre de masse à temps de vol (MALDI-TOF MS) et leurs propriétés technologiques mises en évidence in vitro.Résultats. Les trois isolats de Weissella induisent une baisse du pH initial (principal facteur de sécurité alimentaire) de 6,2 du bouillon MRS (de Man, Rogosa and Sharpe) à une valeur en dessous de 4,2 en moins de 42 h. En plus de présenter une activité alpha-amylase, ces bactéries fermentent le raffinose, un sucre indigeste. Toutes les trois sont osmotolérantes. Par ailleurs, les isolats Wc 114 et Wc 69 sont thermotolérants, tandis que l’isolat Wc 114 présente une activité pectinase nécessaire au ramollissement de la pâte de manioc. Conclusions. Les trois bactéries lactiques isolées (Wc 69, Wc 21 and Wc 114) présentent des propriétés technologiques intéressantes susceptibles de convenir pour contrôler et standardiser la qualité de l’attieké.

International audience; BACKGROUND: Over recent decades, recurring efforts have been devoted to developing testicular cell or tissue cultures for basic and clinical research. However, there remains much confusion, particularly concerning the fate of human germ cells in culture. OBJECTIVE: To reassess the status of human testicular cell types as well as the ability of germ cells to divide and differentiate in organotypic culture. METHODS: Human testicular fragments were maintained for 2 weeks in culture. The viability and functionality of testicular cells were assessed using light and electronic microscopy, apoptotic cell labelling, 5-bromo-2'-deoxyuridine (BrdU) incorporation, immunohistochemistry and quantitative PCR against specific cell markers. RESULTS: A gradual loss of meiotic and post-meiotic germ cells occurred throughout the culture period, irrespective of the presence of gonadotrophins. However, all germ cell types remained traceable for up to 16 days, some still dividing and differentiating at a rate compatible with the in vivo situation. Good maintenance of the general architecture of the explants associated with clearly quantifiable levels of several somatic cell markers was observed. CONCLUSION: Although this culture model is clearly unsuitable for preparing germ cells for therapeutic purposes, it does represent a most valuable tool for testing the effects of biological and chemical agents on testicular tissue.

The effect of hypotonic shock on cultured pavement gill cells from freshwater (FW)- and seawater (SW)-adapted trout was investigated. Exposure to 2/3rd strength Ringer solution produced an increase in cell volume followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also induced a biphasic increase in cytosolic Ca(2+) with an initial peak followed by a sustained plateau. Absence of external Ca(2+) did not modify cell volume under isotonic conditions, but inhibited RVD after hypotonic shock. [Ca(2+)](i) response to hypotonicity was also partially inhibited in Ca-free bathing solutions. Similar results were obtained whether using cultured gill cells prepared from FW or SW fishes. When comparing freshly isolated cells with cultured gill cells, a similar Ca(2+) signalling response to hypotonic shock was observed regardless of the presence or absence of Ca(2+) in the solution. In conclusion, gill pavement cells in primary culture are able to regulate cell volume after a cell swelling and express a RVD response associated with an intracellular calcium increase. A similar response to a hypotonic shock was recorded for cultured gill cells collected from FW and SW trout. Finally, we showed that calcium responses were physiologically relevant as comparable results were observed with freshly isolated cells exposed to hypoosmotic shock.

International audience; Une mammite expérimentale à lipopolysaccharide a été menée en vue d'obtenir le même changement de composition du lait que celui observé dans les cas de mammite clinique, en liminant toutefois toute interaction avec la protolyse bactérienne. Le lactosrum issu d'un lait à fort dénombrement cellulaire (DC) avait un effet mitogénique plus faible sur une culture cellulaire d'hybridomes anti-prolactine humaine (anti-hPL) mais toutefois un effet immuno-stimulant plus important que le sérum foetal de veau. Cet effet immunostimulant a été plus marqué lorsqu'il était exprimé par cellule, avec un effet 2 à 3 fois supérieur à celui observé avec le sérum de veau foetal (P 0,01), traduisant l'efficacité de la complémentation par un lactosérum issu d'un lait à fort DC.

Five types of cellular aggregates have been characterised in embryogenic cell suspensions of banana (#Musa# 'AAA' 'Grande naine' cv.). Type I corresponded to isolated cells or to small cell aggregates. Type II were composed of embryogenic cells. Type III can be distinguished from type II due to the presence of peripheral proliferation zones with embryonic cells. Type IV were composed of protodermic masses histologically comparable to proembryos. Type V were nodules composed of a central zone of meristematic cells and of an external zone of starchy cells. Each culture flask of a cell line contained a majority of one of the above-mentioned aggregate types. Histological studies of somatic embryo developement on semi-solid regeneration medium showed that there were close similarities between the initial steps of ontogenesis of the embryos and the different cell aggregates in liquid multiplication medium. It appeared that aggregates II-IV of the suspension belong to the same development continuum which reproduces the initial phases of somatic embryo ontogenesis on semi-solid medium. Type V resulted from the development of type IV, for which ontogenesis is hindered by direct contact with 2,4-dichlorophenoxyacetic acid and the shaken liquid multiplication medium. Type I aggregates probably do not belong to the development continuum but rather correspond to the degeneration of the other types of aggregates in the suspension. The presence of intermediate types in the liquid medium reinforces the hypothesis of a relationship between the aggregates. The aggregates tended to develop through time from a majority of type II or III at the beginning of their culture to types IV-V for older suspensions.

International audience; Using spectrofluorescence imaging of fura-2 loaded renal A6 cells, we have investigated the generation of the cytosolic Ca2+ signal in response to osmotic shock and localized membrane stretch. Upon hypotonic exposure, the cells began to swell prior to a transient increase in [Ca2+] i and the cells remained swollen after [Ca2+] i had returned towards basal levels. Exposure to 2/3rd strength Ringer produced a cell volume increase within 3 min, followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also produced a transient increase in [Ca2+] after a delay of 22 sec. Both the RVD and [Ca2+] i response to hypotonicity were inhibited in a Ca2+-free bathing solution and by gadolinium (10 μm), an inhibitor of stretch-activated channels. Stretching the membrane by application of subatmospheric pressure (-2 kPa) inside a cell-attached patch-pipette induced a similar global increase in [Ca2+] i as occurred after hypotonic shock. A stretch-sensitive [Ca2+] i increase was also observed in a Ca2+-free bathing solution, provided the patch-pipette contained Ca2+. The mechanosensitive [Ca2+] i response was by gadolinium (10 μm) or Ca2+-free pipette solutions, even when Ca2+ (2 mm) was present in the bath. Long-term (>10 min) pretreatment of the cells with thapsigargin inhibited the [Ca2+] i response to hypotonicity. These results provide evidence that cell swelling or mechanical stimulation can activate a powerful amplification system linked to intracellular Ca2+ release mechanisms.

The growth of three strains of Listeria monocytogenes at 20 degrees C in a meat broth of different pH or water activity was investigated. At inoculation or at the beginning of the exponential phase, cells were exposed to stress by the addition of NaOH or NH4+, acetic acid, NaCl or KCl, in order to reach a pH of either 9.0 or 5.6, or an a(w) of 0.950 or 0.965, respectively. The effects of the exposure to stress on the generation and lag times of each strain were analysed by turbidity measurements for cultures in micro-titer plates. Results were confirmed by conducting the same experiments in a fermentor, except for the maximal population reached. The three strains showed similar behaviour. Cells were able to overcome the alkaline stress rapidly whereas acid and osmotic shocks induced important changes of the growth parameters. Cells exposed to acid or osmotic conditions from the time of inoculation were less affected than cells exposed at the beginning of the mid-exponential phase.

Mammary epithelial cells, obtained from lactating cows, were cultured onto inserts coated with several components of extracellular matrix. The influence of these components upon the maintenance of differentiation has been determinated. Every day, alpha S1-casein secretion was measured by radioimmunoassay (RIA) in apical and basal compartments. Reorganization of functional tight junctions was evaluated by measurement of transepithelial electrical resistance (TER). On EHS matrix, cells underwent alveolar structures and never established TER. alpha S1-casein secretion strongly fluctuated with the day of culture. When plated onto fibronectin, cells reorganized a typical pavement and established TER. Nevertheless, TER and casein secretion highly fluctuated. On laminin-coated inserts, a few cells bound to the substratum, dedifferentiated, and proliferated to confluency within 9 days. TER progressively increased to a stable level after 15 days. Casein was not recovered after 6 days. Cells on type I collagen-coated inserts reorganized an epithelial pavement within 2 days and quickly established a stable TER. They secreted apically high levels of casein during 2 weeks. As cells maintained their biochemical differentiation, the culture on type I collagen-coated inserts seems an efficient model for primary culture of bovine mammary epithelial cells and allows studies of polarized alpha S1-casein secretion.

There are very few reports on the establishment of long-term embryogenic cell cultures of banana, especially of triploid cultivars of commercial interest. Embryogenic cell suspensions were prepared using the cultivar Grand nain, the most widely grown dessert banana in the world. After culture for 5 or 6 months of immature male flowerbuds adjacent to the floral apex, yellow, compact calluses and white, friable embryogenic tissues were induced. Suspension cultures were initiated from embryogenic tissues placed in liquid medium. The packed cell volume (PCV) of the suspensions increased 2- to 5- fold with each monthly culture cycle. Plating of the embryogenic suspensions resulted in approximately 370×103 embryos per ml of PCV. Depending on the size of embryos, 3 to 20% germination was observed. A histological survey of cell suspensions and embryo development was carried out. Cellular aggregates with cells displaying typical embryogenic features were formed. Most of the somatic embryos were probably of unicellular origin.

L'article a pour objet l'induction de l'embryogénèse dans une suspension cellulaire et la définition des phases critiques tout au long du processus. Dans une solution riche en saccharose (source de carbone) on note une augmentation des divisions et la formation de petits amas cellulaires indifférenciés. Le remplacement du saccharose par le galactose induit la différenciation des amas en embryons chlorophylliens. La phase de culture sur saccharose est caractérisée par une forte utilisation du Ca, K, P et du Mg. Seule l'absorption du P augmente (2 fois plus) dans la phase de différenciation (milieu riche en galactose)

International audience; Aurora-C, a member of the Aurora kinase family, is implicated in the regulation of mitosis. In contrast to Aurora-A and Aurora-B its cellular localization and functions are poorly characterized. TACC1 protein belongs to the transforming acidic coiled-coil family shown to interact with the Aurora kinases. In the present study we analyzed the interaction between Aurora-C and TACC1 by means of immunofluorescence (IF), co-immunoprecipitation (IP) and in vitro phosphorylation experiments. We demonstrated that Aurora-C and TACC1 proteins co-localize to the midbody of HeLa cells during cytokinesis. Immunoprecipitated TACC1 from HeLa cell extracts was associated with Aurora-C. In addition, the interaction of the two proteins was tested by analyzing the phosphorylation of TACC1 in vitro. The results demonstrated that TACC1 is phosphorylated by Aurora-C on a serine at position 228. In conclusion, the study demonstrated that TACC1 localizes at the midbody during cytokinesis and interacts with and is a substrate of Aurora-C, which warrant further investigation in order to elucidate the functional significance of this interaction.

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We have previously succeeded in infecting normal human hepatocyte primary cultures with hepatitis B virus (HBV). However, infection was subject to individual variations even in the presence of dimethyl sulfoxide (DMSO), which appeared to increase the amounts of viral DNA associated with the cells. In this study, we have defined conditions which enhance hepatitis B virus penetration into the cells, and we show that, under these conditions, infection of hepatocytes is always possible, regardless of their individual origin. We have found that addition of polyethylene glycol (PEG) to the cultures maintained in the presence of 2% DMSO at the time of infection markedly increased the infection process and made it highly reproducible. Moreover, both the tissue and species specificity were preserved. This increased HBV infection was correlated to increased amount of internalized HBV DNA and to enhanced attachment of the virions. From these results it may be assumed that PEG could favor a better interaction between virions and cells, resulting in an activated internalization of bound viral particles. Data also show that adult human hepatocyte primary cultures, which are not equally susceptible to HBV infection, are consistently capable of viral replication when the viral genome has entered the cells. This suggests that the main limitation of the in vitro HBV infection lies in the ability of human hepatocytes to specifically bind the viral particles.

La rickettsie Cowdria ruminantium a été cultivée avec succès dans des lignées de cellules endothéliales bovines (ombilicales, BUEC, et de microvasculature, BMC), ainsi que dans des cultures primaires de cellules endothéliales d'aorte de bovin (BAEC), mais de manière plus surprenante, également dans des cellules endothéliales d'origine humaine : de veine ombilicale (HUVEC) et de microvasculature (HEMEC). Cette première preuve de pathogénicité de cette rickettsie bovine pour le système cellulaire humain provoque un nouvel intérêt concernant sa signification possible pour la santé humaine. Elle indique également d'autres possibilités pour l'atténuation d'isolats de Cowdria ruminantium et donc de nouvelles perspectives pour le développement d'un vaccin. Pour la production de vaccin, la culture en grand de cellules est essentielle. Les résultats montrent que les cellules endothéliales s'attachent de façon efficace sur des microsphères de collagène. Les BAEC se multiplient bien par lots sur ces billes, et si le processus pouvait être optimisé pour les différents types de cellules, cela faciliterait le développement futur d'une production de vaccin contre la cowdriose dans le réacteur à lit fluide VERAX Système un, qui donne des conditions de culture facilement contrôlables

It was shown in a previous study that rat ventricular myocytes in culture do incorporate in their phospholipids the cyclic fatty acid monomers (CFAM), produced from linolenic acid by heating linseed oil. In the present study, the functional consequences of this incorporation were investigated. As compared to control cells, CFAM containing cells displayed altered electromechanical properties evidenced by decreased maximal diastolic potential, upstroke velocity and rate and a shortened action potential duration. Moreover, oxygen depletion exerted much more adverse effects on CFAM-treated cells than in control cells, such as spontaneous rhythm abnormalities, cell shortening failure and earlier and more pronounced Lactate dehydrogenase release.

Whey acidic protein gene transcription is induced in the mammary gland under the influence of lactogenic hormones: prolactin, insulin and cortisol. The rabbit WAP gene has already been isolated and sequenced in a previous work. In the present study, we have evaluated the role of the 5' flanking region of the rabbit WAP gene in the transcriptional regulation of the WAP gene by using a reporter CAT gene. Chimeric genes containing the upstream region of the WAP gene have been linked to the bacterial CAT gene and transfected into rabbit primary mammary cells. The results reported here show that two regions carrying important regulatory elements of the rabbit WAP gene are located between -6300 and -3000 bp, and between -3000 and -1800 bp upstream from the WAP transcription start point, respectively. The contribute to the high level of expression of the rabbit WAP gene in the mammary cell.

International audience; Adrenoleukodystrophy-related protein, a peroxisomal ABC transporter encoded by ABCD2, displays functional redundancy with the disease-associated X-linked adrenoleukodystrophy protein, making pharmacological induction Of ABCD2 a potentially attractive therapeutic approach. Sterol regulatory element (SRE)-binding proteins (SREBPs) induce ABCD2 through an SRE overlapping with a direct repeat (DR-4) element. Here we show that thyroid hormone (T-3) receptor (TR)alpha and TR beta bind this motif thereby modulating SREBP1-dependent activation of ABCD2. Unliganded TR beta, but not TR alpha, represses ABCD2 induction independently of DNA binding. However, activation by TR alpha and derepression of TR beta are T-3-dependent and require intact SRE/DR-4 motifs. Electrophoretic mobility shift assays with nuclear extracts support a direct interaction of TR and SREBP1 at the SRE/DR-4. In the liver, Abcd2 expression is high in young mice (with high T-3 and TR alpha levels) but downregulated in adults (with low T-3 and TR alpha but elevated TR beta levels). This temporal repression of Abcd2 is blunted in TR beta-deficient mice, and the response to manipulated T-3 states is abrogated in TR alpha-deficient mice. These findings show that TR alpha and TR beta differentially modulate SREBP1-activated ABCD2 expression at overlapping SRE/DR-4 elements, suggesting a novel mode of cross-talk between TR and SREBP in gene regulation.

Mass production of Ehrlichia ruminantium variants from different regions of sub- Saharan Africa is one of the difficulties that must be overcome in producing a heartwater vaccine. Vaccine productivity can be limited by endogenous induction of interferon (IFN), which inhibits the propagation of Ehrlichia ruminantium (ER) in cell culture. Different kinds of endothelial cells, in which ER multiply efficiently, could be grown in a scalable way in VueLife Teflon bags on Cytodex 3 microcarriers where bead-to-bead transfer of cells occurs. The digital holographic microscope designed at the Universit´e Libre de Bruxelles allows detection of the most appropriate time to harvest intracellular microorganisms for vaccine production.

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International audience; Embryogenic cell suspension cultures of garlic (Allium sativum L.) were initiated in liquid medium from friable embryogenic tissue. The optimal parameters for culture maintenance were: (1) an initial cell density of 1–4% (v/v); (2) medium renewal every 14 days and subculturing every 28 days; (3) a low 2,4-dichlorophenoxyacetic acid concentration (0.1–0.3 mg/l). Cultures regenerated during a 14-month period. The cell suspension cultures differentiated embryos following transfer to a semi-solid embryo induction medium, with histological studies confirming and characterising the embryogenic nature of the process. Forty percent of these embryos converted into plantlets, which produced micro bulbs in vitro. The composition of the sulphur compounds of the micro bulbs obtained from cell suspension embryo-derived plantlets differed slightly from those produced by in vitro shoot proliferation-derived plantlets, but after two cycles of multiplication in the field these differences had disappeared.

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The indole alkaloids brucine and yohimbine, just like hypaphorine, counteract indole-3-acetic acid (IAA) activity in seedling roots, root hairs and shoots, but do not appear to alter auxin transport in roots or in cultured cells. In roots, the interactions between IAA and these three alkaloids appear competitive and specific since these molecules interact with IAA but with neither 1-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D), two synthetic auxins. The data reported further support the hypothesis that hypaphorine brucine and yohimbine compete with IAA on some auxin-binding proteins likely to be auxin receptors and that 2,4-D and NAA are not always perceived by the same receptor as IAA or the same component of that receptor. At certain steps of plant development and in certain cells, endogenous indole alkaloids could be involved in IAA activity regulation together with other well-described mechanisms such as conjugation or degradation. Hypaphorine with other active indole alkaloids remaining to be identified, might be regarded as a new class of IAA antagonists.

International audience; We report here on the characterization of tissue-culture cell lines derived from primary cultures of the mouse caput epididymidis epithelium. The cell lines were spontaneously immortalized without the use of transforming oncogenes. In defined conditions, our epididymal cells adopted various morphological features that resembles that of the in vivo epididymis epithelium such as a polarized organization and the presence of junctional structures at their apical/lateral membranes as revealed by electron microscopy analyses. Flow cytometry analysis revealed that we were dealing with homogenous cell populations that had reached a near-tetraploid state. RT-PCR assays were used in order to show that several genes that can be considered as markers of in vivo caput epididymidis epithelium activity were expressed in our cell lines confirming that these cells were indeed in a differentiated state close to their endogenous state.

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International audience; Contamination of food and feeds by mycotoxins is a major problem of human and animals health concern which is also extremely detrimental to economy. Mycotoxins producing moulds may produce a diversity of toxins such as aflatoxins, ochratoxins, trichothecenes, zearalenone, fumonisins, tremorgenic toxins and ergot alkaloids. Although toxicological, environmental and epidemiological studies have addressed the problem of these toxins one by one, more than one mycotoxin are found usually in the same contaminated commodities. That rises the incommensurable problem of multi-toxicosis in which the respective metabolites are also involved. These mycotoxins bear potential toxicity leading to acute and chronic effects in humans and animals, depending on species. The mechanisms that lead to toxic effects, such as immune toxicity, and carcinogenicity are complexe. The risk assessment for humans potentially exposed to multi-mycotoxins suffers very much from the lack of adequate food consumption data. Furthermore, for a given mycotoxin synergism and antagonism with other mycotoxins found in the same food commodities are not taken into account. The case of combination of ochratoxin A (OTA) and fumonisin B1 (FB1) has been addressed in the present paper with the purpose of predicting the in vivo toxicity using a simple in vitro test, i.e. neutral red uptake, in three different cell-lines, C6 glioma cells, Caco-2 cells and Vero cells. Using the equation of [ATLA 27 (1999) 957], in vivo toxicity (LD50) is in adequation with the in vitro data, (IC50 values) for both toxins as well as for the combination of 10 microM OTA and variable concentrations of FB1 (10-50 microM). A synergistic effect is prouved in vitro that is in line with some in vivo data from the literature. Such simple in vitro test may thus help predicting in vivo toxicity of combinations of mycotoxins naturally occurring in foodstuffs.

PRL (prolactin) has been implicated in the proliferation and differentiation of numerous tissues, including the prostate gland. However, the PRL-R (PRL receptor) signal transduction pathway, leading to the stimulation of cell proliferation, remains unclear and has yet to be mapped. The present study was undertaken to develop a clear understanding of the mechanisms involved in this pathway and, in particular, to determine the role of K(+) channels. We used androgen-sensitive prostate cancer (LNCaP) cells whose proliferation is known to be stimulated by PRL. Reverse transcriptase PCR analysis showed that LNCaP cells express a long form of PRL-R, but do not produce its intermediate isoform. Patch-clamp techniques showed that the application of 5 nM PRL increased both the macroscopic K(+) current amplitude and the single K(+)-channel open probability. This single-channel activity increase was reduced by the tyrosine kinase inhibitors genistein, herbimycin A and lavandustine A, thereby indicating that tyrosine kinase phosphorylation is required in PRL-induced K(+) channel stimulation. PRL enhances p59( fyn ) phosphorylation by a factor of 2 after a 10 min application in culture. In addition, where an antip59( fyn ) antibody is present in the patch pipette, PRL no longer increases K(+) current amplitude. Furthermore, the PRL-stimulated proliferation is inhibited by the K(+) channel inhibitors alpha-dendrotoxin and tetraethylammonium. Thus, as K(+) channels are known to be involved in LNCaP cell proliferation, we suggest that K(+) channel modulation by PRL, via p59( fyn ) pathway, is the primary ionic event in PRL signal transduction, triggering cell proliferation.

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To study molecules secreted from cultured plant cells that promote development, maize microspores were transferred into culture and the conditioned media were collected over time and analysed. Electrophoresis indicated that both non-glycosylated and glycosylated proteins including arabinogalactan proteins (AGPs) appeared in the medium and their concentration increased during the time of culture. The development of embryos was correlated with the presence of specific extracellular proteins, using an experimental system based on a tunicamycin inhibition test. In addition, a precise protein analysis was conducted using MALDI-TOF and ESI-MS-MS techniques. These approaches have allowed the identification of 5 other types of proteins: a cell wall invertase, two thaumatin isoforms, one 1-3 beta-glucanase and two chitinase isoforms. Altogether these experiments and results open ways for research aimed at understanding which molecules stimulate embryo formation. Moreover, AGPs may be used to stimulate the development of microspores (pollen embryogenesis) prepared from non-responsive genotypes.

Ovine leptin was cloned in the methylotrophic yeast Pichia pastoris using a pPIC9K vector. Leptin was produced and secreted into the culture medium using the Saccharomyces cerevisiae α-mating factor prepro signal by five clones. Expression levels of leptin varied from clone to clone, depending on the copy number of the ob gene. Highest expression was observed with the single-copy clone S27 (250 mg/l). The modifications of culture conditions in batch and fed-batch culture increase the yield of protein. The use of higher cell concentration (63 g/l) before induction of oLept associate with a regulation of pH at 3.2, which decreases the effects of proteolysis, increases the expression level of the oLept to 402 mg/l. Moreover, compared with the non-producer clone, we observed a drastic decrease in growth rate and biomass yield in the leptin-producing clones. At the end of the fed-batch phase at pH 3.2 with clone S27, mortality rate reached 17.3%. Results showed that recombinant leptin production induced metabolic stress, and a negative impact on biomass yield and growth rate. We characterized the recombinant leptin produced by clone S27. It exhibited a molecular mass of 16 kDa, an N-terminal amino acid sequence identical to that of ovine leptin but with an additional tyrosine introduced by the cloning site. Moreover, it was found to be biologically active in vitro. The available production of a large quantity of oLept will strengthen the functional study for theorical and practical purposes. Copyright © 2004 John Wiley & Sons, Ltd.

Food would seem to be one of the main ways of animal and human contamination with polycyclic aromatic hydrocarbons (PAHs). In vivo studies suggest a transfer in intestinal epithelium by diffusion, which appears extensively governed by the physicochemical properties of PAHs, particularly lipophilicity. However, other mechanisms, such as metabolism, are considered to intervene. Our work aimed at testing in vitro intestinal metabolism and defining its impact on transepithelial transport of PAHs. Caco-2 cells were cultivated on permeable filters and incubated with 14C-labeled benzo[a]pyrene (BaP), pyrene (Pyr), and phenanthrene (Phe), which differ in their physicochemical properties. The results showed that the cells were able to metabolize the compounds. In basal media, Phe appeared to be the least hydroxylated molecule (45% after a 6-h exposure), followed by Pyr (65%) and finally BaP (96%). Inhibition of PAH metabolism showed a determinant effect on kinetics profiles. Transfer in the basal compartment of BaP, Pyr, and Phe radioactivities was, respectively, 26, 4, and 2 times lower with inhibitors, corroborating that intestinal metabolism of PAHs would have a positive impact on their transfer, an impact that increased with their lipophilicity. Furthermore, after a 6-h incubation, metabolites were also detected in apical medium. These findings suggested that intestinal metabolism might play a key role in intestinal barrier permeability and thus in the bioavailability of tested micropollutants.

A study was undertaken to determine whether the interval between thawing and transfer influences both biological and clinical outcomes of cryopreserved blastocysts, using supernumerary embryos cultured in sequential media. One hundred and seventy-two patients who underwent blastocyst thawing without any exclusion criteria were included in this single center prospective study of blastocyst thawing cycles. Outcome of 338 blastocysts originating from culture of supernumerary embryos in sequential media was analyzed after 4 or 20 h of culture between thawing and transfer. Survival rate, re-expansion and hatching rates for surviving blastocysts, implantation rates (IRs), pregnancy and miscarriage rates were studied. Blastocyst survival was not influenced by the incubation time after thawing; however both re-expansion and hatching rates were increased after 20-h incubation. Moreover, the IR per thawed or transferred blastocyst was increased three-fold after 20-h incubation compared to 4-h incubation. Increasing the interval between thawing and transfer appears to be beneficial in order to better select for transfer frozen-thawed blastocysts.

La technique des SCE n'est pas encore opérationnelle comme méthode de micropropagation de masse malgré l'important potentiel de régénération qu'elle permet. La principale raison est l'incidence plus élevée de variation somaclonale observée avec les techniques d'embryogenèse somatique comparativement à celle observée avec la technique classique de multiplication d'apex. Les SCE sont toutefois d'ores et déjà utilisées pour la transformation génétique ou la fusion de protoplastes de bananiers. Les plantes régénérées à partir d'une SCE sont souvent issues d'une cellule unique. Ceci permet d'éviter, dans le cas des plantes transformées, le problème des plantes chimériques, (plantes comprenant des cellules génétiquement modifiées et des cellules non transformées) qui survient lorsque des méristèmes sont utilisés comme matériel de départ pour la transformation. Quatre méthodes d'embryogenèse somatique ont été testées chez le bananier. Chacune utilise un type d'explant différent: des embryons zygotiques (Cronauer et Krikorian 1988, Escalant et Teisson 1989), des tranches de rhizome et des gaines de feuilles (Novak et al. 1989), des fleurs mâles/femelles immatures (Ma 1991, Escalant et al. 1994, Grapin et al. 1996, Grapin et al. 1998) et des cultures de méristèmes proliférantes (scalps) (Dhed'a et al. 1991, Schoofs 1997). Le plus souvent, les SCE sont initiées à partir de fleurs mâles immatures ou de scalps. Une percée majeure en matière d'embryogenèse somatique des bananiers, qui a d'ailleurs inspiré de nombreuses études, revient au professeur Ma de l'Université de Taiwan qui a mis au point le milieu de culture et la méthodologie pour obtenir des SCE à partir de fleurs mâles (Ma 1991). Les premières étapes, jusqu'à la formation du cal embryogène, ont été décrites par Escalant et al. (1994). Par ailleurs, Grapin et al. (1996) et Côte et al. (1996) décrivent l'initiation et la maintenance des suspensions cellulaires ainsi que les étapes de régénération. La méthode de Ma a également été utilisée avec des fleurs immatures femelles pour les cultivars ne produisant pas de fleurs mâles (Grapin et al. 2000). La méthode des scalps utilisée à la Katholieke Universiteit Leuven (KULeuven) repose sur des cultures hautement proliférantes initiées à partir d'apex. Cette méthode a été décrite pour la première fois par Dhed'a (1992) et optimisée par Schoofs (1997). Dans ce guide technique, seules les méthodes des fleurs mâles immatures et des scalps sont décrites car elles s'appuient sur de nombreuses publications et ont été reproduites dans plusieurs laboratoires. Ils sont suivis par des sections expliquant les critères utilisés pour évaluer la qualité d'une suspension embryogène et les difficultés et limites de développement de l'embryogenèse somatique chez les bananiers.

International audience; Une mammite expérimentale à lipopolysaccharide a été menée en vue d'obtenir le même changement de composition du lait que celui observé dans les cas de mammite clinique, en liminant toutefois toute interaction avec la protolyse bactérienne. Le lactosrum issu d'un lait à fort dénombrement cellulaire (DC) avait un effet mitogénique plus faible sur une culture cellulaire d'hybridomes anti-prolactine humaine (anti-hPL) mais toutefois un effet immuno-stimulant plus important que le sérum foetal de veau. Cet effet immunostimulant a été plus marqué lorsqu'il était exprimé par cellule, avec un effet 2 à 3 fois supérieur à celui observé avec le sérum de veau foetal (P 0,01), traduisant l'efficacité de la complémentation par un lactosérum issu d'un lait à fort DC.

Ammi majus and Ruta graveolens plants were chosen to establish a co-culture due to their abilities to produce secondary metabolites from the furanocoumarin (FC) family. The conditions for cultivating A. majus hairy roots and R. graveolens shoots separately were established earlier. In continuation, the conditions of the co-culture of A. majus and R. graveolens were determined in this work. Two systems of co-cultivation were tested: A. majus hairy roots with R. graveolens cell suspension and A. majus hairy roots with R. graveolens shoots. The second model was chosen for further experiments, namely to test different tissues inoculation ratios as well as the influence of the photoperiod and darkness conditions on A. majus and R. graveolens tissues growth and the accumulation of secondary metabolites. The growth index ( t 25 / t 1 ) of R. graveolens shoots was higher in the co-cultures; moreover, 2.5 times higher concentration of xanthotoxin was observed in the co-culture in light conditions.