Caracterização química e mineralogia de solos antrópicos (terras pretas de índio) na amazônia central

Na Amazônia brasileira é comum a ocorrência de sítios arqueológicos. Frequentemente, observa-se que as ações humanas nesses sítios promoveram modificações significativas em muitas das características físicas, morfológicas e químicas dos solos desses ambientes, tornando-os muito diferentes dos solos adjacentes, especialmente nas áreas de terra firme da região. Embora muitos estudos tenham sido conduzidos visando compreender a magnitude dessas modificações e seus reflexos na gênese dos solos influenciados, muitas questões precisam ser mais bem compreendidas, sobretudo as relacionadas com a mineralogia desses solos. Este estudo teve como objetivo avaliar as características químicas, a composição mineralógica das frações argila e areia e o grau de pedogênese de cinco perfis de solos com horizonte A antrópico (Au), em ambientes de terra firme e várzea, localizados na Bacia Sedimentar do Amazonas, entre Coari e Manaus-AM. Amostras de solos foram coletadas, preparadas e submetidas às análises químicas de rotina e caracterizadas quanto aos teores de carbono oxidável e aos teores de Si, Fe, Al e P; adicionalmente, efetuou-se a identificação dos minerais presentes nas frações argila e areia, por meio de difratometria de raios X. Os resultados mostraram que as modificações promovidas pela atividade humana levaram à melhoria da fertilidade do solo, resultando em solos com acidez moderada, elevados teores de Ca2+ e de P disponíveis e baixos teores de Al3+ trocável. Esses resultados também revelaram teores muito elevados de P-total nos horizontes antrópicos, variando entre 1.630 e 8.840 mg kg-1 de P2O5. A mineralogia da fração argila dos solos antrópicos de terra firme revelou dominância de caulinita, além da ocorrência de gibbsita, de goethita e de óxidos de titânio (anatásio). O perfil de várzea, além de caulinita e goethita, apresentou também minerais do tipo 2:1 na fração argila. A mineralogia da fração areia dos solos estudados revelou dominância de quartzo, independentemente do ambiente. A fração magnética é composta por maghemita e hematita. Constatou-se, portanto, significativa riqueza química nos horizontes antrópicos, bem como diferenças na composição mineralógica desses horizontes.

Remplacement valvulaire percutané au moyen de stent-valves : une nouvelle approche thérapeutique [Stents valves for percutaneous valve replacement]

Stents have a long history in traditional valve surgery as both, porcine biological valves as well as pericardial valves are mounted on stents prior to implantation. Recently stent-mounted biological devices have been compressed up to the point, where they can be passed through a catheter. Various routes can be distinguished for implantation: open access, the trans-vascular route in antegrade or retrograde fashion, as well as direct trans-apical or trans-atrial access. Direct access has the potentialforvideo-endoscopic valve replacement. In theory, as well as in the experimental setting, valved stents have been implanted in tricuspid and caval position respectively, as well as in pulmonary, mitral and aortic locations. The largest clinical experience has been achieved in pulmonary position whereas current efforts target the aortic position.

A genetic and biochemical investigation of malondialdehyde production and turnover in Arabidopsis

Malondialdehyde (MDA) is a small reactive molecule which occurs ubiqui¬tous among eukaryotes. Interest in this molecule stems from the fact that it can be highly reactive. In green tissues of plants it is apparently formed pre¬dominantly by reactive oxygen species (ROS)-mediated non-enzymatic oxi¬dation (nLPO) of triunsaturated fatty acids (TFAs). MDA which is formed by nLPO is widely used as a disease marker and is regarded to be a cel-lular toxin. Surprisingly, sites of ROS production like mitochondria and chloroplasts possess membranes which are enriched in nLPO-prone polyun¬saturated fatty acids (PUFAs). In this work we showed that chloroplasts are the major site of MDA production in leaves of adult Arabidopsis thaliana plants, whereas analyses in seedlings revealed accumulation in meristematic tissues like the root tip, lateral roots and the apical meristem region. Char-acterizing the MDA pools in more detail, we could show that MDA in plants was predominantly present in a free, non-reactive enolate form. This might explain why it is tolerated in sites where its protonated form could poten¬tially damage the genome and proteome. Analyzing the biological fate of MDA in leaves using labeled MDA-isotopes. we were able to show that MDA is metabolized and used to assemble lipids. The major end-point metabolite was identified as 18:3-16:3-monqgalactosyldiacylglycerol (MGDG), which is the most abundant lipid in chloroplasts. We hypothesize that PUFAs in sites of ROS production, like at PS II in chloroplasts, might act as buffers pre¬venting damage of proteins, thereby generating molecules such as MDA. The MDA produced in this way appears predominantly in a non-reactive enolate form in the cell until it fulfills a biological function or until it is metabo¬lized in order to assemble polyunsaturated MGDGs. Additionally, nLPO has been reported to increase in pathogenesis and we challenged seedlings and adult plants with necrotrophic fungi. Monitoring MDA during the in¬fections, we found MDA pools in seedlings were highly inducible although they were tightly controlled in the leaves of adult plants. - Malondialdehyde (MDA) est une petite molecule réactive présente de manière ubiquitaire dans les eucaryotes. L'intérêt de cette molécule vient du fait que celle-ci pourrait être très réactive. Dans les tissus verts des plantes, la majorité du MDA est apparement formée par l'oxydation non-enzymatique (nLPO) des acides gras polyinsaturés (PUFAs) transmis par des espèces ac¬tives d'oxygène (ROS). Le MDA formé par nLPO est souvent utilisé comme marqueur de maladies et il est considéré comme une toxine cellulaire. Etonnament, les sites de production comme les mitochondries et les chloro- plastes sont riches en PUFAs qui sont sensibles à la nLPO. Dans cette thèse nous montrons que les chloroplastes répresentent le site de production de MDA dans les feuilles adultes d'Arabidopsis thaliana. Les analyses de MDA dans les plantules ont révélé que le MDA s'accumule dans les tissus meris- tematiques comme celles de la pointe de la racine, des racines latéralles et du meristème apical. Par la caractérisation du MDA présent nous avons pu montrer que la majorité du MDA était présent sous la forme d'un énolate non-réactif. Ceci pourrait expliquer pourquoi le MDA est toléré dans les sites où il pourrait casser le genome ou le protéome s'il est présent sous sa forme protonée. Les analyses du devenir du MDA dans les feuilles par des isotopes du MDA ont montré que celui-ci est metabolisé et utilisé pour assembler des lipides. Le lipide majoritairement métabolisé a été identifié comme étant le 18:3-16:3-monogalactosyldiacylglycerole (MGDG); le lipide le plus abondant dans les chloroplastes. Nous supposons que la présence des PUFAs dans les sites de production du ROS, tout comme le PS II dans les chloroplastes, pourrait jouer un rôle de tampon pour prevenir les protéines de différentes dégradations et ainsi générer des molécules telle que le MDA. La majorité du MDA produit par cette réaction est présente dans la cellule sous la forme d'énolate non-réactif, jusqu'au moment de son utilisation ou lorsqu'il serra metabolisé pour produire des MGDGs polyinsaturés. De plus, il a été décrit que nLPO pourait augmenter dans la pathogenèse, et nous avons testé des plantes adultes et des plantules en présence de champignons nécrotrophiques. L'observation du MDA pendant les infections a montré que les concentrations en MDA sont fortement induites dans les plantules mais contrôlées dans les plantes adultes.

Human Prominin-1 (CD133) Is Detected in Both Neoplastic and Non-Neoplastic Salivary Gland Diseases and Released into Saliva in a Ubiquitinated Form.

Prominin-1 (CD133) is physiologically expressed at the apical membranes of secretory (serous and mucous) and duct cells of major salivary glands. We investigated its expression in various human salivary gland lesions using two distinct anti-prominin-1 monoclonal antibodies (80B258 and AC133) applied on paraffin-embedded sections and characterized its occurrence in saliva. The 80B258 epitope was extensively expressed in adenoid cystic carcinoma, in lesser extent in acinic cell carcinoma and pleomorphic adenoma, and rarely in mucoepidermoid carcinoma. The 80B258 immunoreactivity was predominately detected at the apical membrane of tumor cells showing acinar or intercalated duct cell differentiation, which lined duct- or cyst-like structures, and in luminal secretions. It was observed on the whole cell membrane in non-luminal structures present in the vicinity of thin-walled blood vessels and hemorrhagic areas in adenoid cystic carcinoma. Of note, AC133 labeled only a subset of 80B258-positive structures. In peritumoral salivary gland tissues as well as in obstructive sialadenitis, an up-regulation of prominin-1 (both 80B258 and AC133 immunoreactivities) was observed in intercalated duct cells. In most tissues, prominin-1 was partially co-expressed with two cancer markers: carcinoembryonic antigen (CEA) and mucin-1 (MUC1). Differential centrifugation of saliva followed by immunoblotting indicated that all three markers were released in association with small membrane vesicles. Immuno-isolated prominin-1-positive vesicles contained CEA and MUC1, but also exosome-related proteins CD63, flotillin-1, flotillin-2 and the adaptor protein syntenin-1. The latter protein was shown to interact with prominin-1 as demonstrated by its co-immunoisolation. A fraction of saliva-associated prominin-1 appeared to be ubiquitinated. Collectively, our findings bring new insights into the biochemistry and trafficking of prominin-1 as well as its immunohistochemical profile in certain types of salivary gland tumors and inflammatory diseases.

Comunidade de fungos micorrízicos arbusculares: diversidade, composição e glomalina em área revegetada com sesbânia

A composição de comunidades e diversidade de fungos micorrízicos arbusculares (FMAs) pode ser influenciada por diversos fatores como o clima, a biota do solo e as plantas hospedeiras. Este estudo objetivou avaliar a influência da revegetação de uma cava de extração de argila com Sesbania virgata (SV) em plantios puro e consorciado com Eucalyptus camaldulensis (EC) e Acacia mangium (AM), na composição e diversidade de fungos micorrízicos arbusculares (FMAs), bem como na quantidade da proteína do solo relacionada à glomalina (PSRG). O delineamento experimental utilizado foi em blocos casualizados com quatro tratamentos (plantio puro de SV - 100SV; consórcio de SV + EC - 50SV:50EC; consórcio de SV + AM - 50SV:50AM; e área degradada com vegetação espontânea - ADVE), com três repetições. A revegetação da cava de extração de argila com SV, em plantios puro ou consorciado, reduziu a abundância de esporos e a dominância de espécies (Índice de Simpson - IDS) e aumentou a riqueza de espécies de FMAs e o índice de diversidade de Shannon e Wiener. Além disso, aumentou a quantidade de proteína do solo relacionada à glomalina, quando comparada a área degradada com vegetação espontânea.

Caracterização de vertissolos da ilha de Fernando de Noronha, Pernambuco

O Arquipélago de Fernando de Noronha (FN) possui solos pouco desenvolvidos das classes dos Cambissolos, Vertissolos e Neossolos, com características peculiares relacionadas ao material de origem vulcânico, clima tropical com franco domínio oceânico e relevo ondulado a forte ondulado. Os Vertissolos de FN são formados a partir de rochas básicas, tufos vulcânicos e sedimentos aluvionares e estão associados às superfícies rebaixadas com drenagem impedida do planalto central ou da baixada litorânea. Tendo em vista a necessidade de preservação ambiental, uso agrícola ou geotécnico e características peculiares desse ambiente insular, os Vertissolos de FN precisam de melhor entendimento de seus atributos. O objetivo deste trabalho foi, portanto, caracterizar morfológica, física, química e mineralogicamente Vertissolos de ocorrência comum na ilha de Fernando de Noronha. Três perfis de Vertissolos derivados de diferentes materiais de origem, classes de drenagem e níveis de salinidade e sodicidade foram descritos e coletados para realização das análises de caracterização. Os solos estudados foram: Vertissolo Háplico órtico solódico (P01), Vertissolo Háplico sálico gleissólico (P02) e Vertissolo Háplico sódico gleissólico (P03). Os Vertissolos de FN apresentam feições típicas da ordem, como a textura argilosa a muito argilosa, as superfícies de fricção (slickensides) e o fendilhamento horizontal e vertical quando secos. Esses solos são imperfeitamente a maldrenados e sofrem alagamento temporário no período de maior concentração das chuvas. Apresentam elevados valores de soma e saturação por bases, além de teores altos a muito altos de P extraível, de distribuição irregular entre perfis e com dominância de formas inorgânicas. Não obstante, esses apresentam problemas de acumulação de sais, o que restringe a utilização agrícola ou geotécnica deles. Os principais minerais da fração argila dos solos são do grupo das esmectitas, seguidos por caulinitas e, ou, haloisitas. A fração silte é constituída por hematita, goethita, magnetita/maghemita, ilmenita, mica e feldspato e por minerais pouco comuns em solos brasileiros como crandalita e holandita.

LKB1 regulates polarity remodeling and adherens junction formation in the Drosophila eye.

The serine-threonine kinase LKB1 regulates cell polarity from Caenorhabditis elegans to man. Loss of lkb1 leads to a cancer predisposition, known as Peutz-Jeghers Syndrome. Biochemical analysis indicates that LKB1 can phosphorylate and activate a family of AMPK- like kinases, however, the precise contribution of these kinases to the establishment and maintenance of cell polarity is still unclear. Recent studies propose that LKB1 acts primarily through the AMP kinase to establish and/or maintain cell polarity. To determine whether this simple model of how LKB1 regulates cell polarity has relevance to complex tissues, we examined lkb1 mutants in the Drosophila eye. We show that adherens junctions expand and apical, junctional, and basolateral domains mix in lkb1 mutants. Surprisingly, we find LKB1 does not act primarily through AMPK to regulate cell polarity in the retina. Unlike lkb1 mutants, ampk retinas do not show elongated rhabdomeres or expansion of apical and junctional markers into the basolateral domain. In addition, nutrient deprivation does not reveal a more dramatic polarity phenotype in lkb1 photoreceptors. These data suggest that AMPK is not the primary target of LKB1 during eye development. Instead, we find that a number of other AMPK-like kinase, such as SIK, NUAK, Par-1, KP78a, and KP78b show phenotypes similar to weak lkb1 loss of function in the eye. These data suggest that in complex tissues, LKB1 acts on an array of targets to regulate cell polarity.

Tempo de Captura de Organismos da Mesofauna do Solo e seus Reflexos na Interpretação de Índices da Comunidade Edáfica

RESUMO A mesofauna edáfica compreende organismos de dimensões entre 100 μm e 2 mm, sendo importante avaliar os métodos utilizados para sua caracterização. Os objetivos deste trabalho foram definir o período de tempo de permanência do material de solo no extrator Berlese-Tüllgren para a captura dos organismos da mesofauna e determinar o tempo mínimo de extração para a correta utilização dos índices de Shannon, Simpson, Pielou e Margalef, empregados na avaliação da diversidade, dominância e equabilidade das comunidades da mesofauna. O estudo foi conduzido no Departamento de Solos do Instituto de Agronomia da Universidade Federal Rural do Rio de Janeiro, Seropédica, Rio de Janeiro, em terreno altamente antropizado. Foram instaladas parcelas experimentais com os seguintes tratamentos: feijão-de-porco (Canavalia ensiformis), mucuna-preta (Mucuna aterrima), feijão-bravo-do-Ceará (Canavalia brasiliensis), guandu (Cajanus cajan) e vegetação espontânea, em delineamento de quadrado latino. Em cada uma das unidades experimentais, foram coletadas amostras formadas por serapilheira e material de solo, nos primeiros 5 cm da camada superficial do solo realizada um dia antes do plantio das leguminosas (outubro) e, posteriormente, por ocasião do corte da parte aérea das plantas (março). As amostras foram submetidas a uma bateria de extratores do tipo Berlese-Tüllgren, por um período de 15 dias, realizando-se a contagem diária dos organismos. A partir da contagem e identificação em nível de grupo (Ordem/Família) dos organismos capturados, foram determinados os índices de Margalef, Simpson, Shannon e Pielou, bem como verificou-se a correlação por ordem de Kendall entre os valores obtidos dia a dia até o 8º dia de extração, usando-se o índice de Shannon como referência. Concluiu-se que é possível a redução do tempo de permanência das amostras de material de solo nos funis Berlese-Tüllgren, não sendo recomendável a adoção de período de tempo inferior a seis dias para se determinar a composição da mesofauna edáfica ou para se obterem dados mais consistentes e confiáveis para se avaliarem os índices de diversidade, quando se utiliza esse procedimento.

The Lorenz-maximal core allocations and the kernel in some classes of assignment games

En aquest treball demostrem que en la classe de jocs d'assignació amb diagonal dominant (Solymosi i Raghavan, 2001), el repartiment de Thompson (que coincideix amb el valor tau) és l'únic punt del core que és maximal respecte de la relació de dominància de Lorenz, i a més coincideix amb la solucié de Dutta i Ray (1989), també coneguda com solució igualitària. En segon lloc, mitjançant una condició més forta que la de diagonal dominant, introduïm una nova classe de jocs d'assignació on cada agent obté amb la seva parella òptima almenys el doble que amb qualsevol altra parella. Per aquests jocs d'assignació amb diagonal 2-dominant, el repartiment de Thompson és l'únic punt del kernel, i per tant el nucleolo.

Spermiogenesis and spermatozoon ultrastructure of the bothriocephalidean cestode Clestobothrium crassiceps (Rudolphi, 1819), a parasite of the teleost fish Merluccius merluccius (Gadiformes: Merlucciidae)

Spermiogenesis and the ultrastructure of the spermatozoon of the bothriocephalidean cestode Clestobothrium crassiceps (Rudolphi, 1819), a parasite of the teleost fish Merluccius merluccius (Linnaeus, 1758), have been studied by means of transmission electron microscopy. Spermiogenesis involves firstly the formation of a differentiation zone. It is characterized by the presence of two centrioles associated with striated rootlets, an intercentriolar body and an electron-dense material in the apical region of this zone. Later, two flagella develop from the centrioles, growing orthogonally in relation to the median cytoplasmic process. Flagella then undergo a rotation of 90° until they become parallel to the median cytoplasmic process, followed by the proximodistal fusion of the flagella with the median cytoplasmic process. The nucleus elongates and afterwards it migrates along the spermatid body. Spermiogenesis finishes with the appearance of the apical cone surrounded by the single helical crested body at the base of the spermatid. Finally, the narrowing of the ring of arched membranes detaches the fully formed spermatozoon. The mature spermatozoon of C. crassiceps is filiform and contains two axonemes of the 9 + '1' trepaxonematan pattern, a parallel nucleus, parallel cortical microtubules, and electron-dense granules of glycogen. The anterior extremity of the gamete exhibits a short electron-dense apical cone and one crested body, which turns once around the sperm cell. The first axoneme is surrounded by a ring of thick cortical microtubules that persist until the appearance of the second axoneme. Later, these thick cortical microtubules disappear and thus, the mature spermatozoon exhibits two bundles of thin cortical microtubules. The posterior extremity of the male gamete presents only the nucleus. Results are discussed and compared particularly with the available ultrastructural data on the former 'pseudophyllideans'. Two differences can be established between spermatozoa of Bothriocephalidea and Diphyllobothriidea, the type of spermatozoon (II vs I) and the presence/absence of the ring of cortical microtubules.

Alterations in the local myocardial motion pattern in patients suffering from pressure overload due to aortic stenosis.

BACKGROUND: MR tissue tagging allows the noninvasive assessment of the locally and temporally resolved motion pattern of the left ventricle. Alterations in cardiac torsion and diastolic relaxation of the left ventricle were studied in patients with aortic stenosis and were compared with those of healthy control subjects and championship rowers with physiological volume-overload hypertrophy. METHODS AND RESULTS: Twelve aortic stenosis patients, 11 healthy control subjects with normal left ventricular function, and 11 world-championship rowers were investigated for systolic and diastolic heart wall motion on a basal and an apical level of the myocardium. Systolic torsion and untwisting during diastole were examined by use of a novel tagging technique (CSPAMM) that provides access to systolic and diastolic motion data. In the healthy heart, the left ventricle performs a systolic wringing motion, with a counterclockwise rotation at the apex and a clockwise rotation at the base. Apical untwisting precedes diastolic filling. In the athlete's heart, torsion and untwisting remain unchanged compared with those of the control subjects. In aortic stenosis patients, torsion is significantly increased and diastolic apical untwisting is prolonged compared with those of control subjects or athletes. CONCLUSIONS: Torsional behavior as observed in pressure- and volume-overloaded hearts is consistent with current theoretical findings. A delayed diastolic untwisting in the pressure-overloaded hearts of the patients may contribute to a tendency toward diastolic dysfunction.

Organisation and role of actin microfilaments during cellular growth and morphogenesis in the moss Physcomitrella patens

ABSTRACT The network of actin cytoskeleton is composed of actin filaments (F-actin) that are made by polymerisation of actin monomers and actin binding proteins. It is required for growth and morphogenesis of eukaryotic cells. The labelling of F-actin with constitutively expressed GFP-Talin (Kost et al., 1998) reveals the organisation of cellular actin networks in plants. Due to the lack of information on actin cytoskeleton through gametophytic development of the model moss plant Physcornitrella patens, stable transgenic lines overexpressing GFP-Talin were generated to detect F-actin structures. It is shown that the 35S promoter driven expression is not suitable for F-actin labelling in all cells. When it is replaced by the inducible heat-shock promoter Gmhsp17.3 from soybean, one hour mild heat stress at 37°C followed by recovery at 25°C is enough to induce efficient and transient labelling in all tissues without altering cellular morphology. The optimal observations of F-actin structures at different stages of moss development can be done between 12-18 hours after the induction. By using confocal microscopy, we demonstrate that stellated actin arrays were densely accumulated at the growing tip in regenerating protoplasts, apical protonemal cells and rhizoids and connected with a fine dispersed F-actin mesh. Following three-dimensional growth, the cortical star-like structures are widespread in the meristematic cells of developing bud and young gametophores. On the contrary, undulating networks of actin cables are found at the final stage of cell differentiation. During redifferentiation of mature leaf cells into protonemal filaments the rather stagnant web of actin cables is replaced by diffuse actin meshwork. In eukaryotes, nucleation of the actin monomers prior to their polymerization is driven by the seven-subunit ARP2/3 complex and formins. We cloned the gene encoding the ARP3 subunit of P. patens and generated arp3 mutants of the moss through gene disruption. The knockout of ARP3 affects the elongation of chloronemal cells and blocks further differentiation of caulonemal cells and rhizoids, and the gametophores are slightly stunted compared to wild-type. The arp mutants were created in the heat-shock inducible GFP-Talin strains allowing us to visualise a disorganised actin network and a lack of star-like actin cytoskeleton arrays. We conclude that ARP2/3 dependent nucleation of actin filaments is critical for the growth of filamentous cells, which in turn influences moss colonization. In complementation assays, the overexpression of Physcomitrella and Arab idopsis ARP3 genes in the moss arp3 mutant results in full recovery of wild type phenotype. In contrast the ARP3 subunit of fission yeast is not able to complement the moss arp3 mutant of moss indicating that regulation of the ARP2/3 dependent actin nucleation diverged in different kingdoms. RESUME Le réseau d'actine est composé de filaments de F-actine et d'un ensemble de protéines s'y attachant (Actin binding proteins). Le réseau d'actine est nécessaire à la croissance et à la morphogenèse de toutes les cellules eucaryotes. Chez les plantes, le marquage ainsi que l'étude de l'organisation du réseau d'actine ont été réalisés en utilisant une fusion GFP-Talin (Kost et al., 1998) exprimée sous le control d'un promoteur constitutif. Afin d'étudier les structures F-actine dans les cellules de Physcomitrella Patens et pour combler le manque d'information sur le développement des gamétophores, des lignées transgéniques stables surexprimant GFP-Talin ont été crées. Nous avons démontré que l'utilisation du promoteur 35S est inadéquate pour le marquage complet et homogène des filaments d'actine dans toutes les cellules de P. patens. Par contre, l'utilisation du promoteur inductible Gmhsp17.3 nous a permis de réaliser un marquage transitoire et général dans tous les tissus de la mousse. Une heure de choc thermique à 37°C suivis d'un temps de récupération de 12-18h à 25°C sont les conditions optimales (sans dommages cellulaires) pour l'observation des structures F-actine à différentes étapes de développement de la mousse. En utilisant la microscopie confocale, nous avons observé l'existence de structures F-actine accumulées en forme d'étoiles. Ces structures, qui sont liées au réseau de microfilaments d'actine, ont été observées dans les protoplastes en régénération, les cellules des protonema apicales ainsi que dans les rhizoïdes. En suivant la croissance tridimensionnelle, ces structures en étoiles ont été observées dans les cellules meristématiques des bourgeons et des jeunes gamétophores. Par contre, dans les cellules différentiées ces structures laissent place à des réseaux de câbles épais. Nous avons également remarqué que durant la redifferentiation des cellules foliaires le réseau de câbles de F-actine est remplacé par un réseau de F-actine diffus. Dans les cellules eucaryotes, la nucléation des filaments d'actirie précédant leur polymérisation est contrôlé par sept sous unités du complexe ARP2/3 et par des formines. Nous avons isolé le gène codant pour la sous unité ARP3 de P. patens et nous avons crée des mutants arp3 par intégration ciblée (Knockout). L'élongation des cellules chloronema est clairement affectée dans les mutants arp3. La différentiation des caulonemata et des rhizoïdes est bloquée et les gametophores sont légèrement plus courts comparé au type sauvage. A fin d'étudier l'organisation des filaments d'actines dans les mutants arp3, nous avons aussi réalisé un arp3-knockout dans la lignée Hsp-GFP-Talin. La nouvelle lignée générée nous a permis de visualiser une désorganisation du réseau d'actine et une absence complète de structures de F-actine accumulée en forme d'étoiles. Les résultats obtenus nous amènent à conclure que la nucléation (ARP2/3 dépendante) des filaments d'actine est indispensable à la croissance des cellules filamenteuses. Par conséquent, les filaments d'actine semblent avoir un rôle dans la colonisation des milieux par les mousses. Nous avons également procédé à des essais de complémentation du mutant arp3. La surexpression des gènes ARP3 de Physcomitrella et d'Arabidopsis dans les cellules du mutant arp3 rétabli complètement le phénotype WT. Par contre, le gène ARP3 des levures n'est pas suffisant pour complémenter la même mutation dans les cellules de mousses. Ce résultat démontre que les mécanismes de régulation de la nucléation des filaments d'actine (ARP2/3 dépendante) sont différents entre les différents groupes d'eucaryotes.

Hypoxia-induced inhibition of epithelial Na(+) channels in the lung. Role of Nedd4-2 and the ubiquitin-proteasome pathway.

Transepithelial sodium transport via alveolar epithelial Na(+) channels (ENaC) and Na(+),K(+)-ATPase constitutes the driving force for removal of alveolar edema fluid. Alveolar hypoxia associated with pulmonary edema may impair ENaC activity and alveolar Na(+) absorption through a decrease of ENaC subunit expression at the apical membrane of alveolar epithelial cells (AECs). Here, we investigated the mechanism(s) involved in this process in vivo in the β-Liddle mouse strain mice carrying a truncation of β-ENaC C-terminus abolishing the interaction between β-ENaC and the ubiquitin protein-ligase Nedd4-2 that targets the channel for endocytosis and degradation and in vitro in rat AECs. Hypoxia (8% O2 for 24 h) reduced amiloride-sensitive alveolar fluid clearance by 69% in wild-type mice but had no effect in homozygous mutated β-Liddle littermates. In vitro, acute exposure of AECs to hypoxia (0.5-3% O2 for 1-6 h) rapidly decreased transepithelial Na(+) transport as assessed by equivalent short-circuit current Ieq and the amiloride-sensitive component of Na(+) current across the apical membrane, reflecting ENaC activity. Hypoxia induced a decrease of ENaC subunit expression in the apical membrane of AECs with no change in intracellular expression and induced a 2-fold increase in α-ENaC polyubiquitination. Hypoxic inhibition of amiloride-sensitive Ieq was fully prevented by preincubation with the proteasome inhibitors MG132 and lactacystin or with the antioxidant N-acetyl-cysteine. Our data strongly suggest that Nedd4-2-mediated ubiquitination of ENaC leading to endocytosis and degradation of apical Na(+) channels is a key feature of hypoxia-induced inhibition of transepithelial alveolar Na(+) transport.

Spore germination and development of young mycelia in some Rhizopogon species

Spore germination in Rhizopogon abietis, R. luteolus, R. roseolus and R. villosulus was induced in the presence of Rhodotorula glutinis and activated charcoal, in agar medium (N6:5). In one R. roseolus sample, 51% of spores germinated within 35 days, allowing observation of the course of spore germination and the different developmental patterns of homokaryotic mycelia. In these plates, spores showed two times of germination. The spores that germinated early produced an apical germ tube. Later other spores germinated in proximity to young mycelium, by forming a germ vesicle. One of the hyphal growth patterns obtained (interruption-swelling-ramification) is similar to that reported for other fungi. With this technique for inducing spore germination, it is possible to obtain enough monosporic cultures to perform mating tests. Key words: Basidiomycotina, Hypogeous, Monosporic Cultures.

Secretory IgA and intestinal epithelial cells at the interface between homeostasis regulation and protection against infection

RESUME DESTINE A UN LARGE PUBLICL'intestin est le siège d'intenses agressions de la part de l'ensemble des aliments ingérés, de bactéries agressives dites pathogènes mais également de bactéries dites commensales peuplant naturellement les surfaces intestinales muqueuses. Pour faire face, notre organisme arbore de nombreux niveaux de protections tant physiques, chimiques, mécaniques mais aussi immunitaires. La présence d'un type particulier de cellules, les cellules épithéliales (IEC) assurant une protection physique, ainsi que la production d'anticorps spécialisés par le système immunitaire appelés immunoglobulines sécrétoires A (SlgA) servent conjointement de première ligne de défense contre ces agressions externes. Néanmoins, comment le dialogue s'articule entre ces deux partenaires reste incomplet.Nous avons donc décidé de mimer ces interactions en modélisant les surfaces muqueuses par une monocouche de cellules différenciées en laboratoire. Des souches bactériennes isolées de l'intestin humain seules ou associées à des SlgA non-spécifiques ont été mises au contact de ce modèle cellulaire nous permettant de conclure quant à la présence effective d'une modulation du dialogue bactérie/lEC impliquant une activation de la réponse cellulaire vers un état de tolérance mutuelle. De façon surprenante, nous avons par ailleurs mis en évidence un type d'interaction nouveau entre ces anticorps et ces bactéries. Une étude biochimique nous a permis de détailler un nouveau rôle des SlgA médié par les sucres présents à leur surface dans le maintien d'une relation pacifique avec les commensaux perpétuellement présents, relations qualifiées d'homésostase intestinale.Le rôle protecteur des SlgA a par ailleurs été abordé pour avoir une meilleure appréhension de leur impact au niveau cellulaire lors d'infection par Shigella flexneri, bactérie causant la Shigellose, diarrhée sanglante responsable de la mort de plus d'un million de personnes chaque année. Basée sur le même modèle cellulaire, cette étude nous a permis de démontrer une nouvelle entrée de ce pathogène directement via les IEC. La présence d'anticorps spécifiques à la surface des bactéries restreint leur champs d'action contre les cibles intracellulaires identifiées que sont les filaments soutenant le squelette de la cellule, les fibres d'actine ainsi que les jonctions serrées, réseaux de protéines clés des interactions entre cellules. Cette ouverture au niveau cellulaire apporte un nouvel élan quant à la compréhension du rôle protecteur des SlgA lors d'attaques de l'intestin, protection semblant dépendante d'une agrégation des bactéries.Pour finir, nous avons mis en évidence la détection directe par les cellules de la présence d'anticorps libres dans l'intestin ajoutant une nouvelle réplique dans le dialogue complexe entre ces deux piliers de l'équilibre intestinal que sont les SlgA et les cellules épithéliales.RESUMELa muqueuse intestinale est dotée d'un réseau complexe de protections physico-chimiques, mécaniques ou immunologiques. Associées à un système immunitaire omniprésent, les cellules épithéliales intestinales {IEC) bordant la lumière intestinale ont la double tâche de protéger l'intérieur de l'organisme stérile contre l'invasion et la dissémination d'agents pathogènes, et de maintenir une relation pacifique avec la flore intestinale, rôles également joués par les immunoglobulines sécrétoires A (SlgA), anticorps les plus abondamment présents à la surface des muqueuses. Tant les IEC que les SlgA sont ainsi décrites comme convergeant vers le même objectif ; néanmoins, les rouages de leurs interactions restent largement inconnus.Pour répondre à cette question, des monocouches épithéliales reconstituées in vitro ont été incubées avec des souches commensales telles que des Lactobacillus ou des Bifodobacteria, seules ou complexées avec des SlgA non-spécifiques, nous permettant de décrypter l'influence des SlgA sur la détection des bactéries par les IEC, favorisant l'adhésion bactérienne et la cohésion cellulaire, augmentant l'activation de la voie NF-κΒ ainsi que la sécrétion de la cytokine thymic stromal lymphopoietin contrairement à celle de médiateurs pro-inflammatoires qui reste inchangée. Par ailleurs, une interaction Fab-indépendante est suggérée dans l'interaction SlgA/bactéries. Comme une interaction de faible affinité a été décrite comme prenant naturellement place au niveau de l'intestin, nous avons donc disséqué les mécanismes sous- jacents en utilisant un large spectre de bactérie associés à des protéines soit recombinantes soit isolées à partir de colostrum, mettant en évidence un rôle crucial des N-glycanes présents sur la pièce sécrétoire et soulignant une nouvelle propriété des SlgA dans l'homéostase intestinale.Intrinsèquement liés aux caractéristiques des SlgA, nous nous sommes également focalisés sur leur rôle protecteur lors d'infection par l'enteropathogène Shigella flexneri reproduites in vitro sur des monocouches polarisées. Nous avons tout d'abord démontré une nouvelle porte d'entrée pour ce pathogène directement via les IEC. L'agrégation des bactéries par les SlgA confère aux cellules une meilleure résistance à l'infection, retardant croissance bactérienne et entrée cellulaire, affectant par ailleurs leur capacité à cibler le cytosquelette et les jonctions serrées. La formation de tels cargos détectés de façon biaisée par les IEC apparaît comme une explication plausible au maintien de la cohésion cellulaire médiée par les SlgA.Enfin, le retrotransport des SlgA à travers les IEC a été abordé soulignant une participation active de ces cellules dans la détection de l'environnement extérieur, les impliquant possiblement dans l'activation d'un état muqueux stable.Conjointement, ces résultats indiquent que les SlgA représentent l'un des éléments-clés à la surface de la muqueuse et soulignent la complexité du dialogue établi avec l'épithélium en vue du maintien d'un fragile équilibre intestinal.ABSTRACTThe intestinal mucosa is endowed with a complex protective network melting physiochemical, mechanical and immunological features. Beyond the ubiquitous intestinal immune system, intestinal epithelial cells (IEC) lying the mucosal surfaces have also the dual task to protect the sterile core against invasion and dissemination of pathogens, and maintain a peaceful relationship with commensal microorganisms, aims also achieved by the presence of high amounts of secretory immunoglobulins A (SlgA), the most abundant immunoglobulin present at mucosal surfaces. Both IEC and SlgA are thus described to converge toward the same goal but how their interplay is orchestrated is largely unknown.To address this question, in vitro reconstituted IEC monolayers were first apically incubated with commensal bacteria such as Lactobacillus or Bifodobacteria strains either alone or in complexes with non-specific SlgA. Favoring the bacterial adhesion and cellular cohesion, SlgA impacts on the cellular sensing of bacteria, increasing NF-κΒ activation, and leading to cytokine releases restricted to the thymic stromal lymphopoietin and unaffected expression of pro-inflammatory mediators. Of main interest, bacterial recognition by SlgA suggested a Fab-independent interaction. As this low affinity, called natural coating occurs in the intestine, we further dissected the underlying mechanisms using a larger spectrum of commensal strains associated with recombinant as well as colostrum-derived proteins and pinpointed a crucial role of N-glycans of the secretory component, emphasizing an underestimated role of carbohydrates and another properties of SlgA in mediating intestinal homeostasis.As mucosal protection is also anchored in SlgA and IEC features, we focused on the cellular role of SlgA. Using IEC apical infection by the enteropathogen Shigella flexneri, we have first demonstrated a new gate of entry for this pathogen directly via IEC. Specific SlgA bacterial aggregation conferred to the cells a better resistance to infection, delaying bacterial growth and cellular entry, affecting their ability to damage both the cytoskeleton and the tight junctions. Formation of such big cargos differentially detected by IEC appears as a plausible explanation sustaining at the cellular level the antibody-mediated mucosal protection.Finally, SlgA retrotransport across IEC has been tackled stressing an active IEC sensing of the external environment possibly involved in the steady-state mucosal activation.All together, these results indicate that SlgA represents one of the pivotal elements at mucosal surfaces highlighting the complexity of the dialogue established with the epithelium sustaining the fragile intestinal balance.The Intestinal mucosa is endowed with a complex protective network melting physiochemical, mechanical and immunological features. Beyond the ubiquitous intestinal immune system, intestinal epithelial cells (IEC) lying the mucosal surfaces have also the dual task to protect the sterile core against invasion and dissemination of pathogens, and maintain a peaceful relationship with commensal microorganisms, aims also achieved by the presence of high amounts of secretory immunoglobulins A (SlgA), the most abundant immunoglobulin present at mucosal surfaces. Both IEC and SlgA are thus described to converge toward the same goal but how their interplay is orchestrated is largely unknown.To address this question, in vitro reconstituted IEC monolayers were first apically incubated with commensal bacteria such as Lactobacillus or Bifodobacteria strains either alone or in complexes with non-specific SlgA. Favoring the bacterial adhesion and cellular cohesion, SlgA impacts on the cellular sensing of bacteria, increasing NF-κΒ activation, and leading to cytokine releases restricted to the thymic stromal lymphopoietin and unaffected expression of pro-inflammatory mediators. Of main interest, bacterial recognition by SlgA suggested a Fab-independent interaction. As this low affinity, called natural coating occurs in the intestine, we further dissected the underlying mechanisms using a larger spectrum of commensal strains associated with recombinant as well as colostrum-derived proteins and pinpointed a crucial role of N-glycans of the secretory component, emphasizing an underestimated role of carbohydrates and another properties of SlgA in mediating intestinal homeostasis.As mucosal protection is also anchored in SlgA and IEC features, we focused on the cellular role of SlgA. Using IEC apical infection by the enteropathogen Shigella flexneri, we have first demonstrated a new gate of entry for this pathogen directly via IEC. Specific SlgA bacterial aggregation conferred to the cells a better resistance to infection, delaying bacterial growth and cellular entry, affecting their ability to damage both the cytoskeleton and the tight junctions. Formation of such big cargos differentially detected by IEC appears as a plausible explanation sustaining at the cellular level the antibody-mediated mucosal protection.Finally, SlgA retrotransport across IEC has been tackled stressing an active IEC sensing of the external environment possibly involved in the steady-state mucosal activation.All together, these results indicate that SlgA represents one of the pivotal elements at mucosal surfaces highlighting the complexity of the dialogue established with the epithelium sustaining the fragile intestinal balance.