New pool of cortical interneuron precursors in the early postnatal dorsal white matter.

The migration of cortical γ-aminobutyric acidergic interneurons has been extensively studied in rodent embryos, whereas few studies have documented their postnatal migration. Combining in vivo analysis together with time-lapse imaging on cortical slices, we explored the origin and migration of cortical interneurons during the first weeks of postnatal life. Strikingly, we observed that a large pool of GAD65-GFP-positive cells accumulate in the dorsal white matter region during the first postnatal week. Part of these cells divides and expresses the transcription factor paired box 6 indicating the presence of local transient amplifying precursors. The vast majority of these cells are immature interneurons expressing the neuronal marker doublecortin and partly the calcium-binding protein calretinin. Time-lapse imaging reveals that GAD65-GFP-positive neurons migrate from the white matter pool into the overlying anterior cingulate cortex (aCC). Some interneurons in the postnatal aCC express the same immature neuronal markers suggesting ongoing migration of calretinin-positive interneurons. Finally, bromodeoxyuridine incorporation experiments confirm that a small fraction of interneurons located in the aCC are generated during the early postnatal period. These results altogether reveal that at postnatal ages, the dorsal white matter contains a pool of interneuron precursors that divide and migrate into the aCC.

Expression of the mammalian Xenotropic Polytropic Virus Receptor 1 (XPR1) in tobacco leaves leads to phosphate export.

Phosphate homeostasis in multicellular eukaryotes depends on both phosphate influx and efflux. The mammalian Xenotropic Polytropic Virus Receptor 1 (XPR1) shares homology to the Arabidopsis PHO1, a phosphate exporter expressed in roots. However, phosphate export activity of XPR1 has not yet been demonstrated in a heterologous system. Here, wedemonstrate that transient expression in tobacco leaves of XPR1-GFP leads to specific phosphate export. Like PHO1-GFP, XPR1-GFP is localized predominantly to the endomembrane system in tobacco cells. These results show that tobacco leaves are a good heterologous system to study the transport activity of members of the PHO1/XPR1 family.

Derivation of traceable and transplantable photoreceptors from mouse embryonic stem cells.

Retinal degenerative diseases resulting in the loss of photoreceptors are one of the major causes of blindness. Photoreceptor replacement therapy is a promising treatment because the transplantation of retina-derived photoreceptors can be applied now to different murine retinopathies to restore visual function. To have an unlimited source of photoreceptors, we derived a transgenic embryonic stem cell (ESC) line in which the Crx-GFP transgene is expressed in photoreceptors and assessed the capacity of a 3D culture protocol to produce integration-competent photoreceptors. This culture system allows the production of a large number of photoreceptors recapitulating the in vivo development. After transplantation, integrated cells showed the typical morphology of mature rods bearing external segments and ribbon synapses. We conclude that a 3D protocol coupled with ESCs provides a safe and renewable source of photoreceptors displaying a development and transplantation competence comparable to photoreceptors from age-matched retinas.

Large T Antigen-Specific Cytotoxic T Cells Protect Against Dendritic Cell Tumors through Perforin-Mediated Mechanisms Independent of CD4 T Cell Help.

Our newly generated murine tumor dendritic cell (MuTuDC) lines, generated from tumors developing in transgenic mice expressing the simian virus 40 large T antigen (SV40LgT) and GFP under the DC specific promoter CD11c, reproduce the phenotypic and functional properties of splenic wild type CD8α(+) conventional DCs. They have an immature phenotype with low co-stimulation molecule expression (CD40, CD70, CD80, and CD86) that is upregulated after activation with toll-like receptor ligands. We observed that after transfer into syngeneic C57BL/6 mice, MuTuDC lines were quickly rejected. Tumors grew efficiently in large T transgene-tolerant mice. To investigate the immune response toward the large T antigen that leads to rejection of the MuTuDC lines, they were genetically engineered by lentiviral transduction to express luciferase and tested for the induction of DC tumors after adoptive transfer in various gene deficient recipient mice. Here, we document that the MuTuDC line was rejected in C57BL/6 mice by a CD4 T cell help-independent, perforin-mediated CD8 T cell response to the SV40LgT without pre-activation or co-injection of adjuvants. Using depleting anti-CD8β antibodies, we were able to induce efficient tumor growth in C57BL/6 mice. These results are important for researchers who want to use the MuTuDC lines for in vivo studies.

Brain dynamics of meal size selection in humans.

Although neuroimaging research has evidenced specific responses to visual food stimuli based on their nutritional quality (e.g., energy density, fat content), brain processes underlying portion size selection remain largely unexplored. We identified spatio-temporal brain dynamics in response to meal images varying in portion size during a task of ideal portion selection for prospective lunch intake and expected satiety. Brain responses to meal portions judged by the participants as 'too small', 'ideal' and 'too big' were measured by means of electro-encephalographic (EEG) recordings in 21 normal-weight women. During an early stage of meal viewing (105-145ms), data showed an incremental increase of the head-surface global electric field strength (quantified via global field power; GFP) as portion judgments ranged from 'too small' to 'too big'. Estimations of neural source activity revealed that brain regions underlying this effect were located in the insula, middle frontal gyrus and middle temporal gyrus, and are similar to those reported in previous studies investigating responses to changes in food nutritional content. In contrast, during a later stage (230-270ms), GFP was maximal for the 'ideal' relative to the 'non-ideal' portion sizes. Greater neural source activity to 'ideal' vs. 'non-ideal' portion sizes was observed in the inferior parietal lobule, superior temporal gyrus and mid-posterior cingulate gyrus. Collectively, our results provide evidence that several brain regions involved in attention and adaptive behavior track 'ideal' meal portion sizes as early as 230ms during visual encounter. That is, responses do not show an increase paralleling the amount of food viewed (and, in extension, the amount of reward), but are shaped by regulatory mechanisms.

The TRAF-interacting protein (TRAIP) is a novel E2F target with peak expression in mitosis.

The TRAF-interacting protein (TRAIP) is an E3 ubiquitin ligase required for cell proliferation. TRAIP mRNA is downregulated in human keratinocytes after inhibition of the PI3K/AKT/mTOR signaling. Since E2F transcription factors are downstream of PI3K/AKT/mTOR we investigated whether they regulate TRAIP expression. E2F1 expression significantly increased the TRAIP mRNA level in HeLa cells. Reporter assays with the 1400bp 5'-upstream promoter in HeLa cells and human keratinocytes showed that E2F1-, E2F2- and E2F4-induced upregulation of TRAIP expression is mediated by 168bp upstream of the translation start site. Mutating the E2F binding site within this fragment reduced the E2F1- and E2F2-dependent promoter activities and protein-DNA complex formation in gel shift assays. Abundance of TRAIP mRNA and protein was regulated by the cell cycle with a peak in G2/M. Expression of GFP and TRAIP-GFP demonstrated that TRAIP-GFP protein has a lower steady-state concentration than GFP despite similar mRNA levels. Cycloheximide inhibition experiments indicated that the TRAIP protein has a half-life of around four hours. Therefore, the combination of cell cycle-dependent transcription of the TRAIP gene by E2F and rapid protein degradation leads to cell cycle-dependent expression with a maximum in G2/M. These findings suggest that TRAIP has important functions in mitosis and tumorigenesis.

Improvements in the Assessment of Bacterial Viability and Killing

It is axiomatic that our planet is extensively inhabited by diverse micro-organisms such as bacteria, yet the absolute diversity of different bacterial species is widely held to be unknown. Different bacteria can be found from the depths of the oceans to the top of the mountains; even the air is more or less colonized by bacteria. Most bacteria are either harmless or even advantageous to human beings but there are also bacteria, which can cause severe infectious diseases or spoil the supplies intended for human consumption. Therefore, it is vitally important not only to be able to detect and enumerate bacteria but also to assess their viability and possible harmfulness. Whilst the growth of bacteria is remarkably fast under optimum conditions and easy to detect by cultural methods, most bacteria are believed to lie in stationary phase of growth in which the actual growth is ceased and thus bacteria may simply be undetectable by cultural techniques. Additionally, several injurious factors such as low and high temperature or deficiency of nutrients can turn bacteria into a viable but non-culturable state (VBNC) that cannot be detected by cultural methods. Thereby, various noncultural techniques developed for the assessment of bacterial viability and killing have widely been exploited in modern microbiology. However, only a few methods are suitable for kinetic measurements, which enable the real-time detection of bacterial growth and viability. The present study describes alternative methods for measuring bacterial viability and killing as well as detecting the effects of various antimicrobial agents on bacteria on a real-time basis. The suitability of bacterial (lux) and beetle (luc) luciferases as well as green fluorescent protein (GFP) to act as a marker of bacterial viability and cell growth was tested. In particular, a multiparameter microplate assay based on GFP-luciferase combination as well as a flow cytometric measurement based on GFP-PI combination were developed to perform divergent viability analyses. The results obtained suggest that the antimicrobial activities of various drugs against bacteria could be successfully measured using both of these methods. Specifically, the data reliability of flow cytometric viability analysis was notably improved as GFP was utilized in the assay. A fluoro-luminometric microplate assay enabled kinetic measurements, which significantly improved and accelerated the assessment of bacterial viability compared to more conventional viability assays such as plate counting. Moreover, the multiparameter assay made simultaneous detection of GFP fluorescence and luciferase bioluminescence possible and provided extensive information about multiple cellular parameters in single assay, thereby increasing the accuracy of the assessment of the kinetics of antimicrobial activities on target bacteria.

Dpp spreading is required for medial but not for lateral wing disc growth.

Drosophila Decapentaplegic (Dpp) has served as a paradigm to study morphogen-dependent growth control. However, the role of a Dpp gradient in tissue growth remains highly controversial. Two fundamentally different models have been proposed: the 'temporal rule' model suggests that all cells of the wing imaginal disc divide upon a 50% increase in Dpp signalling, whereas the 'growth equalization model' suggests that Dpp is only essential for proliferation control of the central cells. Here, to discriminate between these two models, we generated and used morphotrap, a membrane-tethered anti-green fluorescent protein (GFP) nanobody, which enables immobilization of enhanced (e)GFP::Dpp on the cell surface, thereby abolishing Dpp gradient formation. We find that in the absence of Dpp spreading, wing disc patterning is lost; however, lateral cells still divide at normal rates. These data are consistent with the growth equalization model, but do not fit a global temporal rule model in the wing imaginal disc.

Identification of novel factors during the establishment of the Casparian strip in the endodermis

Multicellular organisms rely on specialized tissues that allow for the controlled exchange of matter with their surrounding. In order to function properly, these tissues need to establish a tight connection between the individual cells to prevent uncontrolled passive diffusion across the extracellular space. In animals, these connections are called tight and adherens junctions and are a critical feature of epithelia. These connections, however, rely on direct protein-protein interaction of plasma membrane proteins of adjacent cells. Such a mechanism is not possible in plants due to the cell wall, which encases the individual cells. In order to absorb nutrients, while simultaneously preventing uncontrolled diffusion between cells, land plants have evolved the root endodermis, which is functionally equivalent to animal epithelia. Its cells are surrounded by a precisely localized and aligned, ring-like lignin deposition, called the Casparian strip, and therefore tightly connected between each other. Very little was known about the development of the endodermis and the Casparian strip until recently. In the meantime, however, we have identified a family of endodermis- specific proteins, the CASPs, which recruits extracellular proteins the specific Casparian strip membrane domain (CSD) to locally synthesize lignin in the cell wall. Yet, we hardly knew any specifics on how the CSD is initially defined and how the critically important CASPs are being recruited to it. We therefore conducted a forward genetic screen on the localization of CASPI-GFP in order to identify novel mutants, which lack a defined CSD. We identified 48 mutants, which fell into 15 different complementation groups. While some of the isolated genes had previously been identified through different approaches, nine novel genes, which had never been implicated in CSD development and maintenance, were identified. One of them, LORD OF THE RINGS 2 (.LOTR2) is described to greater detail in this work. LOTR2 encodes for EX070A1, a protein of the evolutionary conserved exocyst complex. This complex has frequently been implicated in various secretory processes across kingdoms. In Arabidopsis, it transiently defines the positioning of CASPI-GFP. We have performed a detailed analysis of the dynamics of EX070A1 and CASPI-GFP, including studies with other markers and propose a mechanism, by which the cytosolic EX070A1 transiently defines a plasma membrane domain to recruit transmembrane proteins, which then recruit extracellular enzymes for localized cell wall modification. Considering the ubiquitous expression of EX070A1, we think that this mechanism is potentially of importance not only for the endodermis and the Casparian strip but also for many other tissues, in which the cell wall becomes locally modified. In fact, many other tissues with secondary cell wall modifications contain proteins very similar to the CASPs. It will be interesting to see to which degree this mechanism is employed in other tissues. As for the endodermis, we have now identified the first gene, which is not specific to the endodermis but shows endodermis-specific dynamics. This might give us a better insight on how the plant modulates this ubiquitously present factor in a cell- or tissue-type specific manner. Considering the knowledge, mutants and tools, which are available to us for investigating the endodermis, the Casparian strip, the exocyst complex and EX070A1 might be just the right experimental system to address these questions. -- Les organismes multicellulaires dépendent des tissues spécialisé pour l'échange contrôlé entre eux et leur environnement. Pour leur bon fonctionnement, les cellules de ces tissus ont besoin d'être très étroitement assemblés afin de prévenir la diffusion non-contrôlée à travers l'espace extracellulaire. Chez les animaux, ces connexions sont appelées jonctions serrées et jonctions adhérentes. Ces jonctions dépendent des interactions directes entre les protéines des cellules voisines. Ceci n'est pas possible chez les plantes à cause de la paroi cellulaire qui recouvre chaque cellule individuellement. Pour absorber les nutriments et en même temps empêcher la diffusion non-contrôlé entre cellules, les plantes ont évolué 1'endoderme dans la racine, qui est fonctionnellement équivalent aux épithéliums des animaux. Les cellules de l'endoderme sont ceinturées par une déposition de lignine très précisément localisées comme un anneau et alignées entre les cellules, et qui, donc, connecte étroitement les cellules avoisinante: Le cadre de Caspary. Peu était connu sur le développement de l'endoderme et le cadre de Caspaiy jusqu'à il y a quelques années. Récemment, pourtant, nous avons identifié une famille de protéines spécifiques à l'endoderme, les CASPs, qui définissent le domaine membranaire du cadre de Caspaiy (CSD). Les CASPs recrutent les protéines extracellulaires nécessaire à la synthèse du cadre de Caspary vers une région limité dans la paroi cellulaire. Pourtant, on connaît très peu les processus spécifiques concernant la définition initiale du CSD et comment les CASPs, qui ont une importance cruciale, sont recrutées vers ce domaine. Par conséquent nous avons mené un crible génétique sur la localisation du CASPI- GFP, qui sert comme marqueur pour le CSD. Notre but étant d'isoler de nouveaux mutants affectés dans l'établissement du CSD. Nous avons identifié 48 mutants, en 15 groupes de complémentation. Bien que certains des gènes isolés étaient déjà impliqué dans la formation du cadre de Caspary, neuf nouveaux gènes n'ayant jamais été impliqués dans le développement ou la maintenance du CSD ont pu être identifiés. Un de ces gènes, LORD OF THE RINGS2 (LOTR2) sera décrit plus en détail dans cette étude. LOTR2 code pour EX070A1, qui est une protéine, du complexe exocyste. Ce complexe de protéines a très bien été conservé au cours de l'évolution. Il était souvent impliqué dans plusieurs processus de sécrétion dans toutes les branches de la vie. Chez Arabidopsis, EX070A1 définit la position du CSD d'une façon transitoire et recrute CASP1- GFP. Nous avons mené une analyse détaillée des dynamiques d'EX070Al et CASPI-GFP ainsi que, des études avec des autres mutants. Nous proposons un mécanisme, d'après lequel EX070A1, recruté du cytosol, définit un domaine dans la membrane plasmique pour localiser des protéines transmembranaires, ces dernières ensuite recruteront des enzymes extracellulaires pour la modification locale de la paroi cellulaire. Vu qu'EX070A1 est exprimé dans toute dans la plante, nous pensons que ce mécanisme est potentiellement important non seulement pour l'endoderme et le cadre de Caspary, mais aussi pour les autres tissus où la paroi cellulaire doit être localement modifiée. En effet, plusieurs autres tissus contiennent des protéines très similaires aux CASPs. Il serait intéressant de voir à quelle dégrée ce mécanisme est également utilisé dans ces tissues. En ce qui concerne l'endoderme, nous avons maintenant identifié le premier gène qui n'est pas exprimé spécifiquement dans l'endoderme, mais qui montre tout de même une dynamique caractéristique dans ce tissu. Il serait intéressant de voir comment la plante peut moduler ce facteur omniprésent d'une façon spécifique. Vu les connaissances, les mutants et les outils qu'on a maintenant à notre disposition, l'endoderme et son cadre de Caspary, le complexe exocyste et EX070A1 sont probablement des bons systèmes expérimentaux pour étudier ces questions. -- Identification des nouveaux facteurs pendant l'établissement du cadre de Caspary dans l'endoderme. Lothar Kalmbach, Département de Biologie Moléculaire Végétale (DBMV), Université de Lausanne. Comme tous les autres organismes multicellulaires, les plantes terrestres dépendent de tissus spécialisés pour l'échange contrôlé avec leur environnement. Ces tissus sont importants pour l'absorption des nutriments mais également pour éviter l'influx de composés toxiques. Chez les plantes, ce tissu se trouve dans la racine. C'est l'endoderme. Grâce au cadre de Caspary, qui permet une forte connexion entre les cellules au niveau de leur paroi, l'endoderme empêche les éléments toxiques d'entrer dans le système vasculaire. Depuis quelques années, nous comprenons de plus en plus la nature et la biosynthèse, ainsi que les protéines impliquées dans l'ancrage des enzymes à la membrane plasmique. Nous n'avons eu, par contre, aucune idée sur le mécanisme qui d'abord définit cet endroit dans la membrane plasmique. Nous avons mené un crible génétique sur la localisation de CASPI-GFP, une protéine, qui recrute les enzymes extracellulaires pour la synthèse du cadre de Caspary. Nous avons identifié plusieurs nouveaux gènes qui sont impliqués dans l'intégrité du cadre de Caspary. L'un de ces gènes est EX070A1, qui est un facteur ayant un rôle important lors de la sécrétion des protéines dans tous les organismes eukaryotes. Ces mutants sont gravement affectés au niveau du cadre de Caspary, mais surtout ils ne sont plus capables de localiser CASPI-GFP. Nous avons suivi la dynamique d'EX070Al et de CASP1-GFP en combinaison avec d'autres marqueurs. Nous avons pu montrer que l'accumulation d'EX070Al est spécifique pour l'endoderme et essentielle pour bien localiser CASPI-GFP et donc, le cadre de Caspary. Ces résultats nous aident à mieux comprendre le développement de l'endoderme mais peuvent potentiellement aussi être utilisés pour étudier les modifications de la paroi cellulaire dans d'autres cellules de la plante.

Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB.

Luminescent Lanthanide Reporters: New Concepts for Use in Bioanalytical Applications

Lanthanides represent the chemical elements from lanthanum to lutetium. They intrinsically exhibit some very exciting photophysical properties, which can be further enhanced by incorporating the lanthanide ion into organic or inorganic sensitizing structures. A very popular approach is to conjugate the lanthanide ion to an organic chromophore structure forming lanthanide chelates. Another approach, which has quickly gained interest, is to incorporate the lanthanide ions into nanoparticle structures, thus attaining improved specific activity and binding capacity. The lanthanide-based reporters usually express strong luminescence emission, multiple narrow emission lines covering a wide wavelength range, and exceptionally long excited state lifetimes enabling timeresolved detection. Because of these properties, the lanthanide-based reporters have found widespread applications in various fields of life. This study focuses on the field of bioanalytical applications. The aim of the study was to demonstrate the utility of different lanthanide-based reporters in homogeneous Förster resonance energy transfer (FRET)-based bioaffinity assays. Several different model assays were constructed. One was a competitive bioaffinity assay that utilized energy transfer from lanthanide chelate donors to fluorescent protein acceptors. In addition to the conventional FRET phenomenon, a recently discovered non-overlapping FRET (nFRET) phenomenon was demonstrated for the first time for fluorescent proteins. The lack of spectral overlap in the nFRET mechanism provides sensitivity and versatility to energy transfer-based assays. The distance and temperature dependence of these phenomena were further studied in a DNA-hybridization assay. The distance dependence of nFRET deviated from that of FRET, and unlike FRET, nFRET demonstrated clear temperature dependence. Based on these results, a possible excitation mechanism operating in nFRET was proposed. In the study, two enzyme activity assays for caspase-3 were also constructed. One of these was a fluorescence quenching-based enzyme activity assay that utilized novel inorganic particulate reporters called upconverting phosphors (UCPs) as donors. The use of UCPs enabled the construction of a simple, rather inexpensive, and easily automated assay format that had a high throughput rate. The other enzyme activity assay took advantage of another novel reporter class, the lanthanidebinding peptides (LBPs). In this assay, energy was transferred from a LBP to a green fluorescent protein (GFP). Using the LBPs it was possible to avoid the rather laborious, often poorly repeatable, and randomly positioned chemical labeling. In most of the constructed assays, time-resolved detection was used to eliminate the interfering background signal caused by autofluorescence. The improved signal-to-background ratios resulted in increased assay sensitivity, often unobtainable in homogeneous assay formats using conventional organic fluorophores. The anti-Stokes luminescence of the UCPs, however, enabled the elimination of autofluorescence even without time-gating, thus simplifying the instrument setup. Together, the studied reporters and assay formats pave the way for increasingly sensitive, simple, and easily automated bioanalytical applications.

Receptors and endocytosis of coxsackievirus A9

Coxsackievirus A9 (CV-A9) belongs to human enteroviruses within family Picornaviridae, which are the main cause of aseptic meningitis. In addition, CV-A9 causes a wide range of other clinical manifestations of acute disease including respiratory infections, myocarditis, encephalitis and severe generalized infections in newborns. In this study, the functions of integrins αVβ6 and αVβ3 in the attachment and cellular entry of CV-A9 were analyzed. Further, virus and cell surface interactions and endocytosis of CV-A9 were studied in specific cell lines. Also, a method for production of GFP-expressing CV-A9 particles by long PCR-mediated mutagenesis and in vivo transcription was developed. The results indicated that RGD-motif (arginine-glycine-asparagine) that resides in the viral capsid is important for CV-A9 infection particularly in cell lines expressing integrin αVβ6 and that this integrin serves as a high affinity attachment receptor for the virus. CV-A9 is also capable of infecting certain cell lines independently of αV-integrins by binding to the cell surface HSPA5 protein. Regardless of the attachment stage, the internalization of the virus occurs via the same entry pathway and is dependent on β2M, dynamin, and Arf6 but independent of clathrin and caveolin-1. Furthermore, the virus internalization occurs within Arf6-containing vesicles suggesting that Arf6 is central mediator of CV-A9 endocytosis. While in this study the results of CV-A9 endocytosis were based on microscopical visualization within individual fixed cells, a rapid method for generation of a virus for real-time imaging was also described.

Caracterização das proteínas caveolinas -1 e -2 na placenta de conceptos bovinos clonados transgênicos

RESUMO:A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende-se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2). As caveolinas -1 foram localizadas nos vilos fetais e maternos, mas sua marcação mais forte foi observada no estroma endometrial. As caveolinas -2 tiveram marcação positiva no trofoblasto e membrana córioalantoide, e, especificamente em célula trofoblástica gigante binucleada. Sendo assim, os resultados mostram que a proteína CAV-1 teve uma maior expressão em relação à proteína CAV-2 e que as proteínas CAV-1 e -2 são parte da composição das cavéolas, sendo estruturas importantes e relacionadas com a transferência de moléculas para o feto, realizando a nutrição do mesmo mediante endocitose e pinocitose.

Fluorescence-based imaging of cellular defect in lysinuric protein intolerance (LPI)

Fluoresenssiperusteiset kuvantamismenetelmät lysinurisen proteiini-intoleranssin (LPI) soluhäiriön tutkimuksessa Lysinurinen proteiini-intoleranssi on suomalaiseen tautiperintöön kuuluva autosomaalisesti peit¬tyvästi periytyvä sairaus, jonka aiheuttaa kationisten aminohappojen kuljetushäiriö munuaisten ja ohutsuolen epiteelisolujen basolateraalikalvolla. Aminohappojen kuljetushäiriö johtaa moniin oirei¬siin, kuten kasvuhäiriöön, osteoporoosiin, immuunijärjestelmän häiriöihin, oksenteluun ja runsaspro¬teiinisen ravinnon nauttimisen jälkeiseen hyperammonemiaan. LPI-geeni SLC7A7 (solute carrier family 7 member 7) koodaa y+LAT1 proteiinia, joka on basolateraali¬nen kationisten ja neutraalien aminohappojen kuljettimen kevyt ketju, joka muodostaa heterodimee¬rin raskaan alayksikön 4F2hc:n kanssa. Tällä hetkellä SLC7A7-geenistä tunnetaan yli 50 LPI:n aiheut¬tavaa mutaatiota. Tässä tutkimuksessa erityyppisiä y+LAT1:n LPI-mutaatiota sekä yhdeksän C-terminaalista polypep¬tidiä lyhentävää deleetiota kuvannettiin nisäkässoluissa y+LAT1:n GFP (green fluorescent protein) -fuusioproteiineina. Tulokset vahvistivat muissa soluissa tehdyt havainnot siitä, että 4F2hc on edel¬lytyksenä y+LAT1:n solukalvokuljetukselle, G54V-pistemutantti sijaitsee solukalvolla samoin kuin vil¬lityyppinen proteiini, mutta lukukehystä muuttavia ja proteiinia lyhentäviä mutantteja ei kuljeteta solukalvoon. Lisäksi havaittiin, että poikkeuksena tästä säännöstä ovat y+LAT1-deleetioproteiinit, joista puuttui korkeintaan 50 C-terminaalista aminohappoa. Nämä lyhentyneet kuljettimet sijaitsevat solukalvolla kuten villityyppiset ja LPI-pistemutanttiproteiinit. Dimerisaation osuutta kuljetushäiriön synnyssä tutkittiin käyttämällä fluorescence resonance energy transfer (FRET) menetelmää. Heterodimeerin alayksiköistä kloonattiin ECFP (cyan) ja EYFP (yellow) fuusioproteiinit, joita ilmennettiin nisäkässoluissa, ja FRET mitattiin virtaussytometri-FRET -menetel¬mällä (FACS-FRET). Tutkimuksissa kaikkien mutanttien havaittiin dimerisoituvan yhtä tehokkaasti. Kul¬jetushäiriön syynä ei siten ole alayksiköiden dimerisaation estyminen mutaation seurauksena. Tutkimuksessa havaittiin, että kaikki mutantti-y+LAT1-transfektiot tuottavat vähemmän transfektoi¬tuneita soluja kuin villityyppisen y+LAT1:n transfektiot. Solupopulaatioissa, joihin oli tranfektoitu lu¬kukehystä muuttava tai stop-kodonin tuottava mutaatio havaittiin suurempi kuolleisuus kuin saman näytteen transfektoitumattomissa soluissa, kun taas villityyppistä tai G54V-pistemutanttia tuottavas¬sa solupopulaatiossa oli pienempi kuolleisuus kuin saman näytteen fuusioproteiinia ilmentämättö¬missä soluissa. Tulos osoittaa mutanttiproteiinien erilaiset vaikutukset niitä ilmentäviin soluihin, joko suoraan y+LAT1:n tai 4F2hc:n kautta aiheutuneina. LPIFin SLC7A7 lähetti-RNA:n määrä ei merkittävästi poikennut villityyppisen määrästä fibroblasteissa ja lymfoblasteissa. SLC7A7:n promoottorianalyysissä oli osoitettavissa säätelyalueita geenin 5’ ei-koo¬daavalla alueella sekä ensimmäisten kahden intronin alueella. LPI-taudin tautimekanismin kannalta keskeisin tekijä on kuitenkin aminohappokuljetuksen häiriö, jonka vaikutuksesta näistä aminohapoista riippuvaiset prosessit elimistössä eivät toimi normaalisti. Havaittu virheellinen y+LAT1/4F2hc kuljetuskompleksin sijainti edellyttää lisätutkimuksia sen mahdol¬lisen kliinisen merkityksen selvittämiseksi.

A biolistic process for in vitro gene transfer into chicken embryos

Chicken embryos kept in culture medium were bombarded using a high helium gas pressure biolistic device. To optimize the factors that affect transformation efficiency, the lacZ gene under control of the human cytomegalovirus immediate early enhancer/promoter was used as a reporter gene. There was an inverse relationship between survival rate and transformation efficiency. The best conditions obtained for high embryo survival and high transformation efficiency were achieved with 800 psi helium gas pressure, 500 mmHg vacuum, gold particles, an 8 cm DNA-coated microparticle flying distance to the embryo and embryo placement 0.5 cm from the center of the particle dispersion cone. Under these conditions, transformation efficiency was 100%, survival rate 25% and the number of expression units in the embryo body cells ranged from 100 to 1,000. Expression of green fluorescent protein was also detected in embryos bombarded under optimal conditions. Based on the results obtained, the biolistic process can be considered an efficient method for the transformation of chicken embryos and therefore can be used as a model system to study transient gene expression and tissue-specific promoters.