Transcriptional regulatory patterns of the myelin basic protein and malic enzyme genes by the thyroid hormone receptors alpha1 and beta1.

While there is evidence that the two ubiquitously expressed thyroid hormone (T3) receptors, TRalpha1 and TRbeta1, have distinct functional specificities, the mechanism by which they discriminate potential target genes remains largely unexplained. In this study, we demonstrate that the thyroid hormone response elements (TRE) from the malic enzyme and myelin basic protein genes (METRE and MBPTRE) respectively, are not functionally equivalent. The METRE, which is a direct repeat motif with a 4-base pair gap between the two half-site hexamers binds thyroid hormone receptor as a heterodimer with 9-cis-retinoic acid receptor (RXR) and mediates a high T3-dependent activation in response to TRalpha1 or TRbeta1 in NIH3T3 cells. In contrast, the MBPTRE, which consists of an inverted palindrome formed by two hexamers spaced by 6 base pairs, confers an efficient transactivation by TRbeta1 but a poor transactivation by TRalpha1. While both receptors form heterodimers with RXR on MBPTRE, the poor transactivation by TRalpha1 correlates also with its ability to bind efficiently as a monomer. This monomer, which is only observed with TRalpha1 bound to MBPTRE, interacts neither with N-CoR nor with SRC-1, explaining its functional inefficacy. However, in Xenopus oocytes, in which RXR proteins are not detectable, the transactivation mediated by TRalpha1 and TRbeta1 is equivalent and independent of a RXR supply, raising the question of the identity of the thyroid hormone receptor partner in these cells. Thus, in mammalian cells, the binding characteristics of TRalpha1 to MBPTRE (i.e. high monomer binding efficiency and low transactivation activity) might explain the particular pattern of T3 responsiveness of MBP gene expression during central nervous system development.

Application of RNAi to silence tartrate-resistant acid phosphatase: unexpected effects on the monocyte-macrophage lineage

Tartraatti-resistentin happaman fosfataasin hiljentäminen RNAi menetelmällä: odottamaton vaikutus monosyytti-makrofagi linjan soluissa RNA interferenssi (RNAi) eli RNA:n hiljentyminen löydettiin ensimmäisenä kasveissa, ja 2000-luvulla RNAi menetelmä on otettu käyttöön myös nisäkässoluissa. RNAi on mekanismi, jossa lyhyet kaksi juosteiset RNA molekyylit eli siRNA:t sitoutuvat proteiinikompleksiin ja sitoutuvat komplementaarisesti proteiinia koodaavaan lähetti RNA:han katalysoiden lähetti RNA:n hajoamisen. Tällöin RNA:n koodaamaa proteiinia ei solussa tuoteta. Tässä työssä on RNA interferenssi menetelmän avuksi kehitetty uusi siRNA molekyylien suunnittelualgoritmi siRNA_profile, joka etsii lähetti RNA:sta geenin hiljentämiseen sopivia kohdealueita. Optimaalisesti suunnitellulla siRNA molekyylillä voi olla mahdollista saavuttaa pitkäaikainen geenin hiljeneminen ja spesifinen kohdeproteiinin määrän aleneminen solussa. Erilaiset kemialliset modifikaatiot, mm. 2´-Fluoro-modifikaatio, siRNA molekyylin riboosirenkaassa lisäsivät siRNA molekyylin stabiilisuutta veren plasmassa sekä siRNA molekyylin tehokkuutta. Nämä ovat tärkeitä siRNA molekyylien ominaisuuksia kun RNAi menetelmää sovelletaan lääketieteellisiin tarkoituksiin. Tartraatti-resistentti hapan fosfataasi (TRACP) on entsyymi, joka esiintyy luunsyöjäsoluissa eli osteoklasteissa, antigeenejä esittelevissä dendiriittisissä soluissa sekä eri kudosten makrofageissa, jotka ovat syöjäsoluja. TRACP entsyymin biologista tehtävää ei ole saatu selville, mutta oletetaan että TRACP entsyymin kyvyllä tuottaa reaktiivisia happiradikaaleja on tehtävä sekä luuta hajoittavissa osteoklasteissa sekä antigeenia esittelevissä dendriittisissä soluissa. Makrofageilla, jotka yliekpressoivat TRACP entsyymiä, on myös solunsisäinen reaktiivisten happiradikaalien tuotanto sekä bakteerin tappokyky lisääntynyt. TRACP-geenin hiljentämiseen tarkoitetut spesifiset DNA ja siRNA molekyylit aiheuttivat monosyytti-makrofagilinjan soluviljelymallissa TRACP entsyymin tuoton lisääntymistä odotusten vastaisesti. DNA ja RNA molekyylien vaikutusta TRACP entsyymin tuoton lisääntymiseen tutkittiin myös Tolllike reseptori 9 (TLR9) poistogeenisestä hiirestä eristetyissä monosyyttimakrofaagisoluissa. TRACP entsyymin tuoton lisääntyminen todettiin sekvenssistä ja TLR9:stä riippumattomaksi vasteeksi solun ulkopuolisia DNA ja RNA molekyylejä vastaan. Havainto TRACP entsyymin tuoton lisääntymisestä viittaa siihen, että TRACP entsyymillä on tehtävä solun immuunipuolustusjärjestelmässä.

Functions of the human papillomavirus type 16 E4 protein

1. Abstract Cervical cancer is thought to be the consequence of infection by human papillomaviruses (HPV). In the majority of cases, DNA from HPV type 16 (HPV16) is found in malignant cervical lesions. The initial steps leading to transformation of an infected cell are not clearly understood but in most cases, disruption and integration of the episomal viral DNA must take place. As a consequence, the E2 and E4 genes are usually not expressed whereas the E6 and E7 oncogenes are highly expressed. However, in a normal infection in which the viral DNA is maintained as an episome, all viral genes are expressed. The pattern according to which the viral proteins are made, and therefore the life cycle of the virus, is tightly linked to the differentiation process of the host keratinocyte. The study of the viral oncogenes E6 and E7 has revealed crucial functions in the process of malignant transformation such as degradation of the p53 tumor suppressor protein, deregulation of the Retinoblastoma protein pathway and activation of the telomerase ribonucleoprotein. All these steps are necessary for cancerous lesions to develop. However, the loss of the E2 gene product seems to be necessary for sufficient expression of E6 and E7 in order to achieve such effects. In normal infections, the E4 protein is made abundantly in the later stages of the viral life cycle. Though extensive amounts of work have been carried out to define the function of E4, it still remains unclear. In this study, several approaches have been used to try and determine the functions of E4. First, a cell-penetrating fusion protein was designed and produced in order to circumvent the chronic difficulties of expressing E4 in mammalian cells. Unfortunately, this approach was not successful due to precipitation of the purified fusion protein. Second, the observation that E4 accumulates in cells having modified their adhesion properties led to the hypothesis that E4 might be involved in the differentiation process of keratinocytes. Preliminary results suggest that E4 triggers differentiation. Last, as E4 has been reported to collapse the cytokeratin network of keratinocytes, a direct approach using atomic force microscopy has allowed us to test the potential modification of mechanical properties of cells harboring reorganized cytokeratin networks. If so, a potential role for E4 in viral particle release could be hypothesized. 2. Résumé Il a été établi que le cancer du col de l'utérus se développe essentiellement à la suite d'une infection par le virus du papillome humain (HPV). Dans la majorité des cas analysés, de l'ADN du HPV de type 16 (HPV16) est détecté. Les étapes initiales de la transformation d'une cellule infectée sont mal connues mais il semble qu'une rupture du génome viral, normalement épisomal, suivi d'une intégration dans le génome de la cellule hôte soient des étapes nécessaires dans la plupart des cas. Or il semble qu'il y ait une sélection pour les cas où l'expression des oncogènes viraux E6 et E7 soit favorisée alors que l'expression des gènes E2 et E4 est en général impossible. Par contre, dans une infection dite normale où le génome viral n'est pas rompu, il n'y pas développement de cancer et tous les gènes viraux sont exprimés. L'ordre dans lequel les protéines virales sont produites, et donc le cycle de réplication du virus, est intimement lié au processus de différentiation de la cellule hôte. L'étude des protéines oncogènes E6 et E7 a révélé des fonctions clés dans le processus de transformation des cellules infectées telles que la dégradation du suppresseur de tumeur p53, la dérégulation de la voie de signalisation Rb ainsi que l'activation de la télomérase. Toutes ces activités sont nécessaires au développement de lésions cancéreuses. Toutefois, il semble que l'expression du gène E2 doit être empêchée afin que suffisamment des protéines E6 et E7 soient produites. Lorsque le gène E2 est exprimé, et donc lorsque le génome viral n'est pas rompu, les protéines E6 et E7 n'entraînent pas de telles conséquences. Le gène E4, qui se trouve dans la séquence codante de E2, a aussi besoin d'un génome viral intact pour être exprimé. Dans une infection normale, le gène E4 est exprimé abondamment dans les dernières étapes de la réplication du virus. Bien que de nombreuses études aient été menées afin de déterminer la fonction virale à E4, aucun résultat n'apparaît évident. Dans ce travail, plusieurs approches ont été utilisées afin d'adresser cette question. Premièrement, une protéine de fusion TAT-E4 a été produite et purifiée. Cette protéine, pouvant entrer dans les cellules vivantes par diffusion au travers de la membrane plasmique, aurait permis d'éviter ainsi les problèmes chroniques rencontrés lors de l'expression de E4 dans les cellules mammifères. Malheureusement, cette stratégie n'a pas pu être utilisée à cause de la précipitation de la protéine purifiée. Ensuite, l'observation que E4 s'accumule dans les cellules ayant modifié leurs propriétés d'adhésion a suggéré que E4 pourrait être impliqué dans le procédé de différentiation des kératinocytes. Des résultats préliminaires supportent cette possibilité. Enfin, il a été montré que E4 pouvait induire une réorganisation du réseau des cytokératines. Une approche directe utilisant le microscope à force atomique nous a ainsi permis de tester une potentielle modification des propriétés mécaniques de cellules ayant modifié leur réseau de cytokératines en présence de E4. Si tel est le cas, un rôle dans la libération de particules virales peut être proposé pour E4.

Identification of novel HIV restriction factors

To efficiently replicate within mammalian cells, viruses have to manoeuvre through complex host mechanisms, hijacking a network of host proteins to achieve successful propagation. To prevent this invasion, cells have evolved over time to efficiently block the incursing pathogen by direct or indirect targeting. Human immunodeficiency virus (HIV) is a retrovirus of major global public health issue. In the last decade, extensive focus on innate immune proteins has been given, and particularly restriction factors, proteins inhibiting HIV replication by affecting various stages of the viral cycle. Because of the importance of developing new HIV therapies that are associated with reduced side effects and resistances, there is an urge to understand the antiviral response against HIV. Using common features of known restriction factors as a signature to identify new anti-HIV factors, candidates were identified. Particularly multiple members of the apolipoproteins L (APOL) family were found. Cotransfection experiments confirmed very potent inhibitory effects on HIV-1 expression. Further characterization of APOL6, the best candidate, was carried out. APOL6 was not able to inhibit HIV specifically but rather inhibited any gene-encoded DNA that was cotransfected and therefore APOL6 does not classify as a bona fide restriction factor. In addition, we were able to map the activity of APOL6 to the MAD domain and mainly to residue 174. We also found that other members of the family identified in the screen, APOL1 and 3, could have similar mechanism of action as APOL6. Finally, although the complete mechanism of action of APOL6 has yet to be elucidated, it might be blocked during transfections, potentially improving transfection of primary cells. -- Pour se répliquer efficacement dans les cellules de mammifères, les virus doivent manoeuvrer à travers des mécanismes cellulaires complexes et détourner un réseau de protéines de l'hôte. Pour empêcher cette invasion, les gènes de l'hôte ont évolué dans le temps pour cibler efficacement, directement ou indirectement, l'agent pathogène. Le virus de l'immunodéficience humaine (VIH) est un rétrovirus de problème majeur de santé publique mondiale, mais le faible risque de transmission du virus pourrait être expliqué par la présence d'un système antiviral de l'hôte qui, en cas d'échec, conduit à une infection productive. Durant la dernière décennie, il y a eu un intérêt spécial porté sur les protéines immunitaires innées appelé facteurs de restriction présentant des effets inhibiteurs puissants sur la réplication du VIH en affectant différentes étapes du cycle viral. En raison de l'importance de la recherche de nouvelles thérapies anti-VIH associées à des effets secondaires et des résistances réduites comparé aux traitements actuels, il existe un besoin de comprendre la réponse antivirale innée contre le VIH. Basé sur des caractéristiques communes des facteurs de restriction connus, nous avons proposé d'identifier de nouveaux facteurs anti-VIH. Nous avons trouvé une famille de protéines, les apolipoprotéines L (APOL) montrant les effets inhibiteurs très puissants contre l'expression du VIH-1 dans des expériences de co-transfection. Nous avons décidé d'approfondir le rôle de ces protéines dans l'immunité innée et de se concentrer sur le meilleur candidat APOL6. Nous avons en outre établi qu'APOL6 n'a pas d'activité anti-virale spécifique et donc pas classé comme un facteur de bonne foi de restriction. Par ailleurs, APOL6 est capable d'inhiber fortement l'expression de tout Plasmide cotransfecté. En outre, nous avons été en mesure de cartographier l'activité d'APOL6 au domaine MAD et principalement au résidu 174. Nous avons également constaté que d'autres membres de la famille identifiés dans l'étude, APOL1 et 3, pourraient avoir le même mécanisme d'action qu'APOL6. Enfin, bien que le mécanisme d'action complet d'APOL6 reste à être élucidé, il pourrait être d'une importance biotechnologique car il pourrait potentiellement faciliter la transfection de cellules primaires après l'inhibition d'APOL6.

The role of MAR elements in DNA recombination

The untargeted integration of foreign DNA into the mammalian cell genome, extensively used in gene therapy and biotechnology, remains an incompletely understood process. It is believed to be based on cellular DNA double strand break (DSB) repair machinery and to involve two major steps: i) the formation of long gene arrays (concatemers), and ii) recombination of the resulting concatemer with the genome. The main DSB repair pathways in eukaryotes include non-homologous end-joining (NHEJ), homologous recombination (HR), and microhomology-mediated end-joining (MMEJ). However, it is still not clear, which of these pathways are responsible for transgene integration. Here, we show that NHEJ is not the primary pathway used by mammalian cells in the transgene integration process, while the components of the HR pathway seem to be important for genomic integration but not concatemerization. Instead, concatemer formation appears to be mediated by a subset of the MMEJ pathway, termed synthesis-dependent MMEJ (SD-MMEJ). This mechanism also seems to be preferentially used for plasmid integration into the genome, as confirmed by the analysis of plasmid-to-genome junction sequences, which were found to display an SD-MMEJ pattern. Therefore, we propose the existence of two distinct SD-MMEJ subpathways, relying on different subsets of enzymes. One of these mechanisms appears to be responsible for concatemerization, while the other mechanism, partially dependent in HR enzymes, seems to mediate recombination with the genome. Previous studies performed by our group suggested that matrix attachment regions (MARs), which are epigenetic regulatory DNA elements that participate in the formation of chromatin boundaries and augment transcription, may mediate increased plasmid integration into the genome of CHO cells by stimulating DNA recombination. In the present work, we demonstrate that MAR-mediated plasmid integration results from the enhanced SD-MMEJ pathway. Analysis of transgene integration loci and junction DNA sequences validated the prevalent use of this pathway by the MAR elements to target plasmid DNA into gene-rich areas of the CHO genome. We propose that this finding should in the future help to engineer cells for improved recombinant protein production. In addition to investigating the process of transgene integration, we designed recombination assays to better characterize the components of the MMEJ and SD-MMEJ pathways. We also used CHO cells expressing cycle-sensitive reporter genes to demonstrate a potential role of HR proteins in the cell cycle regulation.

Increased voltage-dependent K+ channel Kv1.3 and Kv1.5 expression correlates with leiomyosarcoma aggressiveness

Voltage-dependent K+ channels (Kv) are involved in the proliferation and differentiation of mammalian cells, since Kv antagonists impair cell cycle progression. Although myofibers are terminally differentiated, some myoblasts may re-enter the cell cycle and proliferate. Since Kv1.3 and Kv1.5 expression is remodeled during tumorigenesis and is involved in smooth muscle proliferation, the purpose of this study was to analyze the expression of Kv1.3 and Kv1.5 in smooth muscle neoplasms. In the present study, we examined human samples of smooth muscle tumors together with healthy speci­mens. Thus, leiomyoma (LM) and leiomyosarcoma (LMS) tumors were analyzed. Results showed that Kv1.3 was poorly expressed in the healthy muscle and indolent LM specimens, whereas aggressive LMS showed high levels of Kv1.3 expression. Kv1.5 staining was correlated with malignancy. The findings show a remodeling of Kv1.3 and Kv1.5 in human smooth muscle sarcoma. A correlation of Kv1.3 and Kv1.5 expression with tumor aggressiveness was observed. Thus, our results indicate Kv1.5 and Kv1.3 as potential tumorigenic targets for aggressive human LMS.

Characterization of a MAF1 knockout mouse model

L'ARN polymérase 3 transcrit un petit groupe de gènes fortement exprimés et impliqués dans plusieurs mécanismes moléculaires. Les ARNs de transfert ou ARNt représentent plus ou moins la moitié du transcriptome de l'ARN polymérase 3. Ils sont directement impliqués dans la traduction des protéines en agissant comme transporteurs d'acides aminés qui sont incorporés à la chaîne naissante de polypeptides. Chez des levures cultivées dans un milieu jusqu'à épuisement des nutriments, Maf1 réprime la transcription par l'ARN polymérase 3, favorisant ainsi l'économie énergétique cellulaire. Dans un modèle de cellules de mammifères, MAF1 réprime aussi la transcription de l'ARN polymérase 3 dans des conditions de stress, cependant il n'existe aucune donnée quant à son rôle chez un mammifère vivant. Pendant mon doctorat, j'ai utilisé une souris délétée pour le gène Maf1 afin de connaître les effets de ce gène chez un mammifère. Etonnamment, la souris Maf1-­‐/-­‐ est résistante à l'obésité même si celle-­‐ci est nourrie avec une nourriture riche en matières grasses. Des études moléculaires et de métabolomiques ont montré qu'il existe des cycles futiles de production et dégradation des lipides et des ARNt, ce qui entraîne une augmentation de la dépense énergique et favorise la résistance à l'obésité. En plus de la caractérisation de la souris Maf1-­‐/-­‐, pendant ma thèse j'ai également développé une méthode afin de normaliser les données de ChIP-­‐sequencing. Cette méthode est fondée sur l'utilisation d'un contrôle interne, représenté ici par l'ajout d'une quantité fixe de chromatine provenant d'un organisme différent de celui étudié. La méthode a amélioré considérablement la reproductibilité des valeurs entre réplicas biologiques. Elle a aussi révélé des différences entre échantillons issus de conditions différentes. Une occupation supérieure de l'ARN polymérase 3 sur les gènes Pol 3 chez les souris Maf1 KO entraîne une augmentation du niveau de précurseurs d'ARNt, ayant pour effet probable la saturation de la machinerie de maturation des ARNt. En effet, chez les souris Maf1 KO, le pourcentage d'ARNt modifiés est plus faible que chez les souris type sauvage. Ce déséquilibre entre le niveau de précurseurs et d'ARNt matures entraîne une diminution de la traduction protéique. Ces résultats ont permis d'identifier de nouvelles fonctions pour la protéine MAF1, comme étant une protéine régulatrice à la fois de la transcription mais aussi de la traduction et en étant un cible potentielle au traitement à l'obésité. -- RNA polymerase III (Pol 3) transcribes a small set of highly expressed genes involved in different molecular mechanisms. tRNAs account for almost half of the Pol 3 transcriptome and are involved in translation, bringing a new amino into the nascent polypeptide chain. In yeast, under nutrient deprivation, Maf1 acts for cell energetic economy by repressing Pol 3 transcription. In mammalian cells, MAF1 also represses Pol 3 activity under conditions of serum deprivation or DNA damages but nothing is known about its role in a mammalian organism. During my thesis studies, I used a Maf1 KO mouse model to characterize the effects of Maf1 deletion in a living animal. Surprisingly, the MAF1 KO mouse developed an unexpected phenotype, being resistant to high fat diet-­‐induced obesity and displaying an extended lifespan. Molecular and metabolomics characterizations revealed futile cycles of lipids and tRNAs, which are produced and immediately degraded, which increases energy consumption in the Maf1 KO mouse and probably explains in part the protection to obesity. Additionally to the mouse characterization, I also developed a method to normalize ChIP-­‐seq data, based on the addition of a foreign chromatin to be used as an internal control. The method improved reproducibility between replicates and revealed differences of Pol 3 occupancy between WT and Maf1 KO samples that were not seen without normalization to the internal control. I then established that increased Pol 3 occupancy in the Maf1 KO mouse liver was associated with increased levels of tRNA precursor but not of mature tRNAs, the effective molecules involved in translation. The overproduction of precursor tRNAs associated with the deletion of Maf1 apparently overwhelms the tRNA processing machinery as the Maf1 KO mice have lower levels of fully modified tRNAs. This maturation defect directly impacts on translation efficiency as polysomic fractions and newly synthetized protein levels were reduced in the liver of the Maf1 KO mouse. Altogether, these results indicate new functions for MAF1, a regulator of both transcription and translation as well as a potential target for obesity treatment.

Negative feedback regulation of calcineurin-dependent Prz1 transcription factor by the CaMKK-CaMK1 axis in fission yeast

Calcium signals trigger the translocation of the Prz1 transcription factor from the cytoplasm to the nucleus. The process is regulated by the calciumactivated phosphatase calcineurin, which activates Prz1 thereby maintaining active transcription during calcium signalling. When calcium signalling ceases, Prz1 is inactivated by phosphorylation and exported to the cytoplasm. In budding yeast and mammalian cells, different kinases have been reported to counter calcineurin activity and regulate nuclear export. Here, we show that the Ca2+/calmodulin-dependent kinase Cmk1 is first phosphorylated and activated by the newly identified kinase CaMKK2 homologue, Ckk2, in response to Ca2+. Then, active Cmk1 binds, phosphorylates and inactivates Prz1 transcription activity whilst at the same time cmk1 expression is enhanced by Prz1 in response to Ca2+. Furthermore, Cdc25 phosphatase is also phosphorylated by Cmk1, inducing cell cycle arrest in response to an increase in Ca2+. Moreover, cmk1 deletion shows a high tolerance to chronic exposure to Ca2+, due to the lack of cell cycle inhibition and elevated Prz1 activity. This work reveals that Cmk1 kinase activated by the newly identified Ckk2 counteracts calcineurin function by negatively regulating Prz1 activity which in turn is involved in activating cmk1 gene transcription. These results are the first insights into Cmk1 and Ckk2 function in Schizosaccharomyces pombe.

Síntese, caracterização e estudo da atividade inibitória de novas dialquilfosforilarilidrazonas sobre o crescimento de tripanossomatídeos

A new series of dialkylphosphorylhydrazones was synthesized through the condensation of aromatic aldehydes with different phosphorylhydrazines. All synthesized compounds were characterized by IR, ¹H-NMR, 13C-NMR and 31P-NMR spectroscopies. The in vitro investigation of the activity of these compounds against Leishmania amazonensis promastigotes and epimastigotes of T. cruzi, showed an efficient inhibition of proliferation, at non toxic concentrations to mammalian cells. The results have shown some derivatives as potential antiparasitic agents against trypanosomatids.

Alpha2-adrenoceptors: Structure and ligand binding properties at the molecular level

Alpha₂-Adrenoceptors: structure and ligand binding properties at the molecular level The mouse is the most frequently used animal model in biomedical research, but the use of zebrafish as a model organism to mimic human diseases is on the increase. Therefore it is considered important to understand their pharmacological differences from humans also at the molecular level. The zebrafish Alpha_2-adrenoceptors were expressed in mammalian cells and the binding affinities of 20 diverse ligands were determined and compared to the corresponding human receptors. The pharmacological properties of the human and zebrafish Alpha_2--adrenoceptors were found to be quite well conserved. Receptor models based on the crystal structures of bovine rhodopsin and the human Beta₂-adrenoceptor revealed that most structural differences between the paralogous and orthologous Alpha_2--adrenoceptors were located within the second extracellular loop (XL2). Reciprocal mutations were generated in the mouse and human Alpha_2--adrenoceptors. Ligand binding experiments revealed that substitutions in XL2 reversed the binding profiles of the human and mouse Alpha_2--adrenoceptors for yohimbine, rauwolscine and RS-79948-197, evidence for a role for XL2 in the determination of species-specific ligand binding. Previous mutagenesis studies had not been able to explain the subtype preference of several large Alpha_2--adrenoceptor antagonists. We prepared chimaeric Alpha_2--adrenoceptors where the first transmembrane (TM1) domain was exchanged between the three human Alpha_2--adrenoceptor subtypes. The binding affinities of spiperone, spiroxatrine and chlorpromazine were observed to be significantly improved by TM1 substitutions of the Alpha_2a--adrenoceptor. Docking simulations indicated that indirect effects, such as allosteric modulation, are more likely to be involved in this phenomenon rather than specific side-chain interactions between ligands and receptors.

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.

Genetic responses against nitric oxide toxicity

The threat of free radical damage is opposed by coordinated responses that modulate expression of sets of gene products. In mammalian cells, 12 proteins are induced by exposure to nitric oxide (NO) levels that are sub-toxic but exceed the level needed to activate guanylate cyclase. Heme oxygenase 1 (HO-1) synthesis increases substantially, due to a 30- to 70-fold increase in the level of HO-1 mRNA. HO-1 induction is cGMP-independent and occurs mainly through increased mRNA stability, which therefore indicates a new NO-signaling pathway. HO-1 induction contributes to dramatically increased NO resistance and, together with the other inducible functions, constitutes an adaptive resistance pathway that also defends against oxidants such as H2O2. In E. coli, an oxidative stress response, the soxRS regulon, is activated by direct exposure of E. coli to NO, or by NO generated in murine macrophages after phagocytosis of the bacteria. This response is governed by the SoxR protein, a homodimeric transcription factor (17-kDa subunits) containing [2Fe-2S] clusters essential for its activity. SoxR responds to superoxide stress through one-electron oxidation of the iron-sulfur centers, but such oxidation is not observed in reactions of NO with SoxR. Instead, NO nitrosylates the iron-sulfur centers of SoxR both in vitro and in intact cells, which yields a form of the protein with maximal transcriptional activity. Although nitrosylated SoxR is very stable in purified form, the spectroscopic signals for the nitrosylated iron-sulfur centers disappear rapidly in vivo, indicating an active process to reverse or eliminate them.

Trypanosoma cruzi infection: a continuous invader-host cell cross talk with participation of extracellular matrix and adhesion and chemoattractant molecules

Several lines of evidence have shown that Trypanosoma cruzi interacts with host extracellular matrix (ECM) components producing breakdown products that play an important role in parasite mobilization and infectivity. Parasite-released antigens also modulate ECM expression that could participate in cell-cell and/or cell-parasite interactions. Increased expression of ECM components has been described in the cardiac tissue of chronic chagasic patients and diverse target tissues including heart, thymus, central nervous system and skeletal muscle of experimentally T. cruzi-infected mice. ECM components may adsorb parasite antigens and cytokines that could contribute to the establishment and perpetuation of inflammation. Furthermore, T. cruzi-infected mammalian cells produce cytokines and chemokines that not only participate in the control of parasitism but also contribute to the establishment of chronic inflammatory lesions in several target tissues and most frequently lead to severe myocarditis. T. cruzi-driven cytokines and chemokines may also modulate VCAM-1 and ICAM-1 adhesion molecules on endothelial cells of target tissues and play a key role in cell recruitment, especially of activated VLA-4+LFA-1+CD8+ T lymphocytes, resulting in a predominance of this cell population in the inflamed heart, central nervous system and skeletal muscle. The VLA-4+-invading cells are surrounded by a fine network of fibronectin that could contribute to cell anchorage, activation and effector functions. Since persistent "danger signals" triggered by the parasite and its antigens are required for the establishment of inflammation and ECM alterations, therapeutic interventions that control parasitism and selectively modulate cell migration improve ECM abnormalities, paving the way for the development of new therapeutic strategies improving the prognosis of T. cruzi-infected individuals.

Glutathione and the redox control system trypanothione/trypanothione reductase are involved in the protection of Leishmania spp. against nitrosothiol-induced cytotoxicity

Glutathione is the major intracellular antioxidant thiol protecting mammalian cells against oxidative stress induced by oxygen- and nitrogen-derived reactive species. In trypanosomes and leishmanias, trypanothione plays a central role in parasite protection against mammalian host defence systems by recycling trypanothione disulphide by the enzyme trypanothione reductase. Although Kinetoplastida parasites lack glutathione reductase, they maintain significant levels of glutathione. The aim of this study was to use Leishmania donovani trypanothione reductase gene mutant clones and different Leishmania species to examine the role of these two individual thiol systems in the protection mechanism against S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a nitrogen-derived reactive species donor. We found that the resistance to SNAP of different species of Leishmania was inversely correlated with their glutathione concentration but not with their total low-molecular weight thiol content (about 0.18 nmol/10(7) parasites, regardless Leishmania species). The glutathione concentration in L. amazonensis, L. donovani, L. major, and L. braziliensis were 0.12, 0.10, 0.08, and 0.04 nmol/10(7) parasites, respectively. L. amazonensis, that have a higher level of glutathione, were less susceptible to SNAP (30 and 100 µM). The IC50 values of SNAP determined to L. amazonensis, L. donovani, L. major, and L. braziliensis were 207.8, 188.5, 160.9, and 83 µM, respectively. We also observed that L. donovani mutants carrying only one trypanothione reductase allele had a decreased capacity to survive (~40%) in the presence of SNAP (30-150 µM). In conclusion, the present data suggest that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, participate in protection of Leishmania against the toxic effect of nitrogen-derived reactive species.

The inhibitory role of sympathetic nervous system in the Ca2+-dependent proteolysis of skeletal muscle

Mammalian cells contain several proteolytic systems to carry out the degradative processes and complex regulatory mechanisms to prevent excessive protein breakdown. Among these systems, the Ca2+-activated proteolytic system involves the cysteine proteases denoted calpains, and their inhibitor, calpastatin. Despite the rapid progress in molecular research on calpains and calpastatin, the physiological role and regulatory mechanisms of these proteins remain obscure. Interest in the adrenergic effect on Ca2+-dependent proteolysis has been stimulated by the finding that the administration of β2-agonists induces muscle hypertrophy and prevents the loss of muscle mass in a variety of pathologic conditions in which calpains are activated. This review summarizes evidence indicating that the sympathetic nervous system produces anabolic, protein-sparing effects on skeletal muscle protein metabolism. Studies are reviewed, which indicate that epinephrine secreted by the adrenal medulla and norepinephrine released from adrenergic terminals have inhibitory effects on Ca2+-dependent protein degradation, mainly in oxidative muscles, by increasing calpastatin levels. Evidence is also presented that this antiproteolytic effect, which occurs under both basal conditions and in stress situations, seems to be mediated by β2- and β3-adrenoceptors and cAMP-dependent pathways. The understanding of the precise mechanisms by which catecholamines promote muscle anabolic effects may have therapeutic value for the treatment of muscle-wasting conditions and may enhance muscle growth in farm species for economic and nutritional purposes.