Molekularbiologische Charakterisierung der Argonauten Proteine AgnA und AgnB innerhalb der RNA vermittelten Genregulation in D. discoideum

Argonauten Proteine übernehmen vielfältige Funktionen in RNA vermittelten Signalwegen zur Genregulation und sind in eukaryotischen Organismen hoch konserviert. Obwohl das Repertoire an kleinen regulatorischen RNAs in D. discoideum schon früh untersucht wurde und dabei sowohl siRNAs als auch miRNAs identifiziert werden konnten, war die Funktion der fünf kodierten Argonauten Proteine zu Beginn meiner Arbeit noch völlig unbekannt. Im Fokus meiner Untersuchung standen die zwei Homologe AgnA und AgnB. Die molekularbiologische Charakterisierung von AgnA hat gezeigt, dass das Protein eine essentielle Funktion bei der posttranskriptionellen Regulation des Retrotransposons DIRS-1 hat. AgnA wird für die Generierung von über 90 % der DIRS-1 siRNAs benötigt, wobei unklar ist, ob die Slicer-Aktivität des Proteins relevant ist oder ob AgnA andere Proteine zur Generierung der kleinen RNAs rekrutiert. Mit Hilfe der Deep Sequencing Analyse kleiner RNAs im AgnA KO konnte die Abreicherung der DIRS-1 siRNAs bestätigt werden. Die Anreicherung von DIRS-1 sense und antisense Transkripten weist deutlich auf eine Deregulation des Retrotransposons bei Abwesenheit von AgnA hin. Der Verlust der AgnA abhängigen Regulationsebene ist nicht nur auf RNA- sondern auch auf DNA-Ebene nachweisbar, da im AgnA Knockout einzelsträngige extrachromosomale DIRS-1 Intermediate nachweisbar sind. Die Analyse dieser Strukturen mit Hilfe von Rasterkraftmikroskopie zeigt, dass die extrachromosomale DNA mit Proteinen assoziiert ist. Das Erscheinungsbild legt die Vermutung nahe, dass es sich um Virus ähnliche Partikel handeln könnte. Die Transposition der DIRS-1 Elemente konnte nicht nachgewiesen werden. Sie schlägt vermutlich fehl, da der zur Integration notwendige DNA-Doppelstrang nicht gebildet wird. Auch wenn der genaue Mechanismus der AgnA abhängigen DIRS-1 Regulation nicht vollständig aufgeklärt werden konnte, weisen die Ergebnisse darauf hin, dass AgnA nicht nur an der Biogenese der kleinen DIRS-1 siRNAs beteiligt ist, sondern auch weiter downstream, vermutlich innerhalb von Effektorkomplexen, als Regulator aktiv ist. AgnB ist nicht an der negativen Regulation des DIRS-1 Retrotransposons beteiligt. Im Gegenteil haben Experimente gezeigt, dass das Protein die Transkription des Elementes und die Bildung von DNA-Intermediaten eher positiv beeinflusst. Im Fall des Retrotransposons Skipper ist unklar, ob die wenigen siRNAs, die identifiziert worden sind, tatsächlich für die Regulation dieses Elementes genutzt werden. Der Knockout von AgnA hat eine Anreicherung der Skipper siRNAs zur Folge, wobei diese sehr variabel ist. Es konnten Skipper Transkripte nachgewiesen werden (Hinas et al., 2007), die wahrscheinlich die Vorläufermoleküle der siRNAs darstellen. Die Menge dieser Transkripte unterscheidet sich allerdings im Wildtyp und den untersuchten Knockout-Stämmen nicht. Bei der Untersuchung der miRNAs zeigte sich eine signifikante Anreicherung dieser regulatorischen RNAs im AgnA Knockout. Die Akkumulation kann durch die Expression von rekombinantem AgnA wieder auf Wildtyp Niveau gebracht werden. Die genaue Funktion von AgnA im miRNA Signalweg konnte aber nicht näher spezifiziert werden. Im Fall der beiden miRNAs konnte im Rahmen dieser Arbeit nachgewiesen werden, dass sie keine 2‘-O Methylierung besitzen und fast ausschließlich im Cytoplasma der Zelle vorliegen. Letzteres weist darauf hin, dass die untersuchten miRNAs ihre Zielgene vermutlich posttranskriptionell regulieren. Die Akkumulation von miRNAs im AgnA KO konnte ebenfalls durch Deep Sequencing Analysen verifiziert werden. Weiterhin wurden tRNA Fragmente gefunden, die im AgnA KO wesentlich stärker vertreten sind. Northern Blot Analysen haben gezeigt, dass ein zusätzliches Fragment der tRNA Asp akkumuliert, wenn AgnA nicht exprimiert wird. Möglicherweise ist AgnA am Umsatz der tRNA beteiligt. Die biologische Funktion der tRNA Fragmente in D. discoideum ist jedoch bisher ungeklärt. Bei der Suche nach putativen Interaktionspartnern konnte im Fall von AgnA das Protein DDB_G0268914 mittels Massenspektrometrie als putativer Interaktionspartner identifiziert werden. Dieses Protein zeigt Homologien zu MOV10 aus H. sapiens, das ebenfalls mit Argonauten Proteinen interagiert (Hock et al., 2007) und die Replikation von Retroviren unterdrückt (Burdick et al., 2010). Die Interaktion zwischen AgnA und dem MOV10 Homolog konnte bisher nicht mit anderen Ansätzen bestätigt werden. Darüber hinaus bleibt zu klären, ob der putative Interaktionsparter ebenfalls an der Regulation des Retrotransposons DIRS-1 beteiligt ist.

HelF und sein Interaktionspartner Xrn1: zwei Regulatoren der RNA-Interferenz in D. discoideum

Hauptziel dieser Arbeit ist die Identifizierung, Verifizierung und Charakterisierung von Interaktionspartnern von HelF, einem Negativregulator der RNA-Interferenz in Dictyostelium discoideum (Popova et al. 2006). Es ist gelungen, die Interaktion von HelF und der 5‘ 3‘ Exonuklease Xrn1 nachzu-weisen, aber alle anderen Versuchen, bisher unbekannte Protein-Interaktionspartner zu identifizieren, schlugen fehl. Xrn1 ist in den Organismen D. melanogaster (Orban und Izaurralde 2005), C. elegans (Newbury und Woollard 2004) und A. thaliana (Gazzani et al. 2004) bereits als Regulator der RNA-Interferenz bekannt. Mit Aufreinigungen nach der TAP-Methode und mit dem Nanotrap wurde ebenfalls versucht, RNA-Interaktionspartner von HelF zu identifizieren. Es konnten in einigen Aufreinigungen putative, für HelF spezifische RNAs identifiziert werden, doch entweder es handelte sich nachweislich nicht um RNA oder die Reproduktion der Daten schlug trotz mehrfacher Versuche fehl. Bezüglich der zellulären Lokalisation von HelF und Xrn1 konnte gezeigt werden, dass HelF zusätzlich zur bekannten Lokalisation in Foci im Nukleus (Popova et al. 2006) vermutlich auch im Cytoplasma und dort angeordnet in mehreren Granula zu finden ist. Xrn1 ist nahezu ausschließlich im Cytoplasma lokalisiert, wo es in mehreren Foci organisiert ist. Es wird vermutet, dass es sich bei diesen Foci um Processing-Bodies (P-Bodies) handelt und dass möglicherweise Xrn1 und HelF in eben diesen P-Bodies co-lokalisieren. In der Entwicklung vom Einzeller zum mehrzelligen Organismus zeigen die Xrn1KO- und die HelFKO-Mutante jeweils einen eindeutigen Phänotyp, der vom Wildtyp abweicht. Die Phänotypen der beiden Mutanten unterscheiden sich deutlich voneinander. Beim Mischen von HelF-Knockout-Zellen mit grün fluoreszierenden Wildtyp-Zellen zeigt sich, dass beide Stämme innerhalb des sich entwickelnden Organismus an definierten Stellen lokalisieren. Entgegen den Erwartungen befinden sich die Zellen der Mutante in den Stadien „Finger“ und „Slug“ nicht hauptsächlich im vorderen Teil des Organismus, sondern sind auch im hinteren Teil, der später die Sporenmasse bildet, vertreten. Dies lässt vermuten, dass HelF-Knockout-Mutanten in gleichem Maße wie Wildtypzellen als Sporen in die nächste Generation übergehen. Weitere Mix-Experimente, in denen HelFKO-Zellen und Xrn1KO-Zellen mit grün fluoreszierenden Wildtypzellen gemischt wurden, belegen eindeutig, dass beide Knockoutmutanten in Konkurrenz zum Wildtyp bei der Generierung von Sporen und somit beim Übergang in die nächste Generation benachteiligt sind. Dies steht im Gegensatz zu den Ergebnissen der vorher beschriebenen Mix-Experimente, in denen der Organismus als Ganzes betrachtet wurde. Weiterhin konnte herausgefunden werden, dass Xrn1 ebenso wie HelF (Popova et al. 2006) eine Rolle als Negativregulator in der RNA-Interferenz innehat. Fraglich ist aber, ob HelF wie bisher angenommen auch Einfluss auf den Weg der Generierung von miRNAs nimmt, da in HelFKO für keinen der beiden miRNA-Kandidaten eine Hoch- bzw. Runterregulierung der reifen miRNAs im Vergleich zum Wildtyp beobachtet werden kann. Im Xrn1KO hingegen ist die reife miRNA ddi-mir-1176 im Vergleich zum Wildtyp hochreguliert. In Bezug auf die Generierung von siRNAs konnte herausgefunden werden, dass Xrn1 und HelF im Fall der Generierung von Skipper siRNAs regulierend eingreifen, dass aber nicht alle siRNAs von der negativen Regulierung durch HelF und Xrn1betroffen sind, was am Beispiel der DIRS-1-siRNAs belegt werden kann. Das von B. Popova entwickelte Modell (Popova 2005) bezüglich der Rolle von HelF in der RNA-Interferenz wurde basierend auf den neu gewonnenen Daten weiterentwickelt und um Xrn1 ergänzt, um die Funktionen von HelF und Xrn1 als Antagonisten der RNA-Interferenz näher zu beleuchten. Literatur: Gazzani, S., T. Lawrenson, et al. (2004). "A link between mRNA turnover and RNA interference in Arabidopsis." Science 306(5698): 1046-8. Newbury, S. and A. Woollard (2004). "The 5'-3' exoribonuclease xrn-1 is essential for ventral epithelial enclosure during C. elegans embryogenesis." Rna 10(1): 59-65. Orban, T. I. and E. Izaurralde (2005). "Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome." Rna 11(4): 459-69. Popova, B. (2005). HelF, a suppressor of RNAi mediated gene silencing in Dictyostelium discoideum. Genetik. Kassel, Universität Kassel. PhD: 200. Popova, B., M. Kuhlmann, et al. (2006). "HelF, a putative RNA helicase acts as a nuclear suppressor of RNAi but not antisense mediated gene silencing." Nucleic Acids Res 34(3): 773-84.

Funktion und Biogenese kleiner RNAs in Dictyostelium discoideum

RNA mediated gene silencing pathways are highly conserved among eukaryotes and they have been well investigated in animals and in plants. Longer dsRNA molecules trigger the silencing pathways: RNase III proteins and their dsRNA binding protein (dsRBP) partners recognize those molecules as a substrate and process 21 nucleotide long microRNAs (miRNAs) or small interfering RNAs (siRNAs). Some organisms encode RNA dependent RNA polymerases (RdRPs), which are able to expand the pool of existing siRNAs. Argonaute proteins are able to bind small regulatory RNAs and are subsequently recruited to target mRNAs by base complementary. This leads in turn to transcriptional or posttranscriptional silencing of respective genes. The Dictyostelium discoideum genome encodes two Dicer homologues (DrnA and DrnB), five Argonaute proteins (AgnA to AgnE) and three RdRPs (RrpA to RrpC). In addition, the amoeba is known to express miRNAs and siRNAs, while the latter derive mainly from the DIRS-1 retrotransposon. One part of this work focused on the miRNA biogenesis pathway of D. discoideum. It was shown that the dsRNA binding protein RbdB is a necessary component for miRNA processing in the amoeba. There were no mature miRNAs detectable by Northern blot analysis in rbdB- strains, which is also true for drnB mutants. Moreover, primary miRNA-transcripts (pri-miRNAs) accumulated in rbdB- and drnB- strains. Fluorescence microscopy studies showed a nuclear localization of RbdB. RbdB accumulated in distinct perinucleolar foci. These were reminiscent of plant dicing bodies that contain essential protein components for miRNA processing. It is well known that RNase III enzymes and dsRBPs work together during miRNA processing in higher eukaryotes. This work demonstrated that the same is true for members of the amoebozoa supergroup. In Arabidopsis the nuclear zinc finger protein Serrate (SE) is also necessary for miRNA processing. The D. discoideum homologue SrtA, however, is not relevant which has been shown by the analysis of the respective knockdown strain. MiRNAs are known to be differentially expressed in several RNAi knockout strains. The accumulation of miRNAs in agnA- strains and a strong decrease in rbdB- strains were criteria that could thus be successfully used (among others) to identify and validate new miRNAs candidates by Illumina®-RNA sequencing. In another part of this study, the silencing and amplification of the DIRS-1 retrotransposons was analyzed in more detail. It was already known that DIRS-1 transcripts and extrachromosomal DIRS-1 DNA molecules accumulated in agnA- strains. This phenotype was correlated with the loss of endogenous DIRS-1 siRNAs in the knockout strain. By deep sequencing analysis of small RNAs from the AX2 wild type and the agnA- strain, the strong decrease of endogenous DIRS-1 siRNAs in the mutant strain (accounting for 70 %) could be confirmed. Further analysis of the data revealed an unequal distribution of DIRS-1 derived siRNAs along the retroelement in the wild type strain, since only very few of them matched the inverted terminal repeats (ITRs) and the 5’- half of the first open reading frame (ORF). Besides, sense and antisense siRNAs were asymmetrically distributed, as well. By using different reporter constructs it was shown indirectly that AgnA is necessary for the RrpC mediated production of secondary DIRS-1 siRNAs. These analyses also demonstrated an amplification of siRNAs in 5’- and in 3’-direction. Further analysis of the agnA- strain revealed that not only DIRS-1 sense transcripts but also ORF2 and ORF3 encoded proteins were enriched. In contrast, the ORF1 encoded protein GAG was equally expressed in the mutant and the wild type. This might reflect the unequal distribution of endogenous DIRS-1 siRNAs along the retrotransposon. Southern Blot and PCR-analyses showed that extrachromosomal DIRS-1 DNA molecules are present in the cytoplasm of angA- strains and that they are complementary to sense transcripts of intact DIRS-1 elements. Thus, the extrachromosomal DIRS-1 intermediates are likely incomplete cDNA molecules generated by the DIRS-1 encoded reverse transcriptase. One could hypothesize that virus like particles (VLPs) are the places of DIRS-1 cDNA synthesis. At least, DIRS-1 GAG proteins interact and fluorescence microscopy studies showed that they localize in distinct cytoplasmic foci which accumulate in close proximity to the nuclei.

Global transcript analysis of rice leaf and seed using SAGE technology.

We have compiled two comprehensive gene expression profiles from mature leaf and immature seed tissue of rice (Oryza sativa ssp. japonica cultivar Nipponbare) using Serial Analysis of Gene Expression (SAGE) technology. Analysis revealed a total of 50 519 SAGE tags, corresponding to 15 131 unique transcripts. Of these, the large majority (approximately 70%) occur only once in both libraries. Unexpectedly, the most abundant transcript (approximately 3% of the total) in the leaf library was derived from a type 3 metallothionein gene. The overall frequency profiles of the abundant tag species from both tissues differ greatly and reveal seed tissue as exhibiting a non-typical pattern of gene expression characterized by an over abundance of a small number of transcripts coding for storage proteins. A high proportion ( approximately 80%) of the abundant tags (> or = 9) matched entries in our reference rice EST database, with many fewer matches for low abundant tags. Singleton transcripts that are common to both tissues were collated to generate a summary of low abundant transcripts that are expressed constitutively in rice tissues. Finally and most surprisingly, a significant number of tags were found to code for antisense transcripts, a finding that suggests a novel mechanism of gene regulation, and may have implications for the use of antisense constructs in transgenic technology.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Identification of a serine protease involved with the regulation of adrenal growth

The adrenal cortex is a dynamic organ in which the cells of the outer cortex continually divide. It is well known that this cellular proliferation is dependent on constant stimulation from peptides derived from the ACTH precursor pro-opiomelanocortin (POMC) because disruption of pituitary corticotroph function results in rapid atrophy of the gland. Previous results from our laboratory have suggested that the adrenal mitogen is a fragment derived from the N-terminal of POMC not containing the gamma-MSH sequence. Because such a peptide is not generated during processing of POMC in the pituitary, we proposed that the mitogen is generated from circulating pro-gamma-MSH by an adrenal protease. Using degenerate oligonucleotides, we identified a secreted serine protease expressed by the adrenal gland that we named adrenal secretory protease (ASP). In the adrenal cortex, expression of ASP is limited to the outer zona glomerulosa/fasciculata, the region where cortical cells are believed to be derived, and is significantly up-regulated during compensatory growth. Y1 adrenocortical cells transfected with a vector expressing an antisense RNA (and thus having reduced levels of endogenous ASP) were found to grow slower than sense controls while also losing their ability to utilize exogenous pro-gamma-MSH in the media supporting a role for ASP in adrenal growth. Digestion of an N-POMC peptide substrate encompassing the residues around the dibasic cleavage site at positions 49/50 with affinity-purified ASP showed cleavage not to occur at the dibasic site but two residues downstream leading us to propose the identity of the adrenal mitogen to be N-POMC (1-52).

Drug interaction and location in liposomes: correlation with polar surface areas

An important step in liposome characterization is to determine the location of a drug within the liposome. This work thus investigated the interaction of dipalmitoylphosphatidylcholine liposomes with drugs of varied water solubility, polar surface area (PSA) and partition coefficient using high sensitivity differential scanning calorimetry. Lipophilic estradiol (ES) interacted strongest with the acyl chains of the lipid membrane, followed by the somewhat polar 5-fluorouracil (5-FU). Strongly hydrophilic mannitol (MAN) showed no evidence of interaction but water soluble polymers inulin (IN) and an antisense oligonucleotide (OLG), which have very high PSAs, interacted with the lipid head groups. Accordingly, the drugs could be classified as: hydrophilic ones situated in the aqueous core and which may interact with the head groups; those located at the water-bilayer interface with some degree of penetration into the lipid bilayer; those lipophilic drugs constrained within the bilayer. (c) 2004 Elsevier B.V. All rights reserved.

MARCKS functions as a novel growth/tumour suppressor in cells of melanocyte origin

Protein kinase C (PKC) plays a pivotal role in modulating the growth of melanocytic cells in culture. We have shown previously that a major physiological substrate of PKC, the 80 kDa myristoylated alanine-rich C-kinase substrate (MARCKS), can be phosphorylated in quiescent, non-tumorigenic melanocytes exposed transiently to a biologically active phorbol ester, but cannot be phosphorylated in phorbol ester-treated, syngeneic malignant melanoma cells. Despite its ubiquitous distribution, the function of MARCKS in cell growth and transformation remains to be demonstrated clearly. We report here that MARCKS mRNA and protein levels are down-regulated significantly in the spontaneously derived murine B16 melanoma cell line compared with syngeneic normal Mel-ab melanocytes. In contrast, the tumourigenic v-Ha-ras-transfonned melan-ocytic line, LTR Ras 2, showed a high basal level of MARCKS phosphorylation which was not enhanced by treatment of cells with phorbol ester. Furthermore, protein levels of MARCKS in LTR Ras 2 cells were similar to those expressed in Mel-ab melanocytes. However, in four out of six murine tumour cell lines investigated, levels of MARCKS protein were barely detectable. Transfection of B16 cells with a plasmid containing the MARCKS cDNA in the sense orientation produced two neomycin-resistant clones displaying reduced proliferative capacity and decreased anchorage-independent growth compared with control cells. In contrast, transfection with the antisense MARCKS construct produced many colonies which displayed enhanced growth and transforming potential compared with control cells. Thus, MARCKS appears to act as a novel growth suppressor in the spontaneous transformation of cells of melanocyte origin and may play a more general role in the tumour progression of other carcinomas.

Modification of cell wall properties in lettuce improves shelf life

It is proposed that post-harvest longevity and appearance of salad crops is closely linked to pre-harvest leaf morphology (cell and leaf size) and biophysical structure (leaf strength). Transgenic lettuce plants (Lactuca sativa cv. Valeria) were produced in which the production of the cell wall-modifying enzyme xyloglucan endotransglucosylase/hydrolase (XTH) was down-regulated by antisense inhibition. Independently transformed lines were shown to have multiple members of the LsXTH gene family down-regulated in mature leaves of 6-week-old plants and during the course of shelf life. Consequently, xyloglucan endotransglucosylase (XET) enzyme activity and action were down-regulated in the cell walls of these leaves and it was established that leaf area and fresh weight were decreased while leaf strength was increased in the transgenic lines. Membrane permeability was reduced towards the end of shelf life in the transgenic lines relative to the controls and bacteria were evident inside the leaves of control plants only. Most importantly, an extended shelf-life of transgenic lines was observed relative to the non-transgenic control plants. These data illustrate the potential for engineering cell wall traits for improving quality and longevity of salad crops using either genetic modification directly, or by using markers associated with XTH genes to inform a commercial breeding programme.

Stabilization of multi-stranded nucleic acid structures using 3'-S-phosphorothiolate linkages

3 '-S-Phosphorothiolate linkages incorporated into an oligodeoxynucleotide have been shown to stabilise duplex formation with a complementary RNA strand, but destabilise a duplex formed with a complementary DNA strand. The four-stranded i-motif structure is also stabilised this modification.

Synthesis of 3 '-S-phosphorothiolate oligonucleotides for their potential use in RNA interference

The potency of RNA interference (RNAi) undoubtedly can be improved through chemical modifications to the small interfering RNAs (siRNA). By incorporation of the 3′-S-phosphorothiolate modification into strands of RNA, it is hoped that specific regions of a siRNA duplex can be stabilised to enhance the target binding affinity of a selected antisense strand into the activated RNA-induced silencing complex (RISC*). Oligonucleotides composed entirely of this modification are desirable so unconventional 5′ → 3′ synthesis is investigated, with initial solution-phase testing proving successful. The phosphoroamidite monomer required for solid-phase synthesis has also been produced.

Chronic systemic therapy with low-dose morpholino oligomers ameliorates the pathology and normalizes locomotor behavior in mdx Mice

The administration of antisense oligonucleotides (AOs) to skip one or more exons in mutated forms of the DMD gene and so restore the reading frame of the transcript is one of the most promising approaches to treat Duchenne muscular dystrophy (DMD). At present, preclinical studies demonstrating the efficacy and safety of long-term AO administration have not been conducted. Furthermore, it is essential to determine the minimal effective dose and frequency of administration. In this study, two different low doses (LDs) of phosphorodiamidate morpholino oligomer (PMO) designed to skip the mutated exon 23 in the mdx dystrophic mouse were administered for up to 12 months. Mice treated for 50 weeks showed a substantial dose-related amelioration of the pathology, particularly in the diaphragm. Moreover, the generalized physical activity was profoundly enhanced compared to untreated mdx mice showing that widespread, albeit partial, dystrophin expression restores the normal activity in mdx mice. Our results show for the first time that a chronic long-term administration of LDs of unmodified PMO, equivalent to doses in use in DMD boys, is safe, significantly ameliorates the muscular dystrophic phenotype and improves the activity of dystrophin-deficient mice, thus encouraging the further clinical translation of this approach in humans.

Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators

Nucleotide-based drug candidates such as antisense oligonucleotides, aptamers, immunoreceptor-activating nucleotides, or (anti)microRNAs hold great therapeutic promise for many human diseases. Phosphorothioate (PS) backbone modification of nucleotide-based drugs is common practice to protect these promising drug candidates from rapid degradation by plasma and intracellular nucleases. Effects of the changes in physicochemical properties associated with PS modification on platelets have not been elucidated so far. Here we report the unexpected binding of PS-modified oligonucleotides to platelets eliciting strong platelet activation, signaling, reactive oxygen species generation, adhesion, spreading, aggregation, and thrombus formation in vitro and in vivo. Mechanistically, the platelet-specific receptor glycoprotein VI (GPVI) mediates these platelet-activating effects. Notably, platelets from GPVI function-deficient patients do not exhibit binding of PS-modified oligonucleotides, and platelet activation is fully abolished. Our data demonstrate a novel, unexpected, PS backbone-dependent, platelet-activating effect of nucleotide-based drug candidates mediated by GPVI. This unforeseen effect should be considered in the ongoing development programs for the broad range of upcoming and promising DNA/RNA therapeutics.

Association of NAD(P)H Oxidase with Glucose-Induced Insulin Secretion by Pancreatic beta-Cells

We previously described the presence of nicotinamide adenine dinucleotide phosphate reduced form [NAD(P)H] oxidase components in pancreatic beta-cells and its activation by glucose, palmitic acid, and proinflammatory cytokines. In the present study, the importance of the NAD(P)H oxidase complex for pancreatic beta-cell function was examined. Rat pancreatic islets were incubated in the presence of glucose plus diphenyleneiodonium, a NAD(P)H oxidase inhibitor, for 1 h or with the antisense oligonucleotide for p47(PHOX) during 24 h. Reactive oxygen species (ROS) production was determined by a fluorescence assay using 2,7-dichlorodihydrofluorescein diacetate. Insulin secretion, intracellular calcium responses, [U-(14)C] glucose oxidation, and expression of glucose transporter-2, glucokinase and insulin genes were examined. Antisense oligonucleotide reduced p47(PHOX) expression [an important NAD(P)H oxidase cytosolic subunit] and similarly to diphenyleneiodonium also blunted the enzyme activity as indicated by reduction of ROS production. Suppression of NAD(P)H oxidase activity had an inhibitory effect on intracellular calcium responses to glucose and glucose-stimulated insulin secretion by isolated islets. NAD(P)H oxidase inhibition also reduced glucose oxidation and gene expression of glucose transporter-2 and glucokinase. These findings indicate that NAD(P)H oxidase activation plays an important role for ROS production by pancreatic beta-cells during glucose-stimulated insulin secretion. The importance of this enzyme complex for the beta-cell metabolism and the machinery involved in insulin secretion were also shown. (Endocrinology 150: 2197-2201, 2009)

The role of proliferator-activated receptor gamma coactivator-1 alpha in the fatty-acid -dependent transcriptional control of interleukin-10 in hepatic cells of rodents

Interleukin-10 (IL-10) is an endogenous factor that restrains hepatic insulin resistance in diet-induced steatosis Reducing IL-10 expression increases proinflammatory activity in the steatotic liver and worsens insulin resistance As the transcriptional coactivator proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) plays a central role in dysfunctional hepatocytic activity in diet-induced steatosis, we hypothesized that at least part of the action of PGC-1 alpha could be mediated by reducing the transcription of the IL-10 gene Here, we used immunoblotting, real-time polymerase chain reaction, immunocytochemistry, and chromatin immunoprecipitation assay to investigate the role of PGC-1 alpha in the control of IL-10 expression in hepatic cells First, we show that, in the intact steatotic liver, the expressions of IL-10 and PGC-1 alpha are increased Inhibiting PGC-1 alpha expression by antisense oligonucleotide increases IL-10 expression and reduces the steatotic phenotype. In cultured hepatocytes, the treatment with saturated and unsaturated fatty acids increased IL-10 expression. This was accompanied by increased association of PGC-1 alpha with c-Maf and p50-nuclear factor (NF) kappa B, 2 transcription factors known to modulate IL-10 expression In addition, after fatty acid treatment. PGC-1 alpha, c-Maf, and p50-NF kappa B migrate from the cytosol to the nuclei of hepatocytes and bind to the IL-10 promoter region Inhibiting NF kappa B activation with salicylate reduces IL-10 expression and the association of PGC-1 alpha with p50-NF kappa B Thus, PGC-1 alpha emerges as a potential transcriptional regulator of the inflammatory phenomenon taking place in the steatotic liver (C) 2010 Elsevier Inc All rights reserved