Evolution and genomic organization of muscle microRNAs in fish genomes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

MicroRNAs targeting the immunomodulatory HLA-G gene: a new survey searching for microRNAs with potential to regulate HLA-G

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expressão diferencial de microRNAs no câncer de próstata

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A quantitative view of the transcriptome of Schistosoma mansoni adult-worms using SAGE

Abstract Background Five species of the genus Schistosoma, a parasitic trematode flatworm, are causative agents of Schistosomiasis, a disease that is endemic in a large number of developing countries, affecting millions of patients around the world. By using SAGE (Serial Analysis of Gene Expression) we describe here the first large-scale quantitative analysis of the Schistosoma mansoni transcriptome, one of the most epidemiologically relevant species of this genus. Results After extracting mRNA from pooled male and female adult-worms, a SAGE library was constructed and sequenced, generating 68,238 tags that covered more than 6,000 genes expressed in this developmental stage. An analysis of the ordered tag-list shows the genes of F10 eggshell protein, pol-polyprotein, HSP86, 14-3-3 and a transcript yet to be identified to be the five top most abundant genes in pooled adult worms. Whereas only 8% of the 100 most abundant tags found in adult worms of S. mansoni could not be assigned to transcripts of this parasite, 46.9% of the total ditags could not be mapped, demonstrating that the 3 sequence of most of the rarest transcripts are still to be identified. Mapping of our SAGE tags to S. mansoni genes suggested the occurrence of alternative-polyadenylation in at least 13 gene transcripts. Most of these events seem to shorten the 3 UTR of the mRNAs, which may have consequences over their stability and regulation. Conclusion SAGE revealed the frequency of expression of the majority of the S. mansoni genes. Transcriptome data suggests that alternative polyadenylation is likely to be used in the control of mRNA stability in this organism. When transcriptome was compared with the proteomic data available, we observed a correlation of about 50%, suggesting that both transcriptional and post-transcriptional regulation are important for determining protein abundance in S. mansoni. The generation of SAGE tags from other life-cycle stages should contribute to reveal the dynamics of gene expression in this important parasite.

Identification and expression analysis of microRNAs and targets in the biofuel crop sugarcane

Abstract Background MicroRNAs (miRNAs) are small regulatory RNAs, some of which are conserved in diverse plant genomes. Therefore, computational identification and further experimental validation of miRNAs from non-model organisms is both feasible and instrumental for addressing miRNA-based gene regulation and evolution. Sugarcane (Saccharum spp.) is an important biofuel crop with publicly available expressed sequence tag and genomic survey sequence databases, but little is known about miRNAs and their targets in this highly polyploid species. Results In this study, we have computationally identified 19 distinct sugarcane miRNA precursors, of which several are highly similar with their sorghum homologs at both nucleotide and secondary structure levels. The accumulation pattern of mature miRNAs varies in organs/tissues from the commercial sugarcane hybrid as well as in its corresponding founder species S. officinarum and S. spontaneum. Using sugarcane MIR827 as a query, we found a novel MIR827 precursor in the sorghum genome. Based on our computational tool, a total of 46 potential targets were identified for the 19 sugarcane miRNAs. Several targets for highly conserved miRNAs are transcription factors that play important roles in plant development. Conversely, target genes of lineage-specific miRNAs seem to play roles in diverse physiological processes, such as SsCBP1. SsCBP1 was experimentally confirmed to be a target for the monocot-specific miR528. Our findings support the notion that the regulation of SsCBP1 by miR528 is shared at least within graminaceous monocots, and this miRNA-based post-transcriptional regulation evolved exclusively within the monocots lineage after the divergence from eudicots. Conclusions Using publicly available nucleotide databases, 19 sugarcane miRNA precursors and one new sorghum miRNA precursor were identified and classified into 14 families. Comparative analyses between sugarcane and sorghum suggest that these two species retain homologous miRNAs and targets in their genomes. Such conservation may help to clarify specific aspects of miRNA regulation and evolution in the polyploid sugarcane. Finally, our dataset provides a framework for future studies on sugarcane RNAi-dependent regulatory mechanisms.

High-throughput sequencing of small RNA transcriptomes reveals critical biological features targeted by microRNAs in cell models used for squamous cell cancer research

Abstract Background The implication of post-transcriptional regulation by microRNAs in molecular mechanisms underlying cancer disease is well documented. However, their interference at the cellular level is not fully explored. Functional in vitro studies are fundamental for the comprehension of their role; nevertheless results are highly dependable on the adopted cellular model. Next generation small RNA transcriptomic sequencing data of a tumor cell line and keratinocytes derived from primary culture was generated in order to characterize the microRNA content of these systems, thus helping in their understanding. Both constitute cell models for functional studies of microRNAs in head and neck squamous cell carcinoma (HNSCC), a smoking-related cancer. Known microRNAs were quantified and analyzed in the context of gene regulation. New microRNAs were investigated using similarity and structural search, ab initio classification, and prediction of the location of mature microRNAs within would-be precursor sequences. Results were compared with small RNA transcriptomic sequences from HNSCC samples in order to access the applicability of these cell models for cancer phenotype comprehension and for novel molecule discovery. Results Ten miRNAs represented over 70% of the mature molecules present in each of the cell types. The most expressed molecules were miR-21, miR-24 and miR-205, Accordingly; miR-21 and miR-205 have been previously shown to play a role in epithelial cell biology. Although miR-21 has been implicated in cancer development, and evaluated as a biomarker in HNSCC progression, no significant expression differences were seen between cell types. We demonstrate that differentially expressed mature miRNAs target cell differentiation and apoptosis related biological processes, indicating that they might represent, with acceptable accuracy, the genetic context from which they derive. Most miRNAs identified in the cancer cell line and in keratinocytes were present in tumor samples and cancer-free samples, respectively, with miR-21, miR-24 and miR-205 still among the most prevalent molecules at all instances. Thirteen miRNA-like structures, containing reads identified by the deep sequencing, were predicted from putative miRNA precursor sequences. Strong evidences suggest that one of them could be a new miRNA. This molecule was mostly expressed in the tumor cell line and HNSCC samples indicating a possible biological function in cancer. Conclusions Critical biological features of cells must be fully understood before they can be chosen as models for functional studies. Expression levels of miRNAs relate to cell type and tissue context. This study provides insights on miRNA content of two cell models used for cancer research. Pathways commonly deregulated in HNSCC might be targeted by most expressed and also by differentially expressed miRNAs. Results indicate that the use of cell models for cancer research demands careful assessment of underlying molecular characteristics for proper data interpretation. Additionally, one new miRNA-like molecule with a potential role in cancer was identified in the cell lines and clinical samples.

Mechanism of resistance to tyrosine kinase inhibitors in philadelphia-positive acute lymphblastic leukaemia (all): from genetic alterations to impaired RNA editing

The Ph chromosome is the most frequent cytogenetic aberration associated with adult ALL and it represents the single most significant adverse prognostic marker. Despite imatinib has led to significant improvements in the treatment of patients with Ph+ ALL, in the majority of cases resistance developed quickly and disease progressed. Some mechanisms of resistance have been widely described but the full knowledge of contributing factors, driving both the disease and resistance, remains to be defined. The observation of rapid development of lymphoblastic leukemia in mice expressing altered Ikaros (Ik) isoforms represented the background of this study. Ikaros is a zinc finger transcription factor required for normal hemopoietic differentiation and proliferation, particularly in the lymphoid lineages. By means of alternative splicing, Ikaros encodes several proteins that differ in their abilities to bind to a consensus DNA-binding site. Shorter, DNA nonbinding isoforms exert a dominant negative effect, inhibiting the ability of longer heterodimer partners to bind DNA. The differential expression pattern of Ik isoforms in Ph+ ALL patients was analyzed in order to determine if molecular abnormalities involving the Ik gene could associate with resistance to imatinib and dasatinib. Bone marrow and peripheral blood samples from 46 adult patients (median age 55 yrs, 18-76) with Ph+ ALL at diagnosis and during treatment with imatinib (16 pts) or dasatinib (30 pts) were collected. We set up a fast, high-throughput method based on capillary electrophoresis technology to detect and quantify splice variants. 41% Ph+ ALL patients expressed high levels of the non DNA-binding dominant negative Ik6 isoform lacking critical N-terminal zinc-fingers which display abnormal subcellular compartmentalization pattern. Nuclear extracts from patients expressed Ik6 failed to bind DNA in mobility shift assay using a DNA probe containing an Ikaros-specific DNA binding sequence. In 59% Ph+ ALL patients there was the coexistence in the same PCR sample and at the same time of many splice variants corresponded to Ik1, Ik2, Ik4, Ik4A, Ik5A, Ik6, Ik6 and Ik8 isoforms. In these patients aberrant full-length Ikaros isoforms in Ph+ ALL characterized by a 60-bp insertion immediately downstream of exon 3 and a recurring 30-bp in-frame deletion at the end of exon 7 involving most frequently the Ik2, Ik4 isoforms were also identified. Both the insertion and deletion were due to the selection of alternative splice donor and acceptor sites. The molecular monitoring of minimal residual disease showed for the first time in vivo that the Ik6 expression strongly correlated with the BCR-ABL transcript levels suggesting that this alteration could depend on the Bcr-Abl activity. Patient-derived leukaemia cells expressed dominant-negative Ik6 at diagnosis and at the time of relapse, but never during remission. In order to mechanistically demonstrated whether in vitro the overexpression of Ik6 impairs the response to tyrosine kinase inhibitors (TKIs) and contributes to resistance, an imatinib-sensitive Ik6-negative Ph+ ALL cell line (SUP-B15) was transfected with the complete Ik6 DNA coding sequence. The expression of Ik6 strongly increased proliferation and inhibited apoptosis in TKI sensitive cells establishing a previously unknown link between specific molecular defects that involve the Ikaros gene and the resistance to TKIs in Ph+ ALL patients. Amplification and genomic sequence analysis of the exon splice junction regions showed the presence of 2 single nucleotide polymorphisms (SNPs): rs10251980 [A/G] in the exon2/3 splice junction and of rs10262731 [A/G] in the exon 7/8 splice junction in 50% and 36% of patients, respectively. A variant of the rs11329346 [-/C], in 16% of patients was also found. Other two different single nucleotide substitutions not recognized as SNP were observed. Some mutations were predicted by computational analyses (RESCUE approach) to alter cis-splicing elements. In conclusion, these findings demonstrated that the post-transcriptional regulation of alternative splicing of Ikaros gene is defective in the majority of Ph+ ALL patients treated with TKIs. The overexpression of Ik6 blocking B-cell differentiation could contribute to resistance opening a time frame, during which leukaemia cells acquire secondary transforming events that confer definitive resistance to imatinib and dasatinib.

Knock down von Desmoglein 2 durch induzierbare Cre-spezifische Rekombination in der Maus

Desmosomen sind hoch organisierte adhäsive interzelluläre Verbindungen, die benachbarte Zellen durch Verankerung mit den Intermediärfilamenten des Zytoskeletts miteinander verknüpfen und so Zellen und Geweben Stabilität verleihen. Die Adhäsionsmoleküle der Desmosomen sind die desmosomalen Cadherine. Diese transmembranen Glykoproteine gehen im Interzellulärraum Verbindungen mit den desmosomalen Cadherinen der Nachbarzelle ein und sind im zytoplasmatischen Bereich Anheftungspunkte für weitere an der Desmosomenbildung beteiligte Proteine. Ziel dieser Arbeit war die Untersuchung der Rolle von Desmoglein 2 (Dsg 2), einem in allen Epithelien exprimierten desmosomalen Cadherin. Da der konstitutive knock out von Dsg 2 embryonal letal ist, wurde im Rahmen dieser Doktorarbeit eine transgene Maus generiert, in der die Reduktion von Dsg 2 temporär regulierbar war (konditionaler knock down). Dazu wurde der Mechanismus der RNA Interferenz genutzt, wodurch Sequenz-spezifische, post-transkriptionelle Regulation von Genen möglich ist. Unter Verwendung eines über Cre/lox-induzierbaren Vektors wurden transgene Mäuse generiert, welche nach Induktion Dsg 2 shRNA exprimieren, die in der Zelle in siRNA umgewandelt wird und zum Abbau der Dsg 2 mRNA führt. Durch Verpaarung der generierten Dsg 2 knock down Maus mit der über Tamoxifen induzierbaren Cre Deleter knock in Mauslinie Rosa26CreERT2 konnte deutliche Reduktion der Dsg 2 Proteinmenge in Leber, Darm und Herz erreicht werden. In Immunfärbungen der Leber wurde zudem eine reduzierte Desmosomenbildung durch Expression der Dsg 2 shRNA detektiert. Die für diese Versuche generierte und getestete Rosa26CreERT2 Mauslinie ermöglichte jedoch nicht in allen Zellen eines Gewebes die komplette Aktivierung der Cre Rekombinase und damit die Expression der shRNA. Dadurch entstanden mosaikartige Wildtyp/knock down-Gewebe, in denen noch ausreichend Desmosomen gebildet wurden, um die Gewebestabilität und -struktur zu erhalten. Für eine funktionale Untersuchung von Dsg 2 in Zusammenhang mit der chronisch entzündlichen Darmerkrankung Colitis ulcerosa wurden die Dsg 2 knock down Mäuse mit Darm-spezifischen, induzierbaren Cre Deleter Mäusen (VillinCreERT2) verpaart. Nach Aktivierung der Cre Rekombinase mittels Tamoxifen wurde in bitransgenen Tieren über Gabe von Azoxymethan (AOM) und Dextransodiumsulfat (DSS) Colitis ulcerosa induziert. Diese entzündliche Erkrankung des Darms ist mit der Induktion von Darmtumoren assoziiert. Bereits nach einmaliger Induktion mit AOM/DSS wurde in der ersten endoskopischen Untersuchung eine starke Entzündung des Darmgewebes und die Ausbildung von flächig wachsenden Tumoren in den Dsg 2 knock down Tieren hervorgerufen. Es ist anzunehmen, dass durch knock down von Dsg 2, und die damit verbundene verminderte Desmosomenbildung und Zelladhäsion, Infiltration von Bakterien durch die epitheliale Barriere des Darms möglich war, und so die Entzündungsreaktion in der Darmmukosa verstärkte. In Zusammenhang mit Verlust der epithelialen Festigkeit durch verringerte Zellkontakte kam es zur Hyperproliferation der Darmmukosa, die sich in Ausbildung von flächigen Tumoren äußerte. In weiteren Experimenten müssen nun die Tumore und das entzündete Gewebe der Colitis-induzierten Mäuse mittels Immunfluoreszenz untersucht werden, um Veränderungen in der Desmosomenformation in situ detektieren zu können. Des Weiteren sind Verpaarungen der Dsg 2 knock down Maus mit anderen Cre Rekombinase exprimierenden Mauslinien möglich, um den Einfluss von Dsg 2 auch in anderen Geweben, beispielsweise im Herzen, zu untersuchen. Die hier vorgelegte Arbeit zeigt also erstmalig den ursächlichen Zusammenhang zwischen Dsg 2 und dem Auftreten von Colitis-assoziierten Tumoren in einem konditionalen RNAi-vermittelten knock down Tiermodell. Die Etablierung dieser Maus ist somit das erste konditionale Mausmodell, welches die bei vielen Krebspatienten gefundenen flachzellig wachsenden Tumore in vivo rekapituliert. Vorausschauend kann man sagen, dass mit Hilfe des im Rahmen dieser Doktorarbeit entwickelten Tiermodells wichtige Erkenntnisses über die Pathologie von Darmtumoren erbracht werden können, die unser Verständnis der Colitis-induzierten Tumorentstehung verbessern.

Fyn kinase targets in oligodendroglial physiology and myelination

In the central nervous system (CNS), oligodendrocytes form the multilamellar and compacted myelin sheath by spirally wrapping around defined axons with their specialised plasma membrane. Myelin is crucial for the rapid saltatory conduction of nerve impulses and for the preservation of axonal integrity. The absence of the major myelin component Myelin Basic Protein (MBP) results in an almost complete failure to form compact myelin in the CNS. The mRNA of MBP is sorted to cytoplasmic RNA granules and transported to the distal processes of oligodendrocytes in a translationally silent state. A main mediator of MBP mRNA localisation is the trans-acting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 which binds to the cis-acting A2 response element (A2RE) in the 3’UTR of MBP mRNA. A signalling cascade had been identified that triggers local translation of MBP at the axon-glial contact site, involving the neuronal cell adhesion molecule (CAM) L1, the oligodendroglial plasma membrane-tethered Fyn kinase and Fyn-dependent phosphorylation of hnRNP A2. This model was confirmed here, showing that L1 stimulates Fyn-dependent phosphorylation of hnRNP A2 and a remodelling of A2-dependent RNA granule structures. Furthermore, the RNA helicase DDX5 was confirmed here acting together with hnRNP A2 in cytoplasmic RNA granules and is possibly involved in MBP mRNA granule dynamics.rnLack of non-receptor tyrosine kinase Fyn activity leads to reduced levels of MBP and hypomyelination in the forebrain. The multiadaptor protein p130Cas and the RNA-binding protein hnRNP F were verified here as additional targets of Fyn in oligodendrocytes. The findings point at roles of p130Cas in the regulation of Fyn-dependent process outgrowth and signalling cascades ensuring cell survival. HnRNP F was identified here as a novel constituent of oligodendroglial cytoplasmic RNA granules containing hnRNP A2 and MBP mRNA. Moreover, it was found that hnRNP F plays a role in the post-transcriptional regulation of MBP mRNA and that defined levels of hnRNP F are required to facilitate efficient synthesis of MBP. HnRNP F appears to be directly phosphorylated by Fyn kinase what presumably contributes to the initiation of translation of MBP mRNA at the plasma membrane.rnFyn kinase signalling thus affects many aspects of oligodendroglial physiology contributing to myelination. Post-transcriptional control of the synthesis of the essential myelin protein MBP by Fyn targets is particularly important. Deregulation of these Fyn-dependent pathways could thus negatively influence disorders involving the white matter of the nervous system.rnrn

Die Bedeutung von KSRP für die pro-inflammatorische Genexpression in Autoimmunerkrankungen - Einfluss des Naturstoffes Resveratrol auf KSRP-vermittelte Regulationsmechanismen

Die Pathogenese chronisch inflammatorischer Erkrankungen ist von einer Dysregulation der pro-inflammatorischen Genexpression geprägt. Dieser liegen wahrscheinlich pathologische Veränderungen der Aktivität von verschiedenen Transkriptionsfaktoren und RNA-bindenden Proteinen zugrunde. In dieser Arbeit konnte die Regulation der KSRP-Expression in einem murinen Modell der rheumatoiden Arthritis (RA) nachgewiesen werden. In humanen Chondrozyten führte eine erhöhte KSRP-Expression zu einer Reduktion der Expression von bekannten KSRP-Zielgenen. Der Vergleich von verschiedenden Microarray-Analysen aus den verwendeten humanen und murinen Modellen der RA führte zur Identifikation von pro-inflammatorischen und pro-angiogenetischen Faktoren (SPARC, MMP2, MMP3, PLA2G2D, GZMA, HPSE, TNMD und IL-18-R), die in der RA eine Rolle spielen und höchstwahrscheinlich durch eine erhöhte KSRP-Expression reguliert werden. Daher könnte eine Modulation der KSRP-Expression bei der Therapie von Autoimmunerkrankungen von Bedeutung sein. In diesem Zusammenhang ist die Detektion der Bindung des cardioprotektiven und anti-inflammatorisch wirkenden Naturstoffs Resveratrol an KSRP zu nennen. Diese spezifische Interaktion führte zu einer Reduktion der p38-MAPK-vermittelten Thr-Phosphorylierung des KSRP-Proteins (in situ und in vivo), was eine Aktivierung der KSRP-vermittelten Mechanismen zur Folge hatte. Somit konnte in situ die mRNA-Stabilität der iNOS reduziert und die miR-155-Expression erhöht werden. Im murinen Atherosklerosemodell führte die Behandlung mit Resveratrol zu einer verringerten Expression bekannter KSRP-Ziel-mRNAs. rnNeben diesem post-translationalen Regulationsmechanismus von KSRP durch Resveratrol konnte die Modulation der KSRP-Expression auf transkriptioneller Ebene durch KSRP selbst gezeigt werden. Dies geschieht möglicherweise über die Bindung von KSRP an das FUSE-analoge Element innerhalb des KSRP-Promotors, welches eine positive Autoregulation der KSRP-Expression bewirkt. Bei der Analyse der post-transkriptionellen Regulation der KSRP-Expression interagierten die mRNA-bindenden Proteine HuR, PABP und die AUF-1-Isoformen p40, p42 und p45 in vitro mit der KSRP-3’UTR. Dabei konnte in Expressionsanalysen nachgewiesen werden, dass die KSRP-mRNA durch PABP positiv und durch p42 negativ reguliert wird.rnZusammenfassend ist zu sagen, dass die KSRP-Expression neben post-translationalen Mechanismen auch auf transkriptioneller und post-transkriptioneller Ebene moduliert wird. Zusätzlich wurde eine Regulation der KSRP-Expression innerhalb entzündlicher Erkrankungen nachgewiesen, die Bedeutung dieser Modulation für die pro-inflammatorischen Genexpression diskutiert und ein möglicher therapeutischer Angriffspunkt durch Resveratrol identifiziert.

Analysen zur Expression proinflammatorischer Mediatoren in humanen Zellkulturmodellen und murinen Tiermodellen chronisch-inflammatorischer Erkrankungen

Die humane induzierbare NO-Synthase (iNOS) spielt bei zahlreichen Erkrankungen wie Asthma, Krebs und der rheumatoiden Arthritis eine entscheidende Rolle. Durch Fehlregulation der iNOS-Expression kommt es häufig zu massiven Gewebeschädigungen. Aus diesem Grund ist es wichtig die Mechanismen der Genregulation der iNOS-Expression zu verstehen. Bei Affinitätschromatographie-Analysen wurde das zytosolische PolyA-bindende Protein (PABP) als direkter Interaktionspartner der 3´UTR der humanen iNOS identifiziert. Weitere Bindungsanalysen konnten eine spezifische Bindestelle für PABP in der 5´UTR und zwei Bindestellen im AU-reichen Bereich der 3´UTR der humanen iNOS nachweisen. Eine siRNA-mediierte Herabregulation von PABP mit Hilfe der stabilen Expression spezifischer siRNAs in DLD-1 Zellen (siPABP Zellen) zeigte eine signifikant verringerte Expression der humanen iNOS und damit einhergehend eine verringerte NO-Produktion nach Zytokinstimulation. Promotoranalysen zeigten keine Veränderung der Induzierbarkeit des humanen 16 kb iNOS-Promotors in siPABP Zellen. RNA-Stabilitätsanalysen zeigten einen verstärkten Abbau der iNOS-mRNA in diesen Zellen, so dass davon auszugehen ist, dass die Regulation der humanen iNOS über die mRNA-Stabilität erfolgt. Reportergen-Analysen mit Plasmiden, welche die 5’ und/oder 3’UTR Sequenzen der humanen iNOS mit den identifizierten PABP-Bindestellen oder Mutationen in diesen Bindestellen enthielten, zeigten, dass PABP die iNOS-mRNA über die 5´UTR stabilisiert und anscheinend über die 3´UTR einen destabilisierenden Effekt auf die mRNA ausübt. Ebenfalls scheint PABP über die 3’UTR dieTranslation der iNOS mRNA zu hemmen. Die Ergebnisse dieser Arbeit zeigen, dass PABP, über seine allgemeinen Funktionen hinaus, eine spezifische Rolle in der Regulation der Expression der humanen iNOS einnimmt.rnDie rheumatoide Arthritis (RA) ist eine chronisch entzündliche Autoimmunerkrankung, welche überwiegend die peripheren Gelenke der Hände und Füße betrifft. Die aktuellen Therapiemöglichkeiten sind immer noch mit einer Vielzahl von Nebenwirkungen behaftet und führen nicht zur vollständigen Remission der Erkrankung, so dass die Entwicklung neuer Medikamente unerlässlich ist. In dieser Arbeit wurden die antiinflammatorischen Substanzen Gallielalacton (Gal) und Oxacyclododecindion (Oxa) im Mausmodell der kollagen-induzierten Arthritis (CIA) getestet. Leider waren beide Substanzen nicht in der Lage die Symptome der CIA zu vermindern, obwohl beide im Modell der LPS-induzierten akuten Entzündung die Expression proinflammatorischer Mediatoren senken konnten. Die Substanz S-Curvularin (SC) hat sich im CIA-Modell bereits bewährt und wurde in dieser Arbeit weiter untersucht. SC war in der Lage die Expression knorpel- und knochendestruktiver Markergene signifikant zu verrindern. rnIn der vorliegenden Arbeit wurden neue microRNAs identifiziert, die in der Pathogenese der CIA eine Dysregulation zeigen. Die Expression dieser microRNAs wurde von SC wieder auf das Normalniveau gebracht, so dass SC eine vielversprechende Substanz in der Therapie chronisch inflammatorische Erkrangungen sein könnte. Die neu identifizierten CIA-relevanten microRNAs könnten als neueRA-Marker oder als Zielstrukturen für neue Medikamente dienen.rn

Different adaptation of IGF-I and its IGFBPs in dairy cows during a negative energy balance in early lactation and a negative energy balance induced by feed restriction in mid-lactation

Control of metabolic pathways is a major task of the somatotropic axis and its constituents. Insulinlike growth-factor binding proteins (IGFBPs) bind IGF-I and -II and act as carriers and regulators of their activities in blood, body fluids and tissues. Over two periods of physiological adaptation, this study investigated the binding pattern of IGF-I to IGFBPs in the plasma of 50 multiparous Holstein dairy cows and identified relationships with the hepatic mRNA abundance of IGFBPs and plasma IGF-I during the lactational negative energy balance (NEB) and during a deliberately induced NEB by feed restriction. Period 1 lasted from week 3 antepartum (a.p.) to week 12 postpartum (p.p.) and period 2, the period of feed restriction, started at around 100 DIM and lasted for three weeks with a control (C) and a restricted group (R). Blood samples and liver biopsies were collected in week 3 a.p., and in weeks 1 and 4 p.p. of period 1 and in weeks 0 and 3 of period 2. For column chromatography of IGFBPs, plasma samples of all animals were pooled by group and time points of sampling. Plasma IGF-I dropped from week 3 a.p. to week 1 p.p. and thereafter increased until week 0 (period 2) and did not change up to week 3 of period 2. The binding of IGF-I to plasma IGFBP-1 and -2 increased in period 1 from week 3 a.p. to week 4 p.p., while at the same time it decreased for IGFBP-3. During period 2, the binding of IGF-I to plasma IGFBP-1 and -2 decreased for both groups, but less for R cows. In C cows, the IGF-I binding to IGFBP-3 in plasma increased from week 0 to week 3 of period 2, whereas R cows showed a slight decrease. In period 1, hepatic mRNA abundance of IGFBP-3 followed the plasma IGFBP-3 binding in contrast to the mRNA abundances of IGFBP-1 and -2. The latter increased from week 3 a.p. to week 1 p.p. and decreased afterwards whereas IGF-I binding to IGFBP-1 and -2 increased. In week 3 of period 2, the binding of IGF-I to IGFBP-1 and -2 and their hepatic mRNA abundance were higher in R cows compared to C cows. Hepatic mRNA abundance of IGF-I was consistently positively correlated with plasma IGF-I, especially pronounced during the NEBs in week 1 p.p. (period 1) and in week 3 (period 2) in R cows. While no distinct relation between mRNA abundance of IGFBP-1 and plasma IGF-I was evident, the mRNA abundance of IGFBP-2 was inversely related to plasma IGF-I over all experimental time points independent of treatment. The mRNA abundance of IGFBP-3 was particularly correlated with plasma IGF-I during the 2 experimental stages of a NEB. Obviously IGFBP-3, but not IGFBP-1 and -2, binding in plasma closely followed the respective pattern of hepatic mRNA abundance during the entire experimental period. The fact that changes in the different plasma IGFBPs during altering metabolic stages in different stages of lactation do not always strictly follow their mRNA abundance in liver suggests tissues other than the liver flexibly contributing to the IGFBP pool in plasma as well as a partially post-transcriptional regulation of IGFBP synthesis.

The Immune Inhibitor A1 Protease of Bacillus anthracis

Bacillus anthracis, an organism ubiquitous in the soil and the causative agent of anthrax, utilizes multiple mechanisms to regulate secreted factors; one example is the activity of secreted proteases. One of the most abundant proteins in the culture supernates of B. anthracis is the Immune Inhibitor A1 (InhA1) protease. Here, I demonstrate that InhA1 modulates the abundance of approximately half of the proteins secreted into the culture supernates, including substrates that are known to contribute to the ability of the organism to cause virulence. For example, InhA1 cleaves the anthrax toxin proteins, PA, LF, and EF. InhA1 also targets a number of additional proteases, including Npr599, contributing to a complex proteolytic regulatory cascade with far-reaching affects on the secretome. Using an intra-tracheal mouse model of infection, I found that an inhA-null strain is attenuated in relation to the parent strain. The data indicate that reduced virulence of the inhA mutant strain may be the result of toxin protein deregulation, decreased association with macrophages, and/or the inability to degrade host antimicrobial peptides. Given the significant modulation of the secretome by InhA1, it is likely that expression of the protease is tightly regulated. To test this I examined inhA1 transcript and protein levels in the parent and various isogenic mutant strains and found that InhA1 expression is regulated by several mechanisms. First, the steady state levels of inhA1 transcript are controlled by the regulatory protein SinR, which inhibits inhA1 expression. Second, InhA1 abundance is inversely proportional to the SinR-regulated protease camelysin, indicating the post-transcriptional regulation of InhA1 by camelysin. Third, InhA1 activity is dependent on a conserved zinc binding motif, suggesting that zinc availability regulates InhA1 activity. The convergence of these regulatory mechanisms signifies the importance of tight regulation of InhA1 activity, activity that substantially affects how B. anthracis interacts with its environment.

Dynamic Remodeling of the Stressed Heart: Role of Protein Degradation Pathways

The heart is a remarkable organ. In order to maintain its function, it remodels in response to a variety of environmental stresses, including pressure overload, volume overload, mechanical or pharmacological unloading and hormonal or metabolic disturbances. All these responses are linked to the inherent capacity of the heart to rebuild itself. Particularly, cardiac pressure overload activates signaling pathways of both protein synthesis and degradation. While much is known about regulators of protein synthesis, little is known about regulators of protein degradation in hypertrophy. The ubiquitin-proteasome system (UPS) selectively degrades unused and abnormal intracellular proteins. I speculated that the UPS may play an important role in both qualitative and quantitative changes in the composition of heart muscle during hypertrophic remodeling. My study hypothesized that cardiac remodeling in response to hypertrophic stimuli is a dynamic process that requires activation of highly regulated mechanisms of protein degradation as much as it requires protein synthesis. My first aim was to adopt a model of left ventricular hypertrophy and determine its gene expression and structural changes. Male Sprague-Dawley rats were submitted to ascending aortic banding and sacrificed at 7 and 14 days after surgery. Sham operated animals served as controls. Effective aortic banding was confirmed by hemodynamic assessment by Doppler flow measurements in vivo. Banded rats showed a four-fold increase in peak stenotic jet velocities. Histomorphometric analysis revealed a significant increase in myocyte size as well as fibrosis in the banded animals. Transcript analysis showed that banded animals had reverted to the fetal gene program. My second aim was to assess if the UPS is increased and transcriptionally regulated in hypertrophic left ventricular remodeling. Protein extracts from the left ventricles of the banded and control animals were used to perform an in vitro peptidase assay to assess the overall catalytic activity of the UPS. The results showed no difference between hypertrophied and control animals. Transcript analysis revealed decreases in transcript levels of candidate UPS genes in the hypertrophied hearts at 7 days post-banding but not at 14 days. However, protein expression analysis showed no difference at either time point compared to controls. These findings indicate that elements of the UPS are downregulated in the early phase of hypertrophic remodeling and normalizes in a later phase. The results provide evidence in support of a dynamic transcriptional regulation of a major pathway of intracellular protein degradation in the heart. The discrepancy between transcript levels on the one hand and protein levels on the other hand supports post-transcriptional regulation of the UPS pathway in the hypertrophied heart. The exact mechanisms and the functional consequences remain to be elucidated.

BTG/TOB factors impact deadenylases.

BTG/TOB factors are a family of antiproliferative proteins whose expression is altered in numerous cancers. They have been implicated in cell differentiation, development and apoptosis. Although proposed to affect transcriptional regulation, these factors interact with CAF1, a subunit of the main eukaryotic deadenylase, and with poly(A)-binding-proteins, strongly suggesting a role in post-transcriptional regulation of gene expression. The recent determination of the structures of BTG2, TOB1 N-terminal domain (TOB1N138) and TOB1N138-CAF1 complexes support a role for BTG/TOB proteins in mRNA deadenylation, a function corroborated by recently published functional characterizations. We highlight molecular mechanisms by which BTG/TOB proteins influence deadenylation and discuss the need for a better understanding of BTG/TOB physiological functions.