cis-Regulation des Drosophila-melanogaster-Gens optomotor-blind

Die zeitliche und räumliche Expression von Genen trägt zu einem entscheidenden Ausmaß zu der Entwicklung eines Organismus bei. Unter vielen Faktoren spielt dabei die transkriptionelle Regulation eine wichtige Rolle. Diese basiert auf Anwesenheit und Binden von regulatorischen Proteinen an cis-regulatorischen Sequenzen (CRMs) und deren Einfluss auf die Transkriptionsmaschinerie am Promotor. Veränderungen der CRMs können zu Veränderungen der Genexpression führen, und somit einen Beitrag zur morphologischen Evolution leisten. rnIn dieser Arbeit wurde die transkriptionelle Regulation des Drosophila melanogaster Gens optomotor-blind insbesondere in den pupalen Tergiten untersucht. In einem Enhancer-Reporter screen wurde eine regulatorische Region in Intron IV, die Reportergen-Expression in den pupalen Tergiten treibt, identifiziert. Große Teile dieser Region (ombTU10 und ombTU11) trieben Reportergen-Expression in einem omb-ähnlichen Muster. Eine weitere Region (ombTU12) trieb Expression in einem für Hh-Zielgene typischen Expressionsmuster. Für ombTU12 konnte eine Hh-Abhängigkeit nachgewiesen werden. Die für Hh-Zielgene typische Enhanceraktivität konnte in dem Subfragment ombTU12Amin lokalisiert werden, welches zwei konservierte Bindestellen des Effektors der Hh-Signaltransduktionskaskase, Cubitus interruptus (Ci), enthält. Eine deutliche Abhängigkeit der Expression dieses Fragments von den Ci-Bindestellen konnte bisher aber noch nicht nachgewiesen werden.rnDeletionen verschiedener Bereiche dieser Tergitenenhancer-Region aus dem endogenen Gen sollten Aufschluss über deren Notwendigkeit in der Regulation von omb geben. Die Deletion des Fragments ombTU10 (ΔombTU10-2) führte zu einer Variabilität in der Pigmentierung der Abdominalsegmente A5 und A6 der Weibchen. Eine Deletion von Teilen des hh-responsiven Fragments ombTU12 (ΔombTU12A) zeigte keinen abdominalen Phänotyp. Dies deutet auf eine redundante Wirkung der Fragmente untereinander, oder mit einem weiteren bisher nicht identifizierten Tergitenenhancer im omb-Locus hin.rnFragmente, die in den pupalen Tergiten Reportergen-Expression trieben, waren zum Teil auch in Imaginalscheiben von Larven aktiv. Desweiteren wurde gezeigt, dass Fragmente, die in Isolation Reportergen-Expression trieben, als Fusionskonstrukt mit benachbarten genomischen Sequenzen keine Expression zeigten und somit im genomischen Kontext inaktiv sein können. Demzufolge sind nicht nur Aktivator- sondern auch Repressorregionen für die korrekte Expression eines Gens von Bedeutung.rnDie Analyse von omb Enhancer-Trap Insertionen zeigte, dass von drei untersuchten Typen (PlacW, PGalW und PGawB) nur Insertionen vom letzteren in den pupalen Tergiten aktiv waren. Von vier PGawB Insertionen waren nur drei aktiv. Es ist denkbar, dass die Orientierung der inaktiven Insertion für die mangelnde Responsivität verantwortlich ist.rn

Charakterisierung und Expression zweier multimerer Hämolympheproteine der Schnecke Biomphalaria glabrata

Die tropische Süsswasserschnecke Biomphalaria glabrata gehört zu der Familie der Planorbidae, welche als einziges Taxon der Gastropoden Hämoglobin als Sauerstofftransportprotein verwenden. Als Zwischenwirt des Bilharzioseerregers Schistosoma mansoni ist B. glabrata von tropenmedizinischer Interesse. Das extrazelluläre BgHb zeigt sich mit einem Anteil von 95% als Hauptprotein in der Hämolymphe. Dieses setzt sich aus Polypeptidketten mit je 240kDa zusammen. Diese wiederrum lassen sich in 13-Häm-Domänen und eine deutlich kleinere N-terminalen nicht Häm-Domäne untergliedern. Die Sequenzierung von zwei der drei Untereinheiten des BgHb (BgHb1, BgHb2) ermöglichte die rekombinante Expression ganzer Untereinheiten in Insektenzellen, und die Expression einiger BgHb2-Konstrukte in E. coli Zellen. Im Rahmen meiner Arbeit gelang es, BgHb1 in biologisch aktiver Form in Insektenzellen zu exprimieren. Das aus dem Überstand der Insektenzellen aufgereinigte rekombinante BgHb1 zeigte eine immunologische Identität mit nativen BgHb. Strukturelle Analysen belegten zudem die Assemblierung des rekombinanten BgHb1 zu einer dem nativen Protein gleichenden Quartärstruktur. Demnach konnte in meiner Arbeit der Nachweis erbracht werden, dass eine einzelne Isoform in der Lage ist, zur Quartärstruktur zu assemblieren. Zusätzlich ergaben Sauerstoffbindungsanalysen, dass das rekombinante BgHb1 reversibel Sauerstoff binden kann.rnIn den restlichen 5% der B. glabrata Hämolymphe zeigt sich ein rudimentäres Hämocyanin, welches für den Sauerstofftransport keine Rolle zu spielen scheint, und ein rosettenförmiges Protein, das es aufzuklären galt. Durch massenspektrometrische Analysen erhaltene Peptidfragmente zeigten eine hohe Sequenzähnlichkeit zu den löslichen Acetylcholin -Bindeproteinen anderer Mollusken. Diese AChBP zeigen eine hohe Sequenzähnlichkeit zur Ligandenbindedomäne von Rezeptoren der Cys-Loop-Proteinfamilie.rnDatenbankrecherchen deckten die Existenz zweier Isoformen auf

Genexpressionsanalyse boviner mesenchymaler Stammzellen und deren in-vitro-differenzierten Folgelinien

Mesenchymale Stamzellen (MSC) sind Vertreter der adulten Stammzellen. Sie bergen durch ihre große Plastizität ein immenses Potential für die klinische Nutzung in Form von Stammzelltherapien. Zellen dieses Typs kommen vornehmlich im Knochenmark der großen Röhrenknochen vor und können zu Knochen, Knorpel und Fettzellen differenzieren. MSC leisten einen wichtigen Beitrag im Rahmen regenerativer Prozesse, beispielsweise zur Heilung von Frakturen. Breite Studien demonstrieren bereits jetzt auch bei komplexeren Erkrankungen (z.B. Osteoporose) therapeutisch vielversprechende Einsatzmöglichkeiten. Oft kommen hierbei aus MSC gezielt differenzierte Folgelinien aus Zellkulturen zum Einsatz. Dies bedingt eine kontrollierte Steuerung der Differenzierungsprozesse in vitro. Der Differenzierung einer Stammzelle liegt eine komplexe Veränderung ihrer Genexpression zugrunde. Genexpressionsmuster zur Erhaltung und Proliferation der Stammzellen müssen durch solche, die der linienspezifischen Differenzierung dienen, ersetzt werden. Die mit der Differenzierung einhergehende, transkriptomische Neuausrichtung ist für das Verständnis der Prozesse grundlegend und wurde bislang nur unzureichend untersucht. Ziel der vorliegenden Arbeit ist eine transkriptomweite und vergleichende Genexpressionsanalyse Mesenchymaler Stammzellen und deren in vitro differenzierten Folgelinien mittels Plasmid - DNA Microarrays und Sequenziertechniken der nächsten Generation (RNA-Seq, Illumina Plattform). In dieser Arbeit diente das Hausrind (Bos taurus) als Modellorganismus, da es genetisch betrachtet eine hohe Ähnlichkeit zum Menschen aufweist und Knochenmark als Quelle von MSC gut verfügbar ist. Primärkulturen Mesenchymaler Stammzellen konnten aus dem Knochenmark von Rindern erfolgreich isoliert werden. Es wurden in vitro Zellkultur - Versuche durchgeführt, um die Zellen zu Osteoblasten, Chondrozyten und Adipozyten zu differenzieren. Zur Genexpressionsanalyse wurde RNA aus jungen MSC und einer MSC Langzeitkultur („alte MSC“), sowie aus den differenzierten Zelllinien isoliert und für nachfolgende Experimente wo nötig amplifiziert. Der Erfolg der Differenzierungen konnte anhand der Genexpression von spezifischen Markergenen und mittels histologischer Färbungen belegt werden. Hierbei zeigte sich die Differenzierung zu Osteoblasten und Adipozyten erfolgreich, während die Differenzierung zu Chondrozyten trotz diverser Modifikationen am Protokoll nicht erfolgreich durchgeführt werden konnte. Eine vergleichende Hybridisierung zur Bestimmung differentieller Genexpression (MSC vs. Differenzierung) mittels selbst hergestellter Plasmid - DNA Microarrays ergab für die Osteogenese mit Genen wie destrin und enpp1, für die undifferenzierten MSC mit dem Gen sema3c neue Kandidatengene, deren biologische Funktion aufzuklären in zukünftigen Experimenten vielversprechende Ergebnisse liefern sollte. Die Analyse der transkriptomweiten Genexpression mittels NGS lieferte einen noch umfangreicheren Einblick ins Differenzierungsgeschehen. Es zeigte sich eine hohe Ähnlichkeit im Expressionsprofil von jungen MSC und Adipozyten, sowie zwischen den Profilen der alten MSC (eine Langzeitkultur) und Osteoblasten. Die alten MSC wiesen deutliche Anzeichen für eine spontane Differenzierung in die osteogene Richtung auf. Durch Analyse der 100 am stärksten exprimierten Gene jeder Zelllinie ließen sich für junge MSC und Adipozyten besonders Gene der extrazellulären Matrix (z.B col1a1,6 ; fn1 uvm.) auffinden. Sowohl Osteoblasten, als auch die alten MSC exprimieren hingegen verstärkt Gene mit Bezug zur oxidativen Phosphorylierung, sowie ribosomale Proteine. Eine Betrachtung der differentiellen Genexpression (junge MSC vs. Differenzierung) mit anschließender Pathway Analyse und Genontologie Anreicherungsstatistik unterstützt diese Ergebnisse vor allem bei Osteoblasten, wo nun jedoch zusätzlich auch Gene zur Regulation der Knochenentwicklung und Mineralisierung in den Vordergrund treten. Für Adipozyten konnte mit Genen des „Jak-STAT signaling pathway“, der Fokalen Adhäsion, sowie Genen des „Cytokine-cytokine receptor interaction pathway“ sehr spannende Einsichten in die Biologie dieses Zelltyps erlangt werden, die sicher weiterer Untersuchungen bedürfen. In undifferenzierten MSC konnte durch differentielle Genexpressionsanalyse die Rolle des nicht kanonischen Teils des WNT Signalweges als für die Aufrechterhaltung des Stammzellstatus potentiell äußerst einflussreich ermittelt werden. Die hier diskutierten Ergebnisse zeigen beispielhaft, dass besonders mittels Genexpressionsanalyse im Hochdurchsatzverfahren wertvolle Einblicke in die komplexe Biologie der Stammzelldifferenzierung möglich sind. Als Grundlage für nachfolgende Arbeiten konnten interessante Gene ermittelt und Hypothesen zu deren Einfluss auf Stammzelleigenschaften und Differenzierungsprozesse aufgestellt werden. Um einen besseren Einblick in den Differenzierungsverlauf zu ermöglichen, könnten künftig NGS Analysen zu unterschiedlichen Differenzierungszeitpunkten durchgeführt werden. Zudem wären weitere Anstrengungen zur erfolgreichen Etablierung der chondrogenen Differenzierung zur vollständigen Analyse der Genexpression des trilinearen Differenzierungspotentials von MSC wünschenswert.

Expression of human cathepsin L or human cathepsin V in mouse thymus mediates positive selection of T helper cells in cathepsin L knock-out mice

A genetic deficiency of the cysteine protease cathepsin L (Ctsl) in mice results in impaired positive selection of conventional CD4+ T helper cells as a result of an incomplete processing of the MHC class II associated invariant chain or incomplete proteolytic generation of positively selecting peptide ligands. The human genome encodes, in contrast to the mouse genome, for two cathepsin L proteases, namely cathepsin L (CTSL) and cathepsin V (CTSV; alternatively cathepsin L2). In the human thymic cortex, CTSV is the predominately expressed protease as compared to CTSL or other cysteine cathepsins. In order to analyze the functions of CTSL and CTSV in the positive selection of CD4+ T cells we employed Ctsl knock-out mice crossed either with transgenic mice expressing CTSL under the control of its genuine human promoter or with transgenic mice expressing CTSV under the control of the keratin 14 (K14) promoter, which drives expression to the cortical epithelium. Both human proteases are expressed in the thymus of the transgenic mice, and independent expression of both CTSL and CTSV rescues the reduced frequency of CD4+ T cells in Ctsl-deficient mice. Moreover, the expression of the human cathepsins does not change the number of CD4+CD25+Foxp3+ regulatory T cells, but the normalization of the frequency of conventional CD4+ T cell in the transgenic mice results in a rebalancing of conventional T cells and regulatory T cells. We conclude that the functional differences of CTSL and CTSV in vivo are not mainly determined by their inherent biochemical properties, but rather by their tissue specific expression pattern.

Uromodulin is expressed in renal primary cilia and UMOD mutations result in decreased ciliary uromodulin expression

Uromodulin (UMOD) mutations are responsible for three autosomal dominant tubulo-interstitial nephropathies including medullary cystic kidney disease type 2 (MCKD2), familial juvenile hyperuricemic nephropathy and glomerulocystic kidney disease. Symptoms include renal salt wasting, hyperuricemia, gout, hypertension and end-stage renal disease. MCKD is part of the 'nephronophthisis-MCKD complex', a group of cystic kidney diseases. Both disorders have an indistinguishable histology and renal cysts are observed in either. For most genes mutated in cystic kidney disease, their proteins are expressed in the primary cilia/basal body complex. We identified seven novel UMOD mutations and were interested if UMOD protein was expressed in the primary renal cilia of human renal biopsies and if mutant UMOD would show a different expression pattern compared with that seen in control individuals. We demonstrate that UMOD is expressed in the primary cilia of renal tubules, using immunofluorescent studies in human kidney biopsy samples. The number of UMOD-positive primary cilia in UMOD patients is significantly decreased when compared with control samples. Additional immunofluorescence studies confirm ciliary expression of UMOD in cell culture. Ciliary expression of UMOD is also confirmed by electron microscopy. UMOD localization at the mitotic spindle poles and colocalization with other ciliary proteins such as nephrocystin-1 and kinesin family member 3A is demonstrated. Our data add UMOD to the group of proteins expressed in primary cilia, where mutations of the gene lead to cystic kidney disease.

BCL-2 family member BOK is widely expressed but its loss has only minimal impact in mice

BOK/MTD was discovered as a protein that binds to the anti-apoptotic Bcl-2 family member MCL-1 and shares extensive amino-acid sequence similarity to BAX and BAK, which are essential for the effector phase of apoptosis. Therefore, and on the basis of its reported expression pattern, BOK is thought to function in a BAX/BAK-like pro-apoptotic manner in female reproductive tissues. In order to determine the function of BOK, we examined its expression in diverse tissues and investigated the consequences of its loss in Bok(-/-) mice. We confirmed that Bok mRNA is prominently expressed in the ovaries and uterus, but also observed that it is present at readily detectable levels in several other tissues such as the brain and myeloid cells. Bok(-/-) mice were produced at the expected Mendelian ratio, appeared outwardly normal and proved fertile. Histological examination revealed that major organs in Bok(-/-) mice displayed no morphological aberrations. Although several human cancers have somatically acquired copy number loss of the Bok gene and BOK is expressed in B lymphoid cells, we found that its deficiency did not accelerate lymphoma development in Eμ-Myc transgenic mice. Collectively, these results indicate that Bok may have a role that largely overlaps with that of other members of the Bcl-2 family, or may have a function restricted to specific stress stimuli and/or tissues.

Endothelium-specific replacement of the connexin43 coding region by a lacZ reporter gene

The murine gap junction protein connexin43 (Cx43) is expressed in blood vessels, with vastly different contribution by endothelial and smooth muscle cells. We have used the Cre recombinase under control of TIE2 transcriptional elements to inactivate a floxed Cx43 gene specifically in endothelial cells. Cre-mediated deletion led to replacement of the Cx43 coding region by a lacZ reporter gene. This allowed us to monitor the extent of deletion and to visualize the endothelial expression pattern of Cx43. We found widespread endothelial expression of the Cx43 gene during embryonic development, which became restricted largely to capillaries and small vessels in all adult organs examined. Mice lacking Cx43 in endothelium did not exhibit altered blood pressure, in contrast to mice deficient in Cx40. Our results show that lacZ activation after deletion of the target gene allows us to determine the extent of cell type-specific deletion after phenotypical investigation of the same animal.

The zebrafish, brain-specific, aromatase cyp19a2 is neither expressed nor distributed in a sexually dimorphic manner during sexual differentiation

Differential cyp19 aromatase expression during development leads to sexual dimorphisms in the mammalian brain. Whether this is also true for fish is unknown. The aim of the current study has been to follow the expression of the brain-specific aromatase cyp19a2 in the brains of sexually differentiating zebrafish. To assess the role of cyp19a2 in the zebrafish brain during gonadal differentiation, we used quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry to detect differences in the transcript or protein levels and/or expression pattern in juvenile fish, histology to monitor the gonadal status, and double immunofluorescence with neuronal or radial glial markers to characterize aromatase-positive cells. Our data show that cyp19a2 expression levels during zebrafish sexual differentiation cannot be assigned to a particular sex; the expression pattern in the brain is similar in both sexes and aromatase-positive cells appear to be mostly of radial glial nature.

Increased expression of the auxiliary beta2-subunit of ventricular L-type Ca2+ channels leads to single-channel activity characteristic of heart failure

BACKGROUND: Increased activity of single ventricular L-type Ca(2+)-channels (L-VDCC) is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary beta-subunits as a possible explanation. METHODS AND RESULTS: By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC beta-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1) or beta(3) isoforms, beta(2a) and beta(2b) induce a high-activity channel behavior typical of failing myocytes. In accordance, beta(2)-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac Ca(V)1.2 also reveal increased single-channel activity and sarcolemmal beta(2) expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing ("Adaptive Phase"), reveal the opposite phenotype, viz: reduced single-channel activity accompanied by lowered beta(2) expression. Additional evidence for the cause-effect relationship between beta(2)-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive Ca(V)1.2 and inducible beta(2) cardiac overexpression. Here in non-failing hearts induction of beta(2)-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure. CONCLUSIONS: Our study presents evidence of the pathobiochemical relevance of beta(2)-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.

Follicle-forming cat thyroid cell lines synthesizing extracellular matrix and basal membrane components: a new tool for the study of thyroidal morphogenesis

Interactions between follicular epithelial cells and extracellular matrix (ECM) are supposed to play an important role in the development and maintenance of thyroid tissue architecture. In the present study we have therefore investigated the synthesis of ECM components by a feline thyroid cell line which is able to form follicle-like structures in vitro, and also in v-ras-transfected and control-transfected sublines. Transfections were performed by lipofection with pZSR (viral Harvey ras gene; neo) and pSV2-neo (control, neo only) plasmids. We have adapted a semisolid culture system composed exclusively of polymerized alginate and therefore devoid of ECM components. Feline cells embedded in alginate gels as single cells and cultured for up to 90 days formed cell clusters within 10 days. Follicle-like structures were formed in the original cell lines and also in the v-ras- and control-transfected cells. Differences in proliferation rates were observed, the v-ras-transfected cells growing up to two to three times faster than the non-transfected cells. Immunostaining was done using rabbit first antibodies directed against mouse collagen IV, human fibronectin, laminin (tumor Engelbreth-Holm-Swarm laminin), perlecan and other ECM components. For comparison, immunostaining was also performed on cryosections of nodular goiters of six hyperthyroid cats. The cell lines and their transfected clones stained strongly positive for collagen IV and fibronectin, and positively but less strongly for laminin and perlecan. The cat goiter tissue stained positively for collagen IV, laminin, perlecan, and fibronectin, and positive staining for S-laminin (containing the beta2-chain) was seen in blood vessel walls in this tissue. In conclusion, cat cell lines grow three-dimensionally in alginate beads over several weeks, they form follicle-like structures and express the same ECM components as the native cat goiter tissue. Transfection with v-ras does increase proliferation rate, but does not fundamentally alter formation of follicle-like structures and ECM expression. Alginate gel culture is a promising new tool for the study of follicular morphogenesis, polarity, the expression pattern of ECM components and of the interaction between thyrocytes and ECM. It avoids interference caused by gels composed of ECM components.

Induction of Bim and Bid gene expression during accelerated apoptosis in severe sepsis

ABSTRACT: INTRODUCTION: In transgenic animal models of sepsis, members of the Bcl-2-family of proteins regulate lymphocyte apoptosis and survival of sepsis. This study investigates the gene regulation of pro- and anti-apoptotic members of the Bcl-2-family of proteins in patients with early stage severe sepsis. METHODS: In this prospective case-control study patients were recruited from three intensive care units in a university hospital. Sixteen patients were enrolled as soon as they fulfilled the criteria of severe sepsis. Ten critically ill but non-septic patients and eleven healthy volunteers served as controls. Blood samples were immediately obtained at inclusion. To confirm the presence of accelerated apoptosis in the patient groups, caspase-3 activation and phosphatidylserine (PS) externalization in CD4+, CD8+ and CD19+ lymphocyte subsets were assessed by flow cytometry. Specific mRNA's of Bcl-2 family members were quantified from whole blood by real-time polymerase chain reaction. To test for statistical significance, Kruskal-Wallis testing with Dunn's multiple comparison test for post hoc testing was performed. RESULTS: In all lymphocyte populations caspase-3 (p<0.05) was activated, which was reflected in an increased PS externalization (p<0.05). Accordingly, lymphocyte counts were decreased in early severe sepsis. In CD4+ T-cells (p<005) and in B-cells (p<0.001) the Bcl-2 protein was decreased in severe sepsis. Gene expression of the BH3-only Bim was massively upregulated as compared to critically ill patients (p<0.001) and 51.6 fold as compared to healthy controls (p<0.05). Bid was increased 12.9 fold compared to critically ill (p<0.001). In the group of the mitochondrial apoptosis-inducers, Bak was upregulated 5.6 fold, while the expression of Bax showed no significant variations. By contrast, the pro-survival members Bcl-2 and Bcl-xl were both downregulated in severe sepsis (p<0.001, p<0.05). CONCLUSIONS: In early severe sepsis a gene expression pattern with induction of the pro-apoptotic Bcl-2 family members Bim, Bid and Bak and a downregulation of the anti-apoptotic Bcl-2 and Bcl-xl was observed in peripheral blood. This constellation may affect cellular susceptibility to apoptosis and complex immune dysfunction in sepsis.

Differential expression of ABC transporters and their regulatory genes during lactation and dry period in bovine mammary tissue

ATP-binding cassette (ABC) transporters play a pivotal role in human physiology, and mutations in these genes often result in severe hereditary diseases. ABC transporters are expressed in the bovine mammary gland but their physiological role in this organ remains elusive. Based on findings in the context of human disorders we speculated that candidate ABC transporters are implicated in lipid and cholesterol transport in the mammary gland. Therefore we investigated the expression pattern of selected genes that are associated with sterol transport in lactating and nonlactating mammary glands of dairy cows. mRNA levels from mammary gland biopsies taken during lactation and in the first and second week of the dry period were analysed using quantitative PCR. Five ABC transporter genes, namely ABCA1, ABCA7, ABCG1, ABCG2 and ABCG5, their regulating genes LXRalpha, PPARgamma, SREBP1 and the milk proteins lactoferrin and alpha-lactalbumin were assessed. A significantly enhanced expression in the dry period was observed for ABCA1 while a significant decrease of expression in this period was detected for ABCA7, ABCG2, SREBP1 and alpha-lactalbumin. ABCG1, ABCG5, LXRalpha, PPARgamma and lactoferrin expression was not altered between lactation and dry period. These results indicate that candidate ABC transporters involved in lipid and cholesterol transport show differential mRNA expression between lactation and the dry period. This may be due to physiological changes in the mammary gland such as immigration of macrophages or the accumulation of fat due to the loss of liquid in the involuting mammary gland. The current mRNA expression analysis of transporters in the mammary gland is the prerequisite for elucidating novel molecular mechanisms underlying cholesterol and lipid transfer into milk.

Metalloprotease meprin beta in rat kidney: glomerular localization and differential expression in glomerulonephritis

Meprin (EC 3.4.24.18) is an oligomeric metalloendopeptidase found in microvillar membranes of kidney proximal tubular epithelial cells. Here, we present the first report on the expression of meprin beta in rat glomerular epithelial cells and suggest a potential involvement in experimental glomerular disease. We detected meprin beta in glomeruli of immunostained rat kidney sections on the protein level and by quantitative RT-PCR of laser-capture microdissected glomeruli on the mRNA level. Using immuno-gold staining we identified the membrane of podocyte foot processes as the main site of meprin beta expression. The glomerular meprin beta expression pattern was altered in anti-Thy 1.1 and passive Heymann nephritis (PHN). In addition, the meprin beta staining pattern in the latter was reminiscent of immunostaining with the sheep anti-Fx1A antiserum, commonly used in PHN induction. Using Western blot and immunoprecipitation assays we demonstrated that meprin beta is recognized by Fx1A antiserum and may therefore represent an auto-antigen in PHN. In anti-Thy 1.1 glomerulonephritis we observed a striking redistribution of meprin beta in tubular epithelial cells from the apical to the basolateral side and the cytosol. This might point to an involvement of meprin beta in this form of glomerulonephritis.

Adipogenic and myogenic gene expression in rotator cuff muscle of the sheep after tendon tear

Chronic rotator cuff tendon tears lead to fatty infiltration and muscle atrophy with impaired physiological functions of the affected muscles. However, the cellular and molecular mechanisms of corresponding pathophysiological processes remain unknown. The purpose of this study was to characterize the expression pattern of adipogenic (PPARgamma, C/EBPbeta) and myogenic (myostatin, myogenin, Myf-5) transcription factors in infraspinatus muscle of sheep after tenotomy, implantation of a tension device, refixation of the tendon, and rehabilitation, reflecting a model of chronic rotator cuff tears. In contrast to human patients, the presented sheep model allows a temporal evaluation of the expression of a given marker in the same individual over time. Semiquantitative RT/PCR analysis of PPARgammaã, myostatin, myogenin, Myf-5, and C/EBPbeta transcript levels was carried out with sheep muscle biopsy-derived total RNA. We found a significantly increased expression of Myf-5 and PPARgamma after tenotomy and a significant change for Myf-5 and C/EBPbeta after continuous traction and refixation. This experimental sheep model allows the molecular analysis of pathomechanisms of muscular changes after rotator cuff tear. The results point to a crucial role of the transcription factors PPARgamma, C/EBPbeta, and Myf-5 in impairment and regeneration of rotator cuff muscles after tendon tears in sheep.

Geographic analysis of RKIP expression and its clinical relevance in colorectal cancer

BACKGROUND This study evaluates the geographic expression pattern of Raf-1 Kinase Inhibitor Protein (RKIP) in colorectal cancer (CRC) in correlation with clinicopathological and molecular features, markers of epithelial-mesenchymal transition (EMT) and survival outcome. METHODS Whole-tissue sections of 220 well-characterised CRCs were immunostained for RKIP. NF-κB and E-Cadherin expression was assessed using a matched multi-punch tissue microarray. Analysis of mismatch repair (MMR) protein expression, B-Raf and KRAS mutations was performed. RKIP expression in normal mucosa, tumour centre, invasion front and tumour buds was each assessed for clinical relevance. RESULTS RKIP was diffusely expressed in normal mucosa and progressively lost towards tumour centre and front (P<0.0001). Only 0.9% of tumour buds were RKIP-positive. In the tumour centre, RKIP deficiency predicted metastatic disease (P=0.0307), vascular invasion (P=0.0506), tumour budding (P=0.0112) and an invasive border configuration (P=0.0084). Loss of RKIP correlated with NF-κB activation (P=0.0002) and loss of E-Cadherin (P<0.0001). Absence of RKIP was more common in MMR-deficient cancers (P=0.0191), while no impact of KRAS and B-Raf mutation was observed. RKIP in the tumour centre was identified as a strong prognostic indicator (HR (95% CI): 2.13 (1.27-3.56); P=0.0042) independently of TNM classification and therapy (P=0.0474). CONCLUSION The clinical relevance of RKIP expression as an independent prognostic factor is restricted to the tumour centre. Loss of RKIP predicts features of EMT and correlates with frequent distant metastasis.