Induktion von Pro-Autophagie-Signalen durch einen extra- oder intrazellulären Alpha-Toxin-Angriff

Autophagie ist ein konservierter, kataboler Mechanismus in allen eukaryoten Zellen. Unter anderem wird ihm eine wichtige Rolle als zellautonomer Abwehrmechanismus gegen Mikroorganismen zugeschrieben; von manchen Infektionserregern wird er jedoch unterlaufen oder sogar genutzt. Der stärkste Auslöser der Autophagie ist ein Mangel an Nährstoffen, insbesondere Aminosäuren. Über die Deaktivierung der Kinase mTORC1 und die Phosphorylierung des eukaryoten Translationsinitiationsfaktors eIF2α hemmt die Nährstoffknappheit die Proteinbiosynthese und aktiviert gleichzeitig Autophagie. Wie Mikroorganismen, insbesondere Bakterien, Autophagie auslösen oder manipulieren, ist derzeit Gegenstand intensiver Forschung. Modifikationen an Mikroben oder Phagosomen und Adapterproteine, die diese Veränderungen und Komponenten des Autophagieapparates erkennen, scheinen jedenfalls bei der selektiven Erkennung durch die Autophagie-Maschinerie wichtig zu sein. rnIn der vorliegenden Dissertationsarbeit wird die Rolle des membranporenbildenden α-Toxins von Staphylococcus aureus für die Induktion von Autophagie beleuchtet. Zum einen erwies sich die Akkumulation von (EGFP)-LC3(II), einem Marker der Autophagosomen, um intrazelluläre S. aureus als abhängig von α-Toxin. Zweitens, genügt extrazellulär appliziertes α-Toxin um (EGFP)-LC3(II)-positive Endosomen zu induzieren. Während der Angriff aus dem extrazellulären Raum jedoch binnen kurzer Zeit eine fokale Kumulation von phosphoryliertem eIF2α an der Plasmamembran induziert, die an der Internalisierung des Toxins beteiligt ist, findet sich am phagosomalen Kompartiment keine Toxin-abhängige Anhäufung von p-eIF2α oder proximalen Autophagieregulatoren. Dies impliziert, dass Toxin-Angriff auf die Plasmamembran, nicht aber auf das Phagosom, zu einer Reaktion führt, wie sie bei massivem Nährstoffmangel zu beobachten ist. Obwohl keine α-Toxin-abhängige Kumulation von p-eIF2α bei einem Angriff aus dem Phagosom erfolgt, findet sich um α-Toxin-produzierende Bakterien eine massive Kumulation von LC3 und Adapterprotein p62/Sequestosome1. Dies deutet daraufhin, dass der Ort des Angriffs - Plasmamembran oder Phagosom – für den Autophagie-induzierenden Mechanismus wichtig sein könnte. Der unterschiedliche Effekt auf die zellulären Ionenkonzentrationen, den ein Angriff auf die Plasmamembran oder auf ein Phagosom auslösen würde, bietet hierfür eine mögliche Erklärung. Die Aktivierung der Autophagie über Adapterproteine könnte dann als back-up Mechanismus fungieren, der auch dann greift, wenn eine Invasion ohne Schädigung der Plasmamembran erfolgt. Ein cross-talk der beiden Induktionswege ist angesichts der Bedeutung von p62 für die selektive und die Hunger-assoziierte Autophagie gut möglich; sezerniertes Toxin könnte durch die Aktivierung der basalen Autophagie Adapter-basierte Mechanismen verstärken.

Regulation des Proteins Alpha-Synuclein während der zellulären Alterung

α-Synuclein wird durch Mutationen sowie der Ausbildung von Proteinaggregaten namens Lewy-Körperchen mit der Entstehung der altersassoziierten Parkinson-Krankheit in Verbindung gebracht. Sowohl familiäre als auch sporadische Fälle sind durch erhöhte α-Synuclein-Spiegel gekennzeichnet. In familiären Fällen wurden Multiplikationen des α-Synuclein-Gens als Ursache für die erhöhte Expression aufgedeckt. In sporadischen Fällen stellt die Alterung den entscheidenden Risikofaktor für die Entstehung der Krankheit dar. Daher wurde in der vorliegenden Arbeit die Regulation von α-Synuclein während der zellulären Alterung in humanen Fibroblasten untersucht. In seneszenten Zellen konnte ein Anstieg der α-Synuclein-Expression nachgewiesen werden, der jedoch die Löslichkeit des Proteins nicht veränderte. Damit scheint die zelluläre Alterung per se nicht für die Aggregation des Proteins, wie sie in Form von Lewy-Körperchen bei z. B. Patienten der Parkinson-Krankheit beobachtet wird, verantwortlich zu sein. Möglicherweise ist die Hochregulation von α-Synuclein eine Folge der Akkumulation von DNA-Schäden in den seneszenten Zellen. Diese Korrelation konnte in jungen Zellen nach dem Einsatz verschiedener DNA-schädigender Agenzien bestätigt werden. Die Untersuchung des Regulationsmechanismus ergab, dass die erhöhte Expression von α-Synuclein in Folge von DNA-Schäden über den ERK1/2-MAPK-Signalweg vermittelt wird. In seneszenten Zellen konnte ebenfalls ein Einfluss dieses Signalweges auf die Expression von α-Synuclein beobachtet werden, allerdings scheint dieser nicht alleinig für die Hochregulation verantwortlich zu sein. Des Weiteren ergab die Betrachtung des γH2A.X-Spiegels nach Induktion von DNA-Schäden, dass α-Synuclein möglicherweise eine protektive Funktion besitzt, da dessen Überexpression zu einer verringerten und die Herunterregulation zu einer vermehrten DNA-Schädigung führte. Die Analyse der subzellulären Lokalisation von α-Synuclein ergab außerdem, dass es in jungen Zellen nach der Induktion von DNA-Schäden zu einer Translokation des Proteins in den Zellkern kommt. Diese Translokation war in seneszenten Zellen verringert. Dies lässt vermuten, dass α-Synuclein in jungen Zellen nach DNA-Schädigung durch den ERK1/2-MAPK-Signalweg hochreguliert wird und durch die Translokation in den Zellkern möglicherweise die Transkription von protektiven Genen beeinflusst oder an DNA-Reparatur-Prozessen beteiligt ist. In seneszenten Zellen ist das Protein zwar deutlich stärker exprimiert, der Transport in den Zellkern jedoch verringert, wodurch die protektive Wirkung im Zellkern herabgesetzt wäre.

Preparation and functional characterisation of recombinant silicatein-alpha from the sponge Suberites domuncula

The marine world is an immense source of biodiversity that provides substances with striking potentials in medicinal chemistry and biotechnology. Sponges (Porifera) are marine animals that represent the most impressive example of organisms possessing the ability to metabolise silica through a family of enzymes known as silicateins. Complex skeletal structures (spicules) made of pure biogenic silica (biosilica) are produced under physiological conditions. Biosilica is a natural material comprising inorganic and organic components with unique mechanical, optical, and physico-chemical properties, including promising potential to be used for development of therapeutic agents in regenerative medicine. Unravelling the intimate physiological mechanisms occurring in sponges during the construction of their siliceous spicules is an on-going project, and several questions have been addressed by the studies proposed by our working group. In this doctoral work, the recombinant DNA technology is exploited for functional and structural characterisation of silicatein. Its precursors are produced as fusion proteins with a chaperone tag (named TF-Ps), and a robust method for the overexpression of native soluble proteins in high concentrations has been developed. In addition, it is observed and proven experimentally that the maturation of silicatein is an autocatalytic event that: (i) can be modulated by rational use of protease inhibitors; (ii) is influenced by the temperature of the environment; (iii) only slightly depends on the pH. In the same experimental framework, observations on the dynamics in the maturation of silicateins allow a better understanding of how the axial filaments form during the early stages of spicule construction. In addition, the definition of new distinct properties of silicatein (termed “structure-guiding” and “structure-forming”) is introduced. By homology models and through comparisons with similar proteins (the cathepsins), domains with significant surface hydrophobicity are identified as potential self-assembly mediators. Moreover, a high-throughput screening showed that TF-Ps could generate crystals under certain conditions, becoming promising for further structural studies. With the goal of optimise the properties of the recombinant silicatein, implementation of new production systems are tried for the first time. Success in the expression of silicatein-type proteins in insect and yeast cells, constitute a promising basis for further development, towards the establishment of an efficient method for the production of a high-value pure and soluble protein.

Erkennung der alpha-Kette des GM-CSF-Rezeptors (CSF2RA) durch einen HLA-unabhängigen alpha:beta-T-Zellrezeptor

Aus dem tumorreaktiven T-Zellrepertoire der Melanompatientin Ma-Mel-86/INTH, bei der im Verlauf Lymphknotenmetastasen HLA-Klasse I-negativer Tumorzellen auftraten, wurden durch Stimulation mit autologen Tumorzellen CD8+ T-Zellklone isoliert und expandiert, die auf Melanomzellen der Patientin CSF2RA (engl. GM-CSF receptor alpha chain) in HLA-unabhängiger Weise erkannten. Aus einem der T-Zellklone wurde ein CSF2RA-reaktiver α:β-T-Zellrezeptor (TCR, engl. T-cell receptor) kloniert (Bezeichnung: TCR-1A.3/46). Die α-Kette des TCR enthielt die Domänen TRAV14/DV4*01, TRAJ48*01 und TRAC*01, die β-Kette die Domänen TRBV10-3*01, TRBD2*01, TRBJ2-7*01 und TRBC2*01. Durch Austausch der humanen konstanten gegen die homologen murinen Domänen wurde der TCR optimiert (Bezeichnung: cTCR-1A.3/46) und hinsichtlich seiner Expression und Funktionalität nach retroviralem Transfer in humane PBMC (engl. peripheral blood mononuclear cells) im 51Chromfreisetzungstest, im IFN-γ-ELISpot-Assay und in einem Degranulations-Assay validiert. TCR-transgene T-Zellen lysierten nicht nur spezifisch die HLA-defizienten, CSF2RA+ Melanomlinien des Modells Ma-Mel-86, sondern erkannten auch Zelllinien verschiedener Spezies nach Transfektion von CSF2RA sowie Monozyten, Granulozyten, dendritische Zellen und ein breites Spektrum hämatologischer Malignome myeloiden Ursprungs ungeachtet deren HLA-Phänotypen. Lymphatische Zellen sowie CD34+ Blutstammzellen wurden in In vitro-Untersuchungen nicht erkannt. Der Zusatz von GM-CSF zu Zellen, die CSF2RA und CSF2RB exprimierten, inhibierte die Erkennung durch TCR-transgene PBMC, während die Koexpression der α- und der ß-Kette des GM-CSF-Rezeptors alleine keinen negativen Effekt auf die Erkennung hatte. Daraus war zu schließen, dass CSF2RA präferentiell freistehend und weniger nach Integration in den heteromultimerischen GM-CSF-Rezeptor-Komplex erkannt wurde. In der zweidimensionalen Collier-de-Perles-Visualisierung der IMGT-Datenbank (engl. International immunogenetics information system) wies der CSF2RA-reaktive TCR-1A.3/46 im Vergleich zu TCR von konventionellen, HLA-restringierten T-Zellen keine Besonderheiten auf. Darüber hinaus waren auch die von den HLA-unabhängigen T-Zellen exprimierten CD8-Moleküle identisch zu den CD8-Molekülen HLA-abhängiger CTL (engl. cytotoxic T lymphocytes). Die Präsenz von CD8-Molekülen förderte die HLA-unabhängige Erkennung von CSF2RA, schien aber dafür nicht zwingend erforderlich zu sein, da Antikörper gegen CD8 die Erkennung zu ca. 65 % blockierten und TCR-transgene CD4+ T-Zellen im Vergleich zu TCR-transduzierten CD8+ T-Zellen eine deutlich verringerte, aber noch erhaltene Funktionalität aufwiesen. Es ist derzeit nicht klar, ob HLA-unabhängige T-Zellen gegen CSF2RA im peripheren Blut der Patientin vorkamen, weil sie der im Tiermodell postulierten Thymusselektion MHC-unabhängiger TCR (Tikhonova et al., Immunity 36:79, 2012) entkommen waren, oder weil ein ursprünglich gegen einen HLA-Peptid-Komplex gerichteter TCR eine HLA-unabhängige Kreuzreaktivität aufwies. CSF2RA verbessert die Glucoseutilisation in malignen Zellen, und es wurden ihm embryotrophe Eigenschaften zugeschrieben (Spielholz et al., Blood 85:973, 1995; Sjöblom et al., Biol. Reprod. 67:1817, 2002). Damit kann CSF2RA malignes Wachstum fördern und ist somit ein potentielles Zielmolekül für die Immuntherapie. Seine HLA-unabhängige Erkennung würde sowohl die HLA-Vielfalt als auch den HLA-Verlust als typische Limitationen der T-Zellimmuntherapie umgehen. Zur Überprüfung der In vivo-Spezifität des HLA-unabhängigen TCR gegen CSF2RA und damit zum Ausschluss relevanter off-tumor-/on-target- bzw. off-tumor-/off-target-Effekte ist jedoch eine Testung in einem präklinischen Tiermodell erforderlich.

Amicrobial pustulosis-like rash in a patient with Crohn's disease under anti-TNF-alpha blocker

Amicrobial pustulosis of the folds (APF) is a recently described entity characterized by relapsing pustular lesions predominantly involving the cutaneous flexures and scalp. This disease typically occurs in association with systemic lupus erythematosus and a variety of other autoimmune diseases. We here describe an APF-like pustular eruption predominantly affecting the scalp, face and trunk, occurring during long-term infliximab treatment for Crohn's disease. Immunohistochemical staining of skin biopsy specimens for myxovirus resistance protein A, a marker for type 1 interferon-inducible proteins, showed increased staining in the epidermis and dermal mononuclear inflammatory infiltrate. Our observation further extends the spectrum of cutaneous adverse reactions potentially related to anti-tumor necrosis factor-α, the clinical context in which APF can occur as well as its clinical presentations.

Role of AMP-activated protein kinase on steroid hormone biosynthesis in adrenal NCI-H295R cells

Regulation of human androgen biosynthesis is poorly understood. However, detailed knowledge is needed to eventually solve disorders with androgen dysbalance. We showed that starvation growth conditions shift steroidogenesis of human adrenal NCI-H295R cells towards androgen production attributable to decreased HSD3B2 expression and activity and increased CYP17A1 phosphorylation and 17,20-lyase activity. Generally, starvation induces stress and energy deprivation that need to be counteracted to maintain proper cell functions. AMP-activated protein kinase (AMPK) is a master energy sensor that regulates cellular energy balance. AMPK regulates steroidogenesis in the gonad. Therefore, we investigated whether AMPK is also a regulator of adrenal steroidogenesis. We hypothesized that starvation uses AMPK signaling to enhance androgen production in NCI-H295R cells. We found that AMPK subunits are expressed in NCI-H295 cells, normal adrenal tissue and human as well as pig ovary cells. Starvation growth conditions decreased phosphorylation, but not activity of AMPK in NCI-H295 cells. In contrast, the AMPK activator 5-aminoimidazole-4-carboxamide (AICAR) increased AMPKα phosphorylation and increased CYP17A1-17,20 lyase activity. Compound C (an AMPK inhibitor), directly inhibited CYP17A1 activities and can therefore not be used for AMPK signaling studies in steroidogenesis. HSD3B2 activity was neither altered by AICAR nor compound C. Starvation did not affect mitochondrial respiratory chain function in NCI-H295R cells suggesting that there is no indirect energy effect on AMPK through this avenue. In summary, starvation-mediated increase of androgen production in NCI-H295 cells does not seem to be mediated by AMPK signaling. But AMPK activation can enhance androgen production through a specific increase in CYP17A1-17,20 lyase activity.

A closer look at the high affinity benzodiazepine binding site on GABAA receptors

Ligands of the benzodiazepine binding site of the GABA(A) receptor come in three flavors: positive allosteric modulators, negative allosteric modulators and antagonists all of which can bind with high affinity. The GABA(A) receptor is a pentameric protein which forms a chloride selective ion channel and ligands of the benzodiazepine binding site stabilize three different conformations of this protein. Classical benzodiazepines exert a positive allosteric effect by increasing the apparent affinity of channel opening by the agonist γ-aminobutyric acid (GABA). We concentrate here on the major adult isoform, the α(1)β(2)γ(2) GABA(A) receptor. The classical binding pocket for benzodiazepines is located in a subunit cleft between α(1) and γ(2) subunits in a position homologous to the agonist binding site for GABA that is located between β(2) and α(1) subunits. We review here approaches to this picture. In particular, point mutations were performed in combination with subsequent analysis of the expressed mutant proteins using either electrophysiological techniques or radioactive ligand binding assays. The predictive power of these methods is assessed by comparing the results with the predictions that can be made on the basis of the recently published crystal structure of the acetylcholine binding protein that shows homology to the N-terminal, extracellular domain of the GABA(A) receptor. In addition, we review an approach to the question of how the benzodiazepine ligands are positioned in their binding pocket. We also discuss a newly postulated modulatory site for benzodiazepines at the α(1)/β(2) subunit interface, homologous to the classical benzodiazepine binding pocket.

Two domains of the V protein of virulent canine distemper virus selectively inhibit STAT1 and STAT2 nuclear import

Canine distemper virus (CDV) causes in dogs a severe systemic infection, with a high frequency of demyelinating encephalitis. Among the six genes transcribed by CDV, the P gene encodes the polymerase cofactor protein (P) as well as two additional nonstructural proteins, C and V; of these V was shown to act as a virulence factor. We investigated the molecular mechanisms by which the P gene products of the neurovirulent CDV A75/17 strain disrupt type I interferon (IFN-alpha/beta)-induced signaling that results in the establishment of the antiviral state. Using recombinant knockout A75/17 viruses, the V protein was identified as the main antagonist of IFN-alpha/beta-mediated signaling. Importantly, immunofluorescence analysis illustrated that the inhibition of IFN-alpha/beta-mediated signaling correlated with impaired STAT1/STAT2 nuclear import, whereas the phosphorylation state of these proteins was not affected. Coimmunoprecipitation assays identified the N-terminal region of V (VNT) responsible for STAT1 targeting, which correlated with its ability to inhibit the activity of the IFN-alpha/beta-mediated antiviral state. Conversely, while the C-terminal domain of V (VCT) could not function autonomously, when fused to VNT it optimally interacted with STAT2 and subsequently efficiently suppressed the IFN-alpha/beta-mediated signaling pathway. The latter result was further supported by a single mutation at position 110 within the VNT domain of CDV V protein, resulting in a mutant that lost STAT1 binding while retaining a partial STAT2 association. Taken together, our results identified the CDV VNT and VCT as two essential modules that complement each other to interfere with the antiviral state induced by IFN-alpha/beta-mediated signaling. Hence, our experiments reveal a novel mechanism of IFN-alpha/beta evasion among the morbilliviruses.

Engineering Tocopherol Selectivity in alpha-TTP: A Combined In Vitro/In Silico Study

"We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of a-tocopherol transfer" "protein (a-TTP) towards a-tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for a-tocopherol to a-TTP 8.26+2.13 kcal mol{1 lower than that of c-tocopherol. Our calculations show that c-tocopherol binds to a-TTP in a significantly distorted geometry as compared to that of the natural ligand. Variations in the hydration of the binding pocket and in the protein structure are found as well. We propose a mutation, A156L, which significantly modifies the selectivity properties of a-TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to c-tocopherol, with striking structural similarities to the wild-type- a-tocopherol complex. The affinity properties are confirmed by differential scanning fluorimetry as well as in vitro competitive binding assays. Our data indicate that residue A156 is at a critical position for determination of the selectivity of a-TTP. The engineering of TTP mutants with modulating binding properties can have potential impact at industrial level for easier purification of single tocopherols from vitamin E mixtures coming from natural oils or synthetic processes. Moreover," "the identification of a c-tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for a-tocopherol selection in omnivorous animals."

Structural Analysis of alpha-Fetoprotein (AFP)-like Peptides with Anti-Breast-Cancer Properties

The abundance of alpha-fetoprotein (AFP), a natural protein produced by the fetal yolk sac during pregnancy, correlates with lower incidence of estrogen receptor positive (ER+) breast cancer. The pharmacophore region of AFP has been narrowed down to a four amino acid (AA) region in the third domain of the 591 AA peptide. Our computational study focuses on a 4-mer segment consisting of the amino acids threonine-proline-valine-asparagine (TPVN). We have run replica exchange molecular dynamics (REMD) simulations and used 120 configurational snapshots from the total trajectory as starting configurations for quantum chemical calculations. We optimized structures using semiempirical (PM3, PM6, PM6-D2, PM6-H2, PM6-DH+, PM6-DH2) and density functional methods (TPSS, PBE0, M06-2X). By comparing the accuracy of these methods against RI-MP2 benchmarks, we devised a protocol for calculating the lowest energy conformers of these peptides accurately and efficiently. This protocol screens out high-energy conformers using lower levels of theory and outlines a general method for predicting small peptide structures.

Characterization of the bovine IkappaB kinases (IKK)alpha and IKKbeta, the regulatory subunit NEMO and their substrate IkappaBalpha

The Nuclear factor (NF)-kappaB signalling pathway plays a critical role in the regulation and coordination of a wide range of cellular events such as cell growth, apoptosis and cell differentiation. Activation of the IKK (inhibitor of NF-kappaB kinase) complex is a crucial step and a point of convergence of all known NF-kappaB signalling pathways. To analyse bovine IKKalpha (IKK1), IKKbeta (IKK2) and IKKgamma (or NF-kappaB Essential MOdulator, NEMO) and their substrate IkappaBalpha (Inhibitor of NF-kappaB), the corresponding cDNAs of these molecules were isolated, sequenced and characterized. A comparison of the amino acid sequences with those of their orthologues in other species showed a very high degree of identity, suggesting that the IKK complex and its substrate IkappaBalpha are evolutionarily highly conserved components of the NF-kappaB pathway. Bovine IKKalpha and IKKbeta are related protein kinases showing 50% identity which is especially prominent in the kinase and leucine zipper domains. Co-immunoprecipitation assays and GST-pull-down experiments were carried out to determine the composition of bovine IKK complexes compared to that in human Jurkat T cells. Using these approaches, the presence of bovine IKK complexes harbouring IKKalpha, IKKbeta, NEMO and the interaction of IKK with its substrate IkappaBalpha could be demonstrated. Parallel experiments using human Jurkat T cells confirmed the high degree of conservation also at the level of protein-protein interactions. Finally, a yeast two-hybrid analysis showed that bovine NEMO molecules, in addition to the binding to IKKalpha and IKKbeta, also strongly interact with each other.

"Self" and "nonself" manipulation of interferon defense during persistent infection: bovine viral diarrhea virus resists alpha/beta interferon without blocking antiviral activity against unrelated viruses replicating in its host cells

Bovine viral diarrhea virus (BVDV), together with Classical swine fever virus (CSFV) and Border disease virus (BDV) of sheep, belongs to the genus Pestivirus of the Flaviviridae. BVDV is either cytopathic (cp) or noncytopathic (ncp), as defined by its effect on cultured cells. Infection of pregnant animals with the ncp biotype may lead to the birth of persistently infected calves that are immunotolerant to the infecting viral strain. In addition to evading the adaptive immune system, BVDV evades key mechanisms of innate immunity. Previously, we showed that ncp BVDV inhibits the induction of apoptosis and alpha/beta interferon (IFN-alpha/beta) synthesis by double-stranded RNA (dsRNA). Here, we report that (i) both ncp and cp BVDV block the induction by dsRNA of the Mx protein (which can also be induced in the absence of IFN signaling); (ii) neither biotype blocks the activity of IFN; and (iii) once infection is established, BVDV is largely resistant to the activity of IFN-alpha/beta but (iv) does not interfere with the establishment of an antiviral state induced by IFN-alpha/beta against unrelated viruses. The results of our study suggest that, in persistent infection, BVDV is able to evade a central element of innate immunity directed against itself without generally compromising its activity against unrelated viruses ("nonself") that may replicate in cells infected with ncp BVDV. This highly selective "self" and "nonself" model of evasion of the interferon defense system may be a key element in the success of persistent infection in addition to immunotolerance initiated by the early time point of fetal infection.

Molecular characterization of the equine collagen, type IX, alpha 2 (COL9A2) gene on horse chromosome 2p16-->p15

The mammalian collagen, type IX, alpha 2 gene (COL9A2) encodes the alpha-2 chain of type IX collagen and is located on horse chromosome 2p16-->p14 harbouring a quantitative trait locus for osteochondrosis. We isolated a bacterial artificial chromosome (BAC) clone containing the equine COL9A2 gene and determined the complete genomic sequence of this gene. Cloning and characterization of equine COL9A2 revealed that the equine gene consists of 32 exons spanning approximately 15 kb. The COL9A2 transcript encodes a single protein of 688 amino acids. Thirty two single nucleotide polymorphisms (SNPs) equally distributed in the gene were detected in a mutation scan of eight unrelated Hanoverian warmblood stallions, including one SNP that affects the amino acid sequence of COL9A2. Comparative analyses between horse, human, mouse and rat indicate that the chromosomal location of equine COL9A2 is in agreement with known chromosomal synteny relationships. The comparison of the gene structure and transcript revealed a high degree of conservation towards the other mammalian COL9A2 genes. We chose three informative SNPs for association and linkage disequilibrium tests in three to five paternal half-sib families of Hanoverian warmblood horses consisting of 44 to 75 genotyped animals. The test statistics did not reach the significance threshold of 5% and so we could not show an association of COL9A2 with equine osteochondrosis.

Histamine increases sphingosine kinase-1 expression and activity in the human arterial endothelial cell line EA.hy 926 by a PKC-alpha-dependent mechanism

Sphingosine 1-phosphate (S1P) is a potent mitogenic signal generated from sphingosine by the action of sphingosine kinases (SKs). In this study, we show that in the human arterial endothelial cell line EA.hy 926 histamine induces a time-dependent upregulation of the SK-1 mRNA and protein expression which is followed by increased SK-1 activity. A similar upregulation of SK-1 is also observed with the direct protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, SK-2 activity is not affected by neither histamine nor TPA. The increased SK-1 protein expression is due to stimulated de novo synthesis since cycloheximide inhibited the delayed SK-1 protein upregulation. Moreover, the increased SK-1 mRNA expression results from an increased promoter activation by histamine and TPA. In mechanistic terms, the transcriptional upregulation of SK-1 is dependent on PKC and the extracellular signal-regulated protein kinase (ERK) cascade since staurosporine and the MEK inhibitor U0126 abolish the TPA-induced SK-1 induction. Furthermore, the histamine effect is abolished by the H1-receptor antagonist diphenhydramine, but not by the H2-receptor antagonist cimetidine. Parallel to the induction of SK-1, histamine and TPA stimulate an increased migration of endothelial cells, which is prevented by depletion of the SK-1 by small interfering RNA (siRNA). To appoint this specific cell response to a specific PKC isoenzyme, siRNA of PKC-alpha, -delta, and -epsilon were used to selectively downregulate the respective isoforms. Interestingly, only depletion of PKC-alpha leads to a complete loss of TPA- and histamine-triggered SK-1 induction and cell migration. In summary, these data show that PKC-alpha activation in endothelial cells by histamine-activated H1-receptors, or by direct PKC activators leads to a sustained upregulation of the SK-1 protein expression and activity which, in turn, is critically involved in the mechanism of endothelial cell migration.

Apoptotic neutrophils release macrophage migration inhibitory factor upon stimulation with tumor necrosis factor-alpha

Macrophage migration inhibitory factor (MIF) is an important cytokine involved in the regulation of innate immunity and present at increased levels during inflammatory responses. Here we demonstrate that mature blood and tissue neutrophils constitutively express MIF as a cytosolic protein not associated with azurophil granules. Functionally active MIF, but not proteases stored in azurophil granules, was released from apoptotic neutrophils following short term tumor necrosis factor (TNF)-alpha stimulation in a caspase-dependent manner and prior to any detectable phagocytosis by monocyte-derived macrophages. Moreover, TNF-alpha-mediated MIF release was blocked by glyburide and propenicide, both inhibitors of ATP-binding cassette-type transporters, suggesting that this transporter system is activated during neutrophil apoptosis. Taken together, apoptotic mature neutrophils release MIF upon short term TNF-alpha stimulation. Therefore, apoptosis may not always occur without the induction of pro-inflammatory mechanisms.