JunB is required for endothelial cell morphogenesis by regulating core-binding factor β

The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor β (CBFβ), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFβ into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFβ in EC morphogenesis

Оценка воздействия производства эмалированных проводов на атмосферный воздух

В данной работе объектом исследования являются источники загрязнения атмосферы на эмальобмоточном производстве АО "Сибкабель" . Цель работы - оценка воздействия производства эмалированных проводов на атмосферный воздух. Для проведения оценки были изучены нормативно-правовая база проектов нормирования предельно-допустимых выбросов, технология эмалирования проводов, инвентаризационная ведомость источников загрязнения воздуха на производстве. Расчетным путем были получены значения максимально-разовых и валовых выбросов загрязняющих веществ, а также максимальные примесные концентрации вредных веществ в приземном слое атмосферы, которые свидетельствуют о необходимости внесения изменений в имеющийся проект нормативов ПДВ.In this paper, the subject of the study are sources of air pollution at the enamel winding production of JSC "Sibkabel". The aim of the work is to assess the effect of production of enameled wires on atmospheric air.For the assessment, the regulatory and legal framework for the projects for the standardization of maximum permissible emissions, the technology for enameling wires, and the inventories of sources of air pollution in production were studied. Calculations were made for the values ??of maximum-one-time and gross emissions of pollutants, as well as maximum impurity concentrations of harmful substances in the surface layer of the atmosphere, which indicate the need to amend the existing draft MPE standards

Entwicklung und Analyse von Mausmodellen zur Untersuchung von Glyzinrezeptordefekten in vivo

Die Erhaltung des Muskeltonus, der die Grundlage für die aufrechte Körperstellung und die Feinabstimmung von Bewegungsabläufen bildet, erfordert ein Gleichgewicht der inhibitorischen und exzitatorischen Impulse, die in den neuronalen Regelkreisen des Rückenmarks verarbeitet werden. Im Rückenmark und Stammhirn von Wirbeltieren wird die synaptische Inhibition vom Strychnin-sensitiven Glyzinrezeptor (GlyR) vermittelt. Dieser liganden-gesteuerte Ionenkanal ist ein pentamerer Proteinkomplex aus drei a- und zwei ßUntereinheiten, der durch ein peripheres Membranprotein, das Gephyrin, in der neuronalen Membran verankert ist. Für die ligandenbindende a-Untereinheit konnten eine Vielzahl von Varianten isoliert werden, die für die Bildung verschiedener GlyR-Isoformen verantwortlich sind. Mutationen, die die Gene für die GlyR-Untereinheiten betreffen, sind stets mit chronischen Bewegungsstömngen assoziiert. So sind Punktmutationen im Gen für die GlyR al-Untereinheit für die Hyperekplexie (Startle Disease) verantwortlich, eine humane Erbkrankheit, die durch ausgeprägte Schreckreaktionen und episodische Muskelsteifheit charakterisiert ist. Die spontanen Mausmutanten spastic (spa), spasmodic (spd) und oscillator (ot), die vergleichbare Bewegungsstömngen manifestieren, tragen ebenfalls Mutationen in den Genen für die GlyR-Untereinheiten. Bei der Mausmutante spa führt eine Transposoninsertion, die im Gen für die GlyR ß-Untereinheit lokalisiert ist, zu einer Störung der GlyR ßExpression. Bei den Mausmutanten spd und ot wurden, wie bei Hyperekplexiepatienten, Mutationen im Gen für die a 1-Untereinheit identifiziert. Diese Mutation führt bei der spasmodischen Maus zu veränderten Rezeptoreigenschaften und bei oscillator zum völligen Verlust der al-Untereinheit. Die Analogie der murinen und humanen Erbkrankheiten ermöglicht die Verwendung der Mausmutanten bei der Entwicklung von in vivo Tiermodellen, die zur Erforschung der molekularen Grundlagen der Glyzinrezeptorfunktion und zur Untersuchung von GlyR-Defekten des Menschen geeignet sind. Für die Entwicklung solcher Tiermodelle wurde in der vorliegenden Arbeit versucht, die hereditären Bewegungsstörungen der Mausmutanten spa, spd und ot durch therapeutischen Gentransfer zu komplementieren. Hierbei sollten die in den Mausmutanten defekten Rezeptorstmkturgene durch solche fremder Spezies ersetzt werden. Für die genetische Rettung der spastischen Mausmutante wurden transgene Mäuse entwickelt, die die ß-Untereinheit der Ratte in ihrem Nervensystem überexprirnieren. Durch Einbringen der Transgenallele in den genetischen Hintergrund der spastischen Maus konnte deren Menge an funktionellen GlyR ß-Transkripten vergrößert werden. Hierdurch konnte eine Zunahme an funktionellen GlyR-Molekülen erreicht und die Manifestierung ihres mutanten Phänotyps verhindert werden. Dies liefe11e zum einen den formalen Beweis für den Zusammenhang von identifiziertem Gendefekt und mutantem Phänotyp und zeigte, daß GlyR-Untereinheiten über Speziesbarrieren hinweg wirksam sind. Zum anderen wurde deutlich, daß das Erscheinen der adulten GlyR-Isoform (GlyRA) an der Membranoberfläche in vivo direkt von der Verfügbarkeit funktioneller ß-Untereinheiten abhängig ist. Darüber hinaus konnte zum ersten Mal gezeigt werden, daß die normale Funktion des glyzinergen Systems bereits dann gewährleistet ist, wenn nur 25% an funktionsfähigen ß-Transkripten gebildet werden bzw. wenn nur ca. die Hälfte der im Wildtyp vorhandenen GlyRA-Moleküle die neuronale Membranoberfläche erreichen. Zur genetischen Rettung der Mausmutanten spasmodic und oscillator wurden, in analogen Versuchsansätzen, transgene Mauslinien etabliert, die die GlyR al-Untereinheit des Menschen in ihrem Nervensystem überexprimieren. Nach Einbringen der Transgenallele in den genetischen Hintergrund der ot Maus konnte deren Phänotyp partiell komplementiert werden. Eine vollständige Rettung dieser Mausmutante bzw. eine Komplementation des spasmodischen Phänotyps konnte, vermutlich aufgrund zu niedriger Transgenexpressionsrate, nicht erreicht werden. Dennoch zeigte das Ergebnis, daß die humane al-Untereinheit in der Maus Funktion übernehmen kann, eine Grundvoraussetzung für die Entwicklung von Mausmodellen, die zur Untersuchung des Pathomechanismus mutierter GlyR-Untereinheiten des Menschen geeignet sind. Zweites Ziel der vorliegenden Arbeit war die Entwicklung von transgenen Mäusen, die die rekombinante GlyR-Untereinheit "Chl" in ihrem Nervensystem exprimieren, für die in vitro gezeigt wurde, daß sie eine dominant negative Wirkung auf die GlyR-Aktivität entfaltet. Durch den Einsatz dieser Untereinheit sollte die GlyR-Aktivität in vivo gezielt reduziert werden und damit der Pathomechanismus der al-Untereinheit in Hyperekplexiepatienten, die ebenfalls als dominant negative GlyR-Untereinheit wirkt, simuliert werden. Die molekularbiologischen Analysen der etablierten Chl-transgen Linien zeigten, daß die transgene Untereinheit, anders als erwartet, die Expression der ligandenbindende al-Untereinheit beeinflußt. Diese Erkenntnis steht im Gegensatz zu den Ergebnissen aus entsprechenden Experimenten mit in vitro Systemen und macht deutlich, daß in vitro Modelle die in vivo Situation nicht unbedingt repräsentieren müssen. Dies unterstreicht die Bedeutung von Tiermodellen bei der Untersuchung der molekularen Grundlagen der glyzinergen Nervenübertragung und bei der Erforschung von humanen Glyzinrezeptordefekten.The maintenance of the muscle tone, which is the prerequesite for balance, posture and movement, depends on the balance of excitatory and inhibitory impulses, that are processed by the neuronal circuits of the spinal cord. In vertebrate brain stem and spinal cord, the postsynaptic inhibition is mediated by the strychnine-sensitive glycine receptor (GlyR). This ligand gated ion channel is a pentameric protein composed of three copies of the a subunit and two copies of the ß subunit. The receptor protein is further associated with a peripheral membrane protein, named gephyrin, that is thought to be responsible for anchoring and synaptic localization of the GlyR. Several isoforms of the ligand binding a subunit create regional and developmental heterogeneity of the GlyR. Mutations in the GlyR subunit genes are associated with neuromotor diseases in mouse and man. In particular, pointmutations in the al subunit have been identified in patients suffering from dominant and recessive forms of hereditary hyperekplexia (startle disease), a disease characterized by exaggerated startle reaction and generalized hypertonia. Related recessive mouse mutants exist, which suffer from similar symptoms and carry defects in either their GlyR al or ß subunit gene, respectively. The mutations spasmodic (spd) and oscillator (ot) represent a partial or complete loss of function mutation of the al subunit. The mutant spastic (spa) has an intronie insertion of a transposable element in the GlyR ß subunit gene leading to disturbed GlyR ß expression. Because of the analogy of thesemurirre and human diseases, the GlyR mouse mutants comprise unique experimental models of human hyperekplexia. To exploit this potential, it was attempted to rescue the mutant phenotype of the mouse mutants by introducing functional GlyR al and ß subunit transgenes derived from other species. For the complementation of the spa phenotype, transgenic mice were created that show neuronal overexpression of the GlyR ß subunit gene of the rat. By introducing the transgene allele into the genetic background of homozygous spa mice, their amount of functional ß transcripts and thereby GlyR level was increased. First, these experiments proved that the spa mutation is causal for the phenotype of the spastic mouse as expression of the transgene rescued the mutant phenotype. Second, by using a transgene derived from another species, it was shown that in glycinergic neurotransmission species barriers can be overcome. Third, it became clear that in vivo the adult GlyR subtype (GlyRA) is only stable when complexed with the GlyR ß subunit. Furthermore, it was demonstrated that partial restoration of GlyR ß expression, i.e. 25% of full length ß transcripts, and around 50% of functional GlyRAmolecules are sufficient for proper function of glycinergic neurotransrnission. To rescue the spasmodic and oscillator phenotype, first transgenic mouse lines were generated showing neuronal overexpression of the human GlyR al subunit. Introducing the transgene allele into the genetic background of the homozygous ot mouse allowed the partial rescue of its mutant phenotype. A complete rescue of their phenotype and a complementation of the spasmodic phenotype, respectively, has not been achieved, presumably because of low transgene expression level. However, by these experiments it has been demonstrated, that the human al subunit can substitute for the murine protein, a prerequesite for the development of transgenic mouse models expressing glycine receptors containing the human hyperekplexia mutations. These models will be an excellent tool to examine the precise effects of human startle mutations on the synaptic physiology in vivo. Finally, transgenic mice were created that show neuronal overexpression of the recombinant GlyR subunit 'Chl '. This GlyR subunit was shown to exert a dominant negativ effect on the electrophysiological properties of the native GlyR in vitro. By using this subunit as a transgene, it was attempted to reduce GlyR activity in vivo and therefore to model the pathomechanism of the mutated al subunit of hyperekplexia patients, that also affects GlyR function in a dominant negative manner. Surprisingly, the expression of this molecule in the central nervaus system of the Chl transgenic mice impaired their endogenaus GlyR al expression. This finding is in cantrast to the results obtained from corresponding experiments in vitro, pointing out the importance of in vivo systems to study the molecular aspects of glycinergic neurotransmission and the pathomechanism of human neuromotoric disorders, caused by GlyR defects

Transient neuromotor phenotype in transgenic spastic mice expressing low levels of glycine receptor beta-subunit: an animal model of startle disease

Startle disease or hereditary hyperekplexia has been shown to result from mutations in the alpha1-subunit gene of the inhibitory glycine receptor (GlyR). In hyperekplexia patients, neuromotor symptoms generally become apparent at birth, improve with age, and often disappear in adulthood. Loss-of-function mutations of GlyR alpha or beta-subunits in mice show rather severe neuromotor phenotypes. Here, we generated mutant mice with a transient neuromotor deficiency by introducing a GlyR beta transgene into the spastic mouse (spa/spa), a recessive mutant carrying a transposon insertion within the GlyR beta-subunit gene. In spa/spa TG456 mice, one of three strains generated with this construct, which expressed very low levels of GlyR beta transgene-dependent mRNA and protein, the spastic phenotype was found to depend upon the transgene copy number. Notably, mice carrying two copies of the transgene showed an age-dependent sensitivity to tremor induction, which peaked at approximately 3-4 weeks postnatally. This closely resembles the development of symptoms in human hyperekplexia patients, where motor coordination significantly improves after adolescence. The spa/spa TG456 line thus may serve as an animal model of human startle disease

Th2 cell-specific cytokine expression and allergen-induced airway inflammation depend on JunB

Naïve CD4+ T cells differentiate into effector T helper 1 (Th1) or Th2 cells, which are classified by their specific set of cytokines. Here we demonstrate that loss of JunB in in vitro polarized Th2 cells led to a dysregulated expression of the Th2-specific cytokines IL-4 and IL-5. These cells produce IFN-γ and express T-bet, the key regulator of Th1 cells. In line with the essential role of Th2 cells in the pathogenesis of allergic asthma, mice with JunB-deficient CD4+ T cells exhibited an impaired allergen-induced airway inflammation. This study demonstrates novel functions of JunB in the development of Th2 effector cells, for a normal Th2 cytokine expression pattern and for a complete Th2-dependent immune response in mice

Collagenase-3 (MMP-13) deficiency protects C57BL/6 mice from antibody-induced arthritis

Introduction: Matrix metalloproteinases (MMPs) are important in tissue remodelling. Here we investigate the role of collagenase-3 (MMP-13) in antibody-induced arthritis. Methods: For this study we employed the K/BxN serum-induced arthritis model. Arthritis was induced in C57BL/6 wild type (WT) and MMP-13-deficient (MMP-13–/–) mice by intraperitoneal injection of 200 μl of K/BxN serum. Arthritis was assessed by measuring the ankle swelling. During the course of the experiments, mice were sacrificed every second day for histological examination of the ankle joints. Ankle sections were evaluated histologically for infiltration of inflammatory cells, pannus tissue formation and bone/cartilage destruction. Semi-quantitative PCR was used to determine MMP-13 expression levels in ankle joints of untreated and K/BxN serum-injected mice. Results: This study shows that MMP-13 is a regulator of inflammation. We observed increased expression of MMP-13 in ankle joints of WT mice during K/BxN serum-induced arthritis and both K/BxN serum-treated WT and MMP-13–/– mice developed progressive arthritis with a similar onset. However, MMP-13–/– mice showed significantly reduced disease over the whole arthritic period. Ankle joints of WT mice showed severe joint destruction with extensive inflammation and erosion of cartilage and bone. In contrast, MMP-13–/– mice displayed significantly decreased severity of arthritis (50% to 60%) as analyzed by clinical and histological scoring methods. Conclusions: MMP-13 deficiency acts to suppress the local inflammatory responses. Therefore, MMP-13 has a role in the pathogenesis of arthritis, suggesting MMP-13 is a potential therapeutic target

Altered endochondral bone development in matrix metalloproteinase 13-deficient mice

The assembly and degradation of extracellular matrix (ECM) molecules are crucial processes during bone development. In this study, we show that ECM remodeling is a critical rate-limiting step in endochondral bone formation. Matrix metalloproteinase (MMP) 13 (collagenase 3) is poised to play a crucial role in bone formation and remodeling because of its expression both in terminal hypertrophic chondrocytes in the growth plate and in osteoblasts. Moreover, a mutation in the human MMP13 gene causes the Missouri variant of spondyloepimetaphyseal dysplasia. Inactivation of Mmp13 in mice through homologous recombination led to abnormal skeletal growth plate development. Chondrocytes differentiated normally but their exit from the growth plate was delayed. The severity of the Mmp13-null growth plate phenotype increased until about 5 weeks and completely resolved by 12 weeks of age. Mmp13-null mice had increased trabecular bone, which persisted for months. Conditional inactivation of Mmp13 in chondrocytes and osteoblasts showed that increases in trabecular bone occur independently of the improper cartilage ECM degradation caused by Mmp13 deficiency in late hypertrophic chondrocytes. Our studies identified the two major components of the cartilage ECM, collagen type II and aggrecan, as in vivo substrates for MMP13. We found that degradation of cartilage collagen and aggrecan is a coordinated process in which MMP13 works synergistically with MMP9. Mice lacking both MMP13 and MMP9 had severely impaired endochondral bone, characterized by diminished ECM remodeling, prolonged chondrocyte survival, delayed vascular recruitment and defective trabecular bone formation (resulting in drastically shortened bones). These data support the hypothesis that proper ECM remodeling is the dominant rate-limiting process for programmed cell death, angiogenesis and osteoblast recruitment during normal skeletal morphogenesis