Activation of HIF1α ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex

Mutations in the VHL tumor suppressor gene result in constitutive expression of many hypoxia-inducible genes, at least in part because of increases in the cellular level of hypoxia-inducible transcription factor HIF1α, which in normal cells is rapidly ubiquitinated and degraded by the proteasome under normoxic conditions. The recent observation that the VHL protein is a subunit of an Skp1-Cul1/Cdc53-F-box (SCF)-like E3 ubiquitin ligase raised the possibility that VHL may be directly responsible for regulating cellular levels of HIF1α by targeting it for ubiquitination and proteolysis. In this report, we test this hypothesis directly. We report development of methods for production of the purified recombinant VHL complex and present direct biochemical evidence that it can function with an E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzyme to activate HIF1α ubiquitination in vitro. Our findings provide new insight into the function of the VHL tumor suppressor protein, and they provide a foundation for future investigations of the mechanisms underlying VHL regulation of oxygen-dependent gene expression.

Vascular tumors in livers with targeted inactivation of the von Hippel–Lindau tumor suppressor

von Hippel–Lindau (VHL) disease is a pleomorphic familial tumor syndrome that is characterized by the development of highly vascularized tumors. Homozygous disruption of the VHL gene in mice results in embryonic lethality. To investigate VHL function in the adult we have generated a conditional VHL null allele (2-lox allele) and null allele (1-lox allele) by Cre-mediated recombination in embryonic stem cells. We show here that mice heterozygous for the 1-lox allele develop cavernous hemangiomas of the liver, a rare manifestation of the human disease. Histologically these tumors were associated with hepatocellular steatosis and focal proliferations of small vessels. To study the cellular origin of these lesions we inactivated VHL tissue-specifically in hepatocytes. Deletion of VHL in the liver resulted in severe steatosis, many blood-filled vascular cavities, and foci of increased vascularization within the hepatic parenchyma. These histopathological changes were similar to those seen in livers from mice heterozygous for the 1-lox allele. Hypoxia-inducible mRNAs encoding vascular endothelial growth factor, glucose transporter 1, and erythropoietin were up-regulated. We thus provide evidence that targeted inactivation of mouse VHL can model clinical features of the human disease and underline the importance of the VHL gene product in the regulation of hypoxia-responsive genes in vivo.

DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos SRE and related sequences

Transcription factors control eukaryotic polymerase II function by influencing the recruitment of multiprotein complexes to promoters and their subsequent integrated function. The complexity of the functional ‘transcriptosome’ has necessitated biochemical fractionation and subsequent protein sequencing on a grand scale to identify individual components. As a consequence, much is now known of the basal transcription complex. In contrast, less is known about the complexes formed at distal promoter elements. The c-fos SRE, for example, is known to bind Serum Response Factor (SRF) and ternary complex factors such as Elk-1. Their interaction with other factors at the SRE is implied but, to date, none have been identified. Here we describe the use of mass-spectrometric sequencing to identify six proteins, SRF, Elk-1 and four novel proteins, captured on SRE duplexes linked to magnetic beads. This approach is generally applicable to the characterisation of nucleic acid-bound protein complexes and the post-translational modification of their components.

Target gene search for the metal-responsive transcription factor MTF-1

Activation of genes by heavy metals, notably zinc, cadmium and copper, depends on MTF-1, a unique zinc finger transcription factor conserved from insects to human. Knockout of MTF-1 in the mouse results in embryonic lethality due to liver decay, while knockout of its best characterized target genes, the stress-inducible metallothionein genes I and II, is viable, suggesting additional target genes of MTF-1. Here we report on a multi-pronged search for potential target genes of MTF-1, including microarray screening, SABRE selective amplification, a computer search for MREs (DNA-binding sites of MTF-1) and transfection of reporter genes driven by candidate gene promoters. Some new candidate target genes emerged, including those encoding α-fetoprotein, the liver-enriched transcription factor C/EBPα and tear lipocalin/von Ebner’s gland protein, all of which have a role in toxicity/the cell stress response. In contrast, expression of other cell stress-associated genes, such as those for superoxide dismutases, thioredoxin and heat shock proteins, do not appear to be affected by loss of MTF-1. Our experiments have also exposed some problems with target gene searches. First, finding the optimal time window for detecting MTF-1 target genes in a lethal phenotype of rapid liver decay proved problematical: 12.5-day-old mouse embryos (stage E12.5) yielded hardly any differentially expressed genes, whereas at stage 13.0 reduced expression of secretory liver proteins probably reflected the onset of liver decay, i.e. a secondary effect. Likewise, up-regulation of some proliferation-associated genes may also just reflect responses to the concomitant loss of hepatocytes. Another sobering finding concerns γ-glutamylcysteine synthetasehc (γ-GCShc), which controls synthesis of the antioxidant glutathione and which was previously suggested to be a target gene contributing to the lethal phenotype in MTF-1 knockout mice. γ-GCShc mRNA is reduced at the onset of liver decay but MTF-1 null mutant embryos manage to maintain a very high glutathione level until shortly before that stage, perhaps in an attempt to compensate for low expression of metallothioneins, which also have a role as antioxidants.

Post-transcriptional regulation of vascular endothelial growth factor mRNA by the product of the VHL tumor suppressor gene.

The VHL tumor suppressor gene is inactivated in patients with von Hippel-Lindau disease and in most sporadic clear cell renal carcinomas. Although VHL protein function remains unclear, VHL does interact with the elongin BC subunits in vivo and regulates RNA polymerase II elongation activity in vitro by inhibiting formation of the elongin ABC complex. Expression of wild-type VHL in renal carcinoma cells with inactivated endogenous VHL resulted in unaltered in vitro cell growth and decreased vascular endothelial growth factor (VEGF) mRNA expression and responsiveness to serum deprivation. VEGF is highly expressed in many tumors, including VHL-associated and sporadic renal carcinomas, and it stimulates neoangiogenesis in growing solid tumors. Despite 5-fold differences in VEGF mRNA levels, VHL overexpression did not affect VEGF transcription initiation or elongation as would have been suggested by VHL-elongin association. These results suggest that VHL regulates VEGF expression at a post-transcriptional level and that VHL inactivation in target cells causes a loss of VEGF suppression, leading to formation of a vascular stroma.

Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein.

Inactivation of the von Hippel-Lindau protein (pVHL) has been implicated in the pathogenesis of renal carcinomas and central nervous system hemangioblastomas. These are highly vascular tumors which overproduce angiogenic peptides such as vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). Renal carcinoma cells lacking wild-type pVHL were found to produce mRNAs encoding VEGF/VPF, the glucose transporter GLUT1, and the platelet-derived growth factor B chain under both normoxic and hypoxic conditions. Reintroduction of wild-type, but not mutant, pVHL into these cells specifically inhibited the production of these mRNAs under normoxic conditions, thus restoring their previously described hypoxia-inducible profile. Thus, pVHL appears to play a critical role in the transduction of signals generated by changes in ambient oxygen tension.

Vascular endothelial growth factor B, a novel growth factor for endothelial cells.

We have isolated and characterized a novel growth factor for endothelial cells, vascular endothelial growth factor B (VEGF-B), with structural similarities to vascular endothelial growth factor (VEGF) and placenta growth factor. VEGF-B was particularly abundant in heart and skeletal muscle and was coexpressed with VEGF in these and other tissues. VEGF-B formed cell-surface-associated disulfide-linked homodimers and heterodimerized with VEGF when coexpressed. Conditioned medium from transfected 293EBNA cells expressing VEGF-B stimulated DNA synthesis in endothelial cells. Our results suggest that VEGF-B has a role in angiogenesis and endothelial cell growth, particularly in muscle.

Non-uniqueness through duality in the von Neumann growth models

Duality can be viewed as the soul of each von Neumann growth model. This is not at all surprising because von Neumann (1955), a mathematical genius, extensively studied quantum mechanics which involves a “dual nature” (electromagnetic waves and discrete corpuscules or light quanta). This may have had some influence on developing his own economic duality concept. The main object of this paper is to restore the spirit of economic duality in the investigations of the multiple von Neumann equilibria. By means of the (ir)reducibility taxonomy in Móczár (1995) the author transforms the primal canonical decomposition given by Bromek (1974) in the von Neumann growth model into the synergistic primal and dual canonical decomposition. This enables us to obtain all the information about the steadily maintainable states of growth sustained by the compatible price-constellations at each distinct expansion factor.

Big bang bifurcations in von Bertalanffy’s dynamics with strong and weak Allee effects

The main purpose of this work was to study population dynamic discrete models in which the growth of the population is described by generalized von Bertalanffy's functions, with an adjustment or correction factor of polynomial type. The consideration of this correction factor is made with the aim to introduce the Allee effect. To the class of generalized von Bertalanffy's functions is identified and characterized subclasses of strong and weak Allee's functions and functions with no Allee effect. This classification is founded on the concepts of strong and weak Allee's effects to population growth rates associated. A complete description of the dynamic behavior is given, where we provide necessary conditions for the occurrence of unconditional and essential extinction types. The bifurcation structures of the parameter plane are analyzed regarding the evolution of the Allee limit with the aim to understand how the transition from strong Allee effect to no Allee effect, passing through the weak Allee effect, is realized. To generalized von Bertalanffy's functions with strong and weak Allee effects is identified an Allee's effect region, to which is associated the concepts of chaotic semistability curve and Allee's bifurcation point. We verified that under some sufficient conditions, generalized von Bertalanffy's functions have a particular bifurcation structure: the big bang bifurcations of the so-called box-within-a-box type. To this family of maps, the Allee bifurcation points and the big bang bifurcation points are characterized by the symmetric of Allee's limit and by a null intrinsic growth rate. The present paper is also a significant contribution in the framework of the big bang bifurcation analysis for continuous 1D maps and unveil their relationship with the explosion birth and the extinction phenomena.

Quantifizierung postholsteinzeitlicher Subrosionen am Salzstock Gorleben durch statistische Auswertung von Bohrergebnissen

The Gorleben salt dome is actually investigated for its suitability as a repository for radioactive waste. It is crossed by a subglacial drainage channel, formed during the Elsterian glaciation (Gorleben channel). Some units of its filling vary strongly in niveau and thickness. Lowest positions and/or largest thickness are found above the salt dome. This is interpreted as a result of subrosion during the Saalean glaciation. The rate can be calculated from niveau differences of sediments formed during the Holsteinian interglacial. However, their position might have been influenced by other factors also (relief of the channel bottom, glacial tectonics, settlement of underlying clay-rich sediments). Their relevance was estimated applying statistical techniques to niveau and thickness data from 79 drillings in the Gorleben channel. Two classes of drillings with features caused by either Saalean subrosion or sedimentary processes during the filling of the Gorleben channel can be distinguished by means of factor and discriminant analysis. This interpretation is supported by the results of classwise correlation and regression analysis. Effects of glacial tectonics on the position of Holsteinian sediments cannot be misunderstood as subrosional. The influence of the settlement of underlying clay sediments can be estimated quantitatively. Saalean subrosion rates calculated from niveau differences of Holsteinian sediments between both classes differ with respect to the method applied: maximum values are 0,83 or 0,96 mm/a, average values are 0,31 or 0,41 mm/a.

Analyse des Ermüdungsverhaltens von Beton anhand der Dehnungsentwicklung

Die Weiterentwicklungen in der Betontechnologie führten in den letzten Jahrzehnten zu Hochleistungsbetonen mit immer höheren Festigkeiten. Der Ermüdungsnachweis wurde jedoch kaum weiterentwickelt und beinhaltet immer noch sehr grobe Herangehensweisen bei der Berücksichtigung des Materialwiderstands von Beton. Für eine grundlegende Weiterentwicklung dieses Nachweises fehlt noch das notwendige Wissen zu den Mechanismen der Betonermüdung. Das Ziel dieser Arbeit war es daher, grundlegende Erkenntnisse zum Ermüdungsverhalten hochfester Betone bei unterschiedlichen zyklischen Beanspruchungen zu ermitteln und hierdurch zu einem besseren Verständnis der Mechanismen der Betonermüdung beizutragen. In der vorliegenden Arbeit wurde das Ermüdungsverhalten eines hochfesten Betons bei Druckschwellbeanspruchung anhand der Dehnungs- und Steifigkeitsentwicklungen untersucht. Betrachtet wurden dabei die Einflüsse der bezogenen Oberspannung, der Belastungsfrequenz und der Wellenform. Zusätzlich wurden, ausgehend von in der Literatur dokumentierten Ansätzen, Versuche bei monoton steigender Beanspruchung und Dauerstandbeanspruchung vergleichend durchgeführt. Die Dehnungs- und Steifigkeitsentwicklungen werden durch die untersuchten Belastungsparameter der Ermüdungsbeanspruchung eindeutig beeinflusst. Charakteristische Zusammenhänge zwischen der Beeinflussung einzelner Kenngrößen der Dehnungs- und Steifigkeitsentwicklung und der Beeinflussung der Bruchlastwechselzahlen wurden aufgezeigt. Anhand der Dehnungen und Steifigkeiten an den Phasenübergängen konnten Hinweise auf beanspru-chungsartabhängige Gefügezustände abgeleitet werden. Die vergleichende Auswertung des Dehnungsverhaltens bei monoton steigender Beanspruchung, Ermüdungsbeanspruchung und Dauerstandbeanspruchung zeigte, dass das Ermüdungsverhalten von Beton nicht adäquat in Anlehnung an andere Beanspruchungsarten beschrieben werden kann. Die Untersuchungsergebnisse wurden in eine Modellvorstellung übertragen, die zur Beurteilung der baustofflichen Phänomene bei zyklischen Beanspruchungen geeignet ist. Dabei wurde die Hypothese aufgestellt, dass sich unterschiedlich ausgeprägte Kleinst-Gefügeveränderungen beanspruchungsabhängig einstellen, die die Entstehung und Ausbreitung von Mikrorissen beeinflussen. Die detaillierte Untersuchung der Dehnungs- und Steifigkeitsentwicklungen führte zu neuen und tiefergehenden Erkenntnissen und sollte ergänzt durch die Betrachtungen von Gefügezuständen zukünftig weiterverfolgt werden.

Der Zusammenhang der Theorie des geplanten Verhaltens mit der selbstberichteten Individualisierungspraxis von Lehrpersonen

Einstellungen stellen als Teil der professionellen Handlungskompetenz von Lehrpersonen eine wichtige handlungsleitende Determinante des Unterrichtsgeschehens dar. Hierzu wird auf Basis der Theorie des geplanten Verhaltens ein Erwartungs-mal-Wert-theoretisches Einstellungsinstrument faktoriell validiert und der Zusammenhang der inklusiven Einstellung, Normvorstellung und Lehrerselbstwirksamkeitsüberzeugung mit der selbstberichteten Individualisierungspraxis von Lehrpersonen betrachtet. Die Ergebnisse einer exploratorischen Faktorenanalyse zeigen in Studie I drei Einstellungsfaktoren, die in Studie II konfirmatorisch bestätigt wurden. Studie III zeigt, dass die selbstberichtete Individualisierungspraxis durch die Normvorstellung und die Intention, sich den Herausforderungen eines inklusiven Unterrichtes anzunehmen, vorhergesagt werden kann. Die Intention mediiert dabei den Zusammenhang der selbstberichteten Individualisierungspraxis mit der Einstellung vollständig und mit der Normvorstellung partiell. Die Lehrerselbstwirksamkeitsüberzeugung sagt demgegenüber die selbstberichtete Individualisierungspraxis weder direkt noch indirekt vorher. (DIPF/Orig.)

Fragebogen zur Erfassung von Störungen im Unterricht: die Faktorenstruktur der Schülerinnen- und Schülerversion

In diesem Beitrag wird ein neu entwickelter Schülerinnen- und Schülerfragebogen zur Erfassung aggressiver und nicht aggressiver Schülerstörungen, aggressiven Lehrerverhaltens, Störungen des methodisch-didaktischen Settings sowie Klassenführung und Beziehung vorgestellt und die testtheoretischen Kennwerte diskutiert. Die faktorielle Struktur wurde an einer Stichprobe von N=1341 Schülerinnen und Schüler der fünften und sechsten Klasse ermittelt. Eine explorative Faktorenanalyse mit Oblimin-Rotation ergab sieben eindeutige, gut interpretierbare Faktoren, welche den theoretisch postulierten Konstrukten entsprechen. Vier Faktoren erfassen Störungen und drei Faktoren umfassen störungspräventive Merkmale des Unterrichts. Die internen Konsistenzen der Skalen liegen zwischen .60 und .88. (DIPF/Orig.)