1,721,337 research outputs found
Closing the gap: Identification of human 3-ketosteroid reductase, the last unknown enzyme of mammalian cholesterol biosynthesis
No full textThe protein encoded by the HSD17B7 gene was originally described as a prolactin receptor-associated protein and as 17β-hydroxysteroid dehydrogenase (HSD) type 7. Its ability to synthesize 17β-estradiol in vitro has been reported previously. However, we demonstrate that HSD17B7 is the ortholog of the yeast 3-ketosteroid reductase Erg27p and converts zymosterone to zymosterol in vitro, using reduced nicotinamide adenine dinucleotide phosphate as cofactor. Expression of human and murine HSD17B7 in an Erg27p-deficient yeast strain complements the 3-ketosteroid reductase deficiency of the cells and restores growth on sterol-deficient medium. A fusion of HSD17B7 with green fluorescent protein is located in the endoplasmic reticulum, the site of postsqualene cholesterogenesis. Further critical evidence for a role of HSD17B7 in cholesterol metabolism is provided by the observation that its murine ortholog is a member of the same highly distinct embryonic synexpression group as hydroxymethyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme of sterol biogenesis, and is specifically expressed in tissues that are involved in the pathogenesis of congenital cholesterol-deficiency disorders. We conclude that HSD17B7 participates in postsqualene cholesterol biosynthesis, thus completing the molecular cloning of all genes of this central metabolic pathway. In its function as the 3-ketosteroid reductase of cholesterol biosynthesis, HSD17B7 is a novel candidate for inborn errors of cholesterol metabolism
Identifizierung und Charakterisierung eines Y-chromosom-kodierten Gens das in Zellen der akuten myeloischen Leukemie überexprimiert ist
No full textMale patients with acute myeloid leukaemia (AML) who receive female haematopoietic stem cells show the best therapy outcome. Y chromosome gene expression has been evaluated in male AML M4-M5 cells and in male healthy CD14+ cells. The genes BCORL2, PCDH11, TGIF2L, and VCY were found to be up-regulated in leukaemic cells. After immunological studies, one peptide derived from the gene VCY and able to weakly stimulate female cytotoxic T-cells has been identified. Several aspects of the putative transcription factor TGIF2LY have been characterized, including its subcellular localization and identification of its DNA-binding sequence.Beste Therapieergebnisse bei männlichen Patienten mit akuter myeloischer Leukämie (AML) werden bei der Transplantation von weiblichen hämatopoetischen Stammzellen sichtbar. Expression von Y Chromosom-kodierten Genen wurde in männlichen AML M4-M5 Zellen, sowie in männlichen gesunden CD14+ Zellen, gemessen. Die Gene BCORL2, PCDH11, TGIF2L und VCY waren in den leukämischen Zellen überexprimiert. Nach immunologischen Studien wurde ein Peptid aus der VCY Protein identifiziert, welches weibliche zytotoxische T-Zellen stimulieren kann. Mehrere Aspekte des mutmaßlichen Transkriptionsfaktors TGIF2LY wurden charakterisiert, inklusive seiner subzellulären Lokalisierung und seiner DNA-Bindungssequenz
A functional and putative physiological role of calcitriol in Patched1/Smoothened interaction.
No full textThe Patched1 (Ptch)-mediated inhibition of Smoothened (Smo) is still an open question. However, a direct Ptch/Smo interaction has been excluded, Smo modulators were identified, but the endogenous signal transmitting molecule remains undiscovered. Here, we demonstrate that calcitriol, the hormonally active form of vitamin D3, is an excellent candidate for transmission of Ptch/Smo interaction. Our study reveals that Ptch expression is sufficient to release calcitriol from the cell and that calcitriol inhibits Smo action and ciliary translocation by acting on a site distinct from the 7-transmembrane-domain or the cysteine-rich-domain. Moreover calcitriol strongly synergizes with itraconazole (ITZ) in Smo inhibition which not results from elevated calcitriol bio-availability due to ITZ-mediated 24-hydroxylase inhibition but rather from a direct interaction of the compounds at the level of Smo. Together, we suggest that calcitriol represents a possible endogenous transmitter of Ptch/Smo interaction. Moreover calcitriol or calcitriol derivatives combined with ITZ might be a treatment option of Hedgehog-associated cancers
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Molekulare Wirkung von Metformin auf den hepatozellulären Metabolismus
No full textThis study aimed to extent our understanding of the hepatocellular metformin transport and cellular mode of action. A LC-MS/MS based metformin quantification method and a cell number quantification method for the normalization of metabolomics data were developed. The uptake of metformin by Hep G2 cells was assessed and the impact of glucose and metformin on the metabolism was analyzed in a diabetes and cancer related context.Diese Arbeit setzte es sich zum Ziel, das Verständnis des hepatozellulären Metformintransportes und dessen zellulärer Wirkweise zu erweitern. Eine LC-MS/MS-basierte Metforminquantifizierungsmethode und eine Zellzahlquantifizierungsmethode zur Normalisierung von Metabolomics-Daten wurden entwickelt. Die Aufnahme von Metformin durch Hep G2 Zellen wurde analysiert und der Einfluss von Glukose und Metformin auf den Metabolismus wurde im Zusammenhang mit Diabetes und Krebs untersucht
Charakterisierung der neuen humanen Aldo-Keto Reduktase AKR1B15 und der 17beta-Hydroxysteroiddehydrogenase 17beta-HSD12
No full textEnzyme actions in metabolic pathways play an important role in health and disease and represent potent targets for diagnosis and therapy. This thesis covered the first in-depth characterization of a novel human AKR, called AKR1B15. In addition, it included the development of a procedure for the purification of the membrane-bound human SDR 17β-HSD12 as enzymatically active protein and of a LC-MS/MS method for the simultaneous analysis of up to eight vitamin D metabolites in biological samples.Die Aktivität von Enzymen in Stoffwechselwegen trägt wesentlich zu Gesundheit und Krankheit bei und ist so ein potentes Ziel für die Diagnose und Therapie. Diese Arbeit umfasst die erste detailliertere Charakterisierung einer neuen humanen AKR: AKR1B15. Zudem beinhaltet sie die Entwicklung eines Verfahrens zur Aufreinigung der membranständigen humanen SDR 17β-HSD12 als aktives Enzym und einer LC-MS/MS Methode für die simultane Analyse von bis zu acht Vitamin D Metaboliten in biologischen Proben
SDR and AKR enzymes as a target of rational inhibitor development and research on function of new SDR members.
No full textHydroxysteroiddehydrogenasen (HSDs) spielen eine bedeutende Rolle in Regulierung der Biosynthese von Steroidhormonen und gehören zur zwei großen Familien der Enzymen: short chain dehydogenases (SDR) und der aldo-keto reductases (AKR). Die Fehlregulierung einiger HSD-Aktivitäten führt zu verschiedenen schweren Störungen wie Alzheimer Syndrom oder hormonabhängigem Krebs. Deshalb stellen die HSDs schon seit vielen Jahren interessante Ziele für die der pharmazeutische Industrie für die Entwicklung neuer spezifischer Inhibitoren dar. Zwei der 17beta-Hydroxysteroid dehydrogenasen, die 17beta-Hydroxysteroiddehydrogenase Typ 3 (zur Familie der short chain dehydogenases (SDRs) gehörend) und die 17beta-Hydroxysteroid Dehydrogenase Typ 5 (eine Aldo-Keto Reduktase (AKR)), katalysieren die Testosteron Biosynthese und ihre Überaktivität wird assoziiert mit einigen Krankheiten wie Prostatakrebs.
Die Fortschritte in der Bioinformatik und Computer-basierten Methoden für Molekulare Modellierung ermöglichen Hochdurchsatz-Screening Methoden und erleichtern erheblich die systematische Entwicklung neuer enzym-spezifischer Liganden mit den gewünschten Eigenschaften, die später als Medikamente dienen könnten. Kooperierende Forscher der pharmazeutische Firma BioNetWorks und eines Forschungsteams der Universität Innsbruck entwickelten zwei Ligand- und Struktur -basiert Pharmakophormodelle und wendeten diese an, um neue mögliche Inhibitoren gegen die Aktivitäten der 17beta-Hydroxysteroiddehydrogenasen Typ 3 und 5 zu identifizieren. In der vorliegenden Arbeit wurden dann 35 verschiedene, durch das in silico Screening ausgewählte Chemikalien zur biologischen Prüfung und Bewertung untersucht. Aus dieser Gruppe wurden einige zukunftsträchtige Substanzen als 17beta-HSD3 oder 5 Inhibitoren für weitere mehr detailliert biologische Prüfungen identifiziert. Einer der Inhibitor-Kandidaten gegen die reduktive Aktivität der 17beta-HSD5 zeigte sogar eine Hemmwirkung im nanomolaren Bereich und wurde daher detailierter untersucht. Ein Ki von 180 nM und IC50 Werte in Bereich zwischen 140nM und 290nM, abhängig von der Sorte der enzymatischen Assay, wurden ermittelt.
Darüber hinaus wurden 3 in der Forschungsliteratur kaum annotierte humane SDR-Enzyme, die einige Ähnlichkeiten zu gut bekannten Hydroxysteroiddehydrogenasen zeigen, in dieser Doktorarbeit detaillierter charakterisiert: Hydroxysteroiddehydrogenase Typ 8 (HSD17B8), SDR-O (SDR-Orphan) und die Hydroxysteroiddehydrogenase like 2 (HSDL2). Um die Bedeutung für die mögliche Funktionen der Enzyme im Menschen zu erfassen wurden Experimente wie z.B. subzelluläre Lokalisierung durchgeführt, sowie enzymatische Tests zur Überprüfung der Aktivität mit Steroiden und Retinoiden. Während das bevorzugte Vorkommen für zwei der EnzymeHydroxysteroid dehydrogenases (HSDs) play a key role in the regulation of the steroid hormone metabolism and belong to two protein superfamilies the short-chain dehydrogensases/reductses (SDR) and the aldo-keto reductases (AKR). Up-regulation of their enzymatic activity can be involved in pathogenesis of many serious disorders in humans like Alzheimer’s disease or hormone-dependent cancers. Thus, since many years HSDs constitute interesting targets for the development of specific inhibitors which could be applied as potent therapeutic drugs. Two of the 17beta-hydroxysteroid dehydrogenases, the 17beta-hydroxysteroid dehydrogenase type 3 belonging to the SDRs and 17beta-hydroxysteroid dehydrogenase type 5 (belonging to the aldoketo reductases (AKRs), catalyze reactions of the testosterone biosynthesis and an enhanced activity of the enzymes is linked to several androgen-related illnesses such as for example prostate cancer.
Nowadays due to advances in bioinformatics as well computational methods of molecular modelling high throughput rational approaches for designing new enzyme-specific inhibitors are available. Co-operating researchers of the company BioNetWorks and the University Innsbruck developed two ligand-and structure-based pharmacophore models and applied those to identify potential inhibitors for 17betaHSD3 and 5. By this in silico screen, 35 chemically diverse compounds we found as potent candidate inhibitors. In this PhD work here the 35 candidate compounds were subjected to biological evaluation. This allowed to discover some promising compounds, which could be lead structures for further researching or pharmacological tools in future projects. One of the compounds against the reductive activity of 17betaHSD5 even displayed an inhibitory activity in the nanomolar range and therefore was characterized in more detail. For this compound a Ki of 180 nM and IC50 values ranging between 140nM and 290nM, were found, depending on the kind of enzymatic assay applied.
Moreover, not all recently identified SDRs have been analyzed in detail yet. Three barely annotated human SDR candidates, which reveal some similarities to known hydroxysteroid dehydrogneses, were chosen in this PhD work for further characterization: hydroxysteroid dehydrogenase type 8 HSD17B8, an orphan SDR (SDR-O) and hydroxysteroid dehydrogenase like 2 (HSDL2). In search for their potent functions in human results for e.g., subcellular localization, bioinformatics studies based on sequence analysis of primary amino acid structure as well enzymatic tests checking the activities towards steroid and retinoid substrates are here presented. Two of the enzyme could be assigned to cellular compartments, HSD17B8 to mitochondria, HSDL2 to peroxisomes or mitochondria, while the preferred location of SDR-O remains unclear. Of the three enzymes only HSD17B8 showed an enzymatic activity with a steroid substrate, in that case estradiol, and only SDR-O was able to metabolize a retinoid compound, i.e., weak activity towards retinaldehyde in the presence of NADH as cofactor was observed with the human SDR-O gene
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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