Massenspektrometrische Charakterisierung endogener Wachstumshormonvarianten und Entwicklung einer Methode zum Nachweis rekombinanten Wachstumshormons in humanem Plasma mittels zweidimensionaler Gelelektrophorese zum Einsatz in der Dopinganalytik

Humanes endogenes Wachstumshormon wird in der Hypophyse produziert und ist einer der wichtigsten Wachstumsregulatoren im menschlichen Körper. Außerdem hat Wachstums¬hormon eine Fülle metabolischer Funktionen wie Fettabbau und Muskelaufbau und hat Einfluss auf den Knochen- sowie Mineralstoffhaushalt. Besonders aufgrund der endokrino¬logischen Wirkungen auf Lipolyse und Proteinanabolismus wird Wachstums¬hormon im Sport zur Leistungssteigerung eingesetzt, und dessen Verwendung ist von der Welt Anti-Doping Agentur (WADA) verboten. Endogenes Wachstumshormon ist ein heterogenes Protein und besteht aus vielen Varianten und Fragmenten. Die Hauptform, die etwa 90% des Wachstumshormons im Körper ausmacht, ist 22 kDa groß und entspricht dem rekombinanten Wachstumshormon. Andere bereits identifizierte Formen sind eine 20 kDa Splice-Variante, phosphorylierte und deamidierte Formen und Fragmente sowie Polymere. Eine Testmethode zur Detektion rekombinanten Wachstumshormons muss daher die niederkonzentrierten, für endogenes Wachstumshormon spezifischen Formen detektieren. Im Rahmen der vorliegenden Arbeit wurde eine Dopingkontrollmethode entwickelt, die Wachstumshomon aus humanem Plasma durch Immunoaffinitätsaufreinigung, 2D-Elektrophorese und Immunoblot detektiert und die Unterscheidung von endogenem und rekombinantem Wachstumshormon ermöglicht. Des Weiteren wurden die in der Methode detektierten endogenen Formen mittels hochauflösender/hochakkurater Massenspektrometrie identifiziert. Die Immunoaffinitätsaufreinigung dient zur Isolation des Wachstumshormons und des zugegebenen internen Standards (plazentares Laktogen) mittels eines polyklonalen anti-Wachstumshormon-Antikörpers und, an einen sekundären Antikörper gekoppelten, magnetischen Beads. Nach Auftrennung mittels zweidimensionaler Gelelektrophorese werden die Proteine mittels Immunoblot und Chemilumineszenzreaktion visualisiert. Die Prozedur detektiert bis zu vier endogene Wachstumshormonvarianten, erlaubt die Analyse von sechs Proben in einer Aufarbeitung und nimmt 2 ½ Tage in Anspruch. Die Methode wurde unter Berücksichtigung der analytischen Parameter Spezifität, Linearität, Nachweisgrenze (0,3 ng/ml), Präzision (14%) und Wiederfindung (30%) validiert. Der Einfluss einer belastungsinduzierten Wachstumshormonausschüttung auf die Detektion unterschiedlicher Varianten wurde mittels eines Belastungstests geprüft und ergab keine Diskriminierung endogener Formen. Um Grenzwerte für die Präsenz rekombinanten Wachstumshormons in einer Probe festlegen zu können wurden Proben einer Referenzgruppe gesunder Probanden sowie Proben von Patienten gemessen, die rekombinantes Wachstumshormon applizieren. Aus der Referenzpopulation wurde ein Unterscheidungslimit von 0,52 für das auf den internen Standard normierte Spotvolumen der 22 kDa-Hauptform errechnet. Proben mit einem normierten Spotvolumen der 22 kDa-Hauptform > 0,52 müssen, wenn sie endogenes Wachstumshormon enthalten, mindestens eine weitere Form (die 20 kDa Splice-Variante) zeigen. Ist dies nicht der Fall, wird die Schlussfolgerung gezogen, dass die Probe rekombinantes Wachstumshormon enthält. Die Werte der analysierten Patientenproben lagen weit über dem Unterscheidungslimit und konnten eindeutig als Proben identifiziert werden, die rekombinantes Wachstumshormon enthalten. Der entwickelte Test ist damit eine wertvolle Ergänzung und sollte als Bestätigungsmethode zu dem bisher verwendeten Immunoassay eingesetzt werden. Die massenspektrometrische Identifizierung konzentrierte sich auf die vier endogenen Spots, die in der Dopingkontrollmethode detektiert werden. Diese wurden identifiziert als 1) 22 kDa-Hauptform, 2) 20 kDa Splice-Variante, 3) phosphorylierte Form mit Modifikationen an Serin 106 und 150 und 4) einer glykosylierten Form, die vermutlich an Threonin 60 modifiziert ist. Die Glykosylierung ist eine O-Glykosylierung vom Mucin-Typ und die Strukur wurde als Verknüpfung einer Hexose (Hex) mit einem N-acetylierten Hexosamin (HexNac) und zwei Sialinsäuren (NeuAc) identifiziert. Zusätzlich wurden Fragmente mit 9 und 12 kDa sowie unterschiedliche oxidierte Formen detektiert. Die massenspektrometrische Charakterisierung von Varianten und Isoformen, die in einer auf immunologischen Techniken beruhenden Methode detektiert werden, ist entscheidend, um Ergebnisse bewerten und ungewöhnliche Werte besser einordnen zu können. Des Weiteren kann die Identifizierung neuer Varianten eines Proteins zur Aufklärung von Funktionen und Signalwegen beitragen. Damit sind die hier vorgestellten Ergebnisse sowohl für die Dopinganalytik als auch für die Endokrinologie sehr bedeutsam

Lest We Forget: Foundational Women for Historically and Socially

Accredited teacher education programs include historical and educational psychological foundations. Noticeably missing are women’s voices and theories in both disciplines. Explanations and descriptions of why it is imperative to incorporate historically notable and influential women into foundations coursework is the focus of this article.Accredited teacher education programs include historical and educational psychological foundations. Noticeably missing are women’s voices and theories in both disciplines. Explanations and descriptions of why they missing are imperative. Incorporating historically notable and influential women into foundations coursework is the focus of this article. This historical case study contributes to gender equity by providing distinguished, notable women researchers and theorists that have been marginalized in foundations literature.The featured women are integral to the canon of educational foundations and certainly deserve to be included in the 21st Century scholarship. Myriad other women are overlooked. These seven fundamental voices have been excluded from teacher education courses and courses that support them. Many of the women practiced educational social justice and were connected to men who were credited for their work. Others were simply disregarded. All warrant study; however, this article features the following progressive educators: Ella Flagg Young (1845 – 1918), Anna Julia Cooper (1858 – 1964), Lucy Sprague Mitchell (1878 – 1967), and Charlotte Hawkins Brown (1883 – 1961). Also highlighted are three salient examples from the area of educational psychology, Sabina Spielrein (1884 – 1942), Diana Baumrind, (1927b) and Carol Gilligan (1936b)

Lest We Forget: Foundational Women for Historically and Socially Responsive Women

Accredited teacher education programs include historical and educational psychological foundations. Noticeably missing are women’s voices and theories in both disciplines. Explanations and descriptions of why it is imperative to incorporate historically notable and influential women into foundations coursework is the focus of this article

RNA interference for performance enhancement and detection in doping control

RNA interference represents a comparably new route of regulating and manipulating specific gene expression. Promising results were obtained in experimental therapies aim at the treatment of different kinds of diseases including cancer, diabetes mellitus or Dychenne muscular dystrophy. While studies on down-regulation efficiency are often performed by analyzing the regulated protein, the direct detection of small, interfering RNA molecules and antisense oligonucleotides is of great interest for the investigation of the metabolism and degradation and also for the detection of a putative misuse of these molecules in sports. Myostatin down-regulation was shown to result in increased performance and muscle growth and the regulation of several other proteins could be relevant for performance enhancement. This mini-review summarizes current approaches for the mass spectrometric analysis of siRNA and antisense oligonucleotides from biological matrices and the available data on biodistribution, metabolism, and half-life of relevant substances are discussed. Copyright (C) 2011 John Wiley & Sons, Ltd

Lest We Forget: Foundational Women for Historically and Socially Responsive Women

Accredited teacher education programs include historical and educational psychological foundations. Noticeably missing are women�s voices and theories in both disciplines. Explanations and descriptions of why they missing are imperative. Incorporating historically notable and influential women into foundations coursework is the focus of this article. This historical case study contributes to gender equity by providing distinguished, notable women researchers and theorists that have been marginalized in foundations literature. The featured women are integral to the canon of educational foundations and certainly deserve to be included in the 21st Century scholarship. Myriad other women are overlooked. These seven fundamental voices have been excluded from teacher education courses and courses that support them. Many of the women practiced educational social justice and were connected to men who were credited for their work. Others were simply disregarded. All warrant study; however, this article features the following progressive educators: Ella Flagg Young (1845 � 1918), Anna Julia Cooper (1858 � 1964), Lucy Sprague Mitchell (1878 � 1967), and Charlotte Hawkins Brown (1883 � 1961). Also highlighted are three salient examples from the area of educational psychology, Sabina Spielrein (1884 � 1942), Diana Baumrind, (1927b) and Carol Gilligan (1936b)

Lest We Forget: Foundational Women for Historically and Socially

Accredited teacher education programs include historical and educational psychological foundations. Noticeably missing are women’s voices and theories in both disciplines. Explanations and descriptions of why it is imperative to incorporate historically notable and influential women into foundations coursework is the focus of this article.Accredited teacher education programs include historical and educational psychological foundations. Noticeably missing are women’s voices and theories in both disciplines. Explanations and descriptions of why they missing are imperative. Incorporating historically notable and influential women into foundations coursework is the focus of this article. This historical case study contributes to gender equity by providing distinguished, notable women researchers and theorists that have been marginalized in foundations literature.The featured women are integral to the canon of educational foundations and certainly deserve to be included in the 21st Century scholarship. Myriad other women are overlooked. These seven fundamental voices have been excluded from teacher education courses and courses that support them. Many of the women practiced educational social justice and were connected to men who were credited for their work. Others were simply disregarded. All warrant study; however, this article features the following progressive educators: Ella Flagg Young (1845 – 1918), Anna Julia Cooper (1858 – 1964), Lucy Sprague Mitchell (1878 – 1967), and Charlotte Hawkins Brown (1883 – 1961). Also highlighted are three salient examples from the area of educational psychology, Sabina Spielrein (1884 – 1942), Diana Baumrind, (1927b) and Carol Gilligan (1936b)

Mass spectrometric detection of siRNA in plasma samples for doping control purposes

Small interfering ribonucleic acid (siRNA) molecules can effect the expression of any gene by inducing the degradation of mRNA. Therefore, these molecules can be of interest for illicit performance enhancement in sports by affecting different metabolic pathways. An example of an efficient performance-enhancing gene knockdown is the myostatin gene that regulates muscle growth. This study was carried out to provide a tool for the mass spectrometric detection of modified and unmodified siRNA from plasma samples. The oligonucleotides are purified by centrifugal filtration and the use of an miRNA purification kit, followed by flow-injection analysis using an Exactive mass spectrometer to yield the accurate masses of the sense and antisense strands. Although chromatography and sensitive mass spectrometric analysis of oligonucleotides are still challenging, a method was developed and validated that has adequate sensitivity (limit of detection 0.25-1 nmol mL(-1)) and performance (precision 11-21%, recovery 23-67%) for typical antisense oligonucleotides currently used in clinical studies

Detection of His-tagged Long-R-3-IGF-I in a black market product

Objective: Performance-enhancing substances are illicitly used in elite or amateur sports and may be obtained from the black market due to a cheaper and easier availability. Although various studies have shown that black market products frequently do not contain the declared substances, enormous amounts of illegally produced and/or imported drugs are confiscated from athletes or at customs with alarming results concerning the outcome of the analyses of the ingredients. This case report describes the identification of His-tagged Long-R-3-IGF-I, which is usually produced for biochemical studies, in an injection vial. Design: The ingredients were isolated by immunoaffinity purification and identified by nano-UPLC, high-resolution/high accuracy mass spectrometry of the intact and trypsinated substance and by an enzyme-linked immunosorbent assay. Results: (Tandem) mass spectra characterized the protein as Long-R-3-IGF-I with a His(6)-tag attached to the C-terminus by the linker amino acids Leu-Glu. Conclusion: His-tags are commonly added to proteins during synthesis to allow a convenient and complete purification of the final product and His-tags are subsequently removed by specific enzymes when being attached to the N-terminus. The effects of His-tagged Long-R-3-IGF-I in humans have not been elucidated or described and the product may rather be a by-product from biochemical studies than synthesized for injection purposes. (C) 2010 Growth Hormone Research Society. Published by Elsevier Ltd. All rights reserved