Untersuchungen zur Struktur und Funktion von nicht translatierten Regionen eines Thrombin-Inhibitorgens

Kamp P-B. Untersuchungen zur Struktur und Funktion von nicht translatierten Regionen eines Thrombin-Inhibitorgens. Bielefeld (Germany): Bielefeld University; 1999.Die vorliegende Arbeit beschreibt neue Erkenntnisse zur Organisation und Expression des Heparinkofaktor II-Gens (HCII-Gen). Die Struktur des 5'-gelegenen Bereichs des HCII-Gens von Maus und Ratte wurde aufgeklärt. Der Intron 1-Bereich umfasst beim HCII-Gen der Maus 4,8 kB. Das HCII-Intron 1 der Ratte weist dagegen eine Länge von 4,3 kB auf. Die Intron 1 Sequenz bestätigt, dass die zwei verschiedenen HCII-Transkripte der Ratte auf differentiellem Spleißen beruhen. Darüber hinaus wurde nachgewiesen, dass auch beim HCII-Gen der Maus zwei unterschiedliche HCII-Transkripte im Lebergewebe existieren. Diese Transkripte entsprechen nicht den HCII-mRNAs der Ratte. Sie werden jedoch ebenfalls durch differentielles Spleißen gebildet. Eine Gegenüberstellung der ermittelten HCII-Sequenzen und der entsprechenden menschlichen Sequenz legt nahe, dass das zusätzliche Exon 1B der Ratte vor allem durch einfache Punktmutationen de novo aus Intronsequenzen entstand. Das differentielle Spleißen im 5'-Bereich des HCII-Gens der Maus kann durch Punktmutationen, durch Rekombinationsereignisse von (CA)n-Wiederholungen oder durch unterschiedliche Enden eines Retroposons verursacht sein. Die Exon/Intronstuktur innerhalb der [alpha]1-Antitrypsin-Serpinfamilie ist stark konserviert. Ein Vergleich der bekannten HCII-cDNA-Sequenzen zeigt die übereinstimmende Genstruktur im kodierenden Sequenzbereich. Variabel scheint dagegen das 5'-Ende zu sein, vergleichbar dem [alpha]1-Antitrypsin. Der Transkriptionsstartbereich menschlicher 2,3 kB HCII-Transkripte ließ sich über die T-PCR-Methodik identifizieren. Die Startpunkte der häufigsten Transkripte erstrecken sich über einen Bereich von 45 Bp vor Exon 1. Die Suche nach bekannten proximalen Promotorelementen in einer Region vor dem Startbereich der mRNAs legt nahe, dass die Transkription nicht von einer TATA-Box, sondern von INR-Elementen gesteuert wird. Weitere Untersuchungen ergaben, dass HCII- und auch Antithrombin-Transkripte nicht nur in menschlicher Leber vorkommen, sondern auch in unterschiedlichen Konzentrationen im Gewebe des Gehirns, des Herzens, der Lunge, der Niere und der Plazenta vorliegen. In poly(A)+ -RNAs der genannten Gewebe ließen sich längere HCII-Transkripte nachweisen, die Schmidt zuvor in einer Lungen-cDNA-Bank entdeckte. Eine Charakterisierung dieser Transkripte ergab, dass das Exon 1 der 2,3 kB-Transkripte nicht vorhanden ist. Die mRNAs beginnen in einem über 1 kB großen Bereich vor dem Exon 2. Mit Hilfe eines Reportergenkonstruktes wurde gezeigt, dass ein 2,2 kB großer, 5'-gelegener Bereich des menschlichen HCII-Gens keine Expression der Transkriptionseinheit in einer Leberzellinie bewirkt

Heparin cofactor II, antithrombin-beta and their complexes with thrombin in human tissues

Kamp P-B, Strathmann A, Ragg H. Heparin cofactor II, antithrombin-beta and their complexes with thrombin in human tissues. THROMBOSIS RESEARCH. 2001;101(6):483-491.In the presence of glycosaminoglycans, thrombin is rapidly inactivated by two natural inhibitors secreted from liver: antithrombin (AT) is presumed to be the principal thrombin inhibitor in circulating blood, while for heparin cofactor II (HCII), a role outside circulation has been proposed. In this study, we show that HCII and AT differ with respect to their association with human tissues. Aside from brain, each of these inhibitors was found in sodium dodecyl sulphate (SDS) soluble extracts of various human organs, with a preponderance of HCII in placenta. AT levels, however, predominated in liver. Compared to plasma, the beta -variant of AT was found to be strongly enriched in human organs, while tissue-resident HCII did not differ in its electrophoretic mobility from the circulating form. In placenta, comparable amounts of HCII/thrombin and AT/thrombin complexes were detected, indicating that HCII may exert a thrombin regulating role in that organ under conditions of tissue or blood vessel damage. Transcripts coding for HCII and AT were detected in all tissues examined. The low levels of their mRNAs suggest that most of the tissue-associated thrombin inhibitor molecules originate from circulation and are retained in organs, possibly by specific receptors. The differential presence of HCII and AT in organs is in accordance with individual physiological roles of these inhibitors. (C) 2001 Elsevier Science Ltd. All rights reserved

Magnetic particles as markers and carriers of biomolecules

Brückl H, Panhorst M, Schotter J, Kamp P-B, Becker A. Magnetic particles as markers and carriers of biomolecules. IEE Proceedings - Nanobiotechnology. 2005;152(1):41.The detection and manipulation of biomolecules on a common platform is of considerable interest not only for application in devices such as diagnostic tools but also for basic research in biological and medical systems. A promising approach is the utilisation of magnetic particles as markers and carriers for biomolecules. The principle functionality of this approach is demonstrated by the authors. Magnetic particles used as markers can be detected by highly sensitive magnetoresistive sensors resulting in a purely electronic signal. A direct comparison with the standard fluorescence method reveals the advantages of using the magnetic particles. In addition, magnetic particles used as carriers can be manipulated on-chip via currents running through especially designed line patterns. Some current drawbacks and future aspects are discussed. The combination of sensing and manipulating magnetic particles is a promising choice for future integrated lab-on-a-chip systems

Vertebrate serpins: Construction of a conflict-free phylogeny by combining exon-intron and diagnostic site analyses

Ragg H, Lokot T, Kamp P-B, Atchley WR, Dress A. Vertebrate serpins: Construction of a conflict-free phylogeny by combining exon-intron and diagnostic site analyses. MOLECULAR BIOLOGY AND EVOLUTION. 2001;18(4):577-584.A combination of three independent biological features, genomic organization, diagnostic amino acid sites, and rare indels, was used to elucidate the phylogeny of the vertebrate serpin (serine protease inhibitor) superfamily. A strong correlation between serpin gene families displaying (1) a conserved exon-intron pattern and (2) family-specific combinations of amino acid residues at specific sites suggests that present-day vertebrates encompass six serpin gene families which evolved from primordial genes by massive intron insertion before or during early vertebrate radiation. Introns placed at homologous positions in the gene sequences in combination with diagnostic sequence characters may also constitute a reliable kinship indicator for other protein superfamilies

Comparison of a prototype magnetoresistive biosensor to standard fluorescent DNA detection

Schotter J, Kamp P-B, Becker A, Pühler A, Reiss G, Brückl H. Comparison of a prototype magnetoresistive biosensor to standard fluorescent DNA detection. BIOSENSORS & BIOELECTRONICS. 2004;19(10):1149-1156.We present a comparative analysis of a magnetoresistive biosensor to standard fluorescent DNA detection. The biosensor consists of giant magnetoresistive (GMR) type Cu/Ni80Fe20 multilayers in the second antiferromagnetic coupling maximum. Each of the 206 elements of the magnetoresistive biosensor is patterned into a spiral-shaped line that can cover the area of a typical DNA spot (70 mum diameter). The probe DNA is assembled on top of the sensor elements in different concentrations ranging from 16 pg/mul to 10 ng/mul. Complementary biotin-labeled analyte DNA is hybridized to the probe DNA at a concentration of 10 ng/mul. A number of different commercially available magnetic microspheres are investigated to determine the most appropriate markers. The experimental comparison shows that the relative sensitivity of the magnetoresistive biosensor is superior to the fluorescent detection at low probe DNA concentrations. (C) 2003 Elsevier B.V. All rights reserved

Detection and manipulation of biomolecules by magnetic carriers

Brzeska M, Panhorst M, Kamp P-B, et al. Detection and manipulation of biomolecules by magnetic carriers. JOURNAL OF BIOTECHNOLOGY. 2004;112(1-2):25-33.The detection and manipulation of single molecules on a common platform would be of great interest for basic research of biological or chemical systems. A promising approach is the application of magnetic carriers. The principles are demonstrated in this contribution. It is shown that paramagnetic beads can be detected by highly sensitive magnetoresistive sensors yielding a purely electronic signal. Different configurations are discussed. The capability of the sensors to detect even single markers is demonstrated by a model experiment. In addition, the paramagnetic beads can be used as carriers for biomolecules. They can be manipulated on-chip via currents running through specially designed line patterns. Thus, magnetic markers in combination with magnetoresistive sensors are a promising choice for future integrated lab-on-a-chip systems. (C) 2004 Elsevier B.V. All rights reserved

A biochip based on magnetoresistive sensors

Schotter J, Kamp P-B, Becker A, et al. A biochip based on magnetoresistive sensors. In: IEEE Transactions on Magnetics. IEEE TRANSACTIONS ON MAGNETICS. Vol 38. IEEE-INST ELECTRICAL ELECTRONICS ENGINEERS INC; 2002: 3365-3367.The signal response of Bangs Laboratories magnetic markers with a mean diameter of 0.86 mum on a spiral-shaped giant magnetoresistance sensor with a diameter of 70 mum is investigated. The data show a linear dependence of the sensor signal on the surface coverage of the magnetic markers. The detection limit is reached at a coverage of about 5%, which corresponds to about 200 markers distributed across the surface of the sensor. The corresponding molecular binding of the analyte DNA and the magnetic markers show good selectivity and sensitivity

Magnetoresistive logic and biochip

Brückl H, Brzeska M, Brinkmann D, et al. Magnetoresistive logic and biochip. In: Journal of Magnetism and Magnetic Materials. JOURNAL OF MAGNETISM AND MAGNETIC MATERIALS. Vol 282. ELSEVIER SCIENCE BV; 2004: 219-224.While some magnetoresistive devices based on giant magnetoresistance or spin-dependent tunneling are already commercialized, a new branch of development is evolving towards magnetoresistive logic with magnetic tunnel junctions. Furthermore, the new magnetoelectronic effects show promising properties in magneto resistive biochips, which are capable of detecting even single molecules (e.g. DNA) by functionalized magnetic markers. The unclear limits of this approach are discussed with two model systems. (C) 2004 Elsevier B.V. All rights reserved

Molecular Detection with Magnetic Labels and Magnetoresistive Sensors

Schotter J, Panhorst M, Brzeska M, et al. Molecular Detection with Magnetic Labels and Magnetoresistive Sensors. In: Gross R, Sidorenko A, Tagirov L, eds. Nanoscale Devices - Fundamentals and Applications. NATO Science Series. Vol 233. Dordrecht: Springer Netherlands; 2007: 35-46