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Small-subunit (SSU) rRNA genes (rDNA) were amplified by PCR from a hot pool environmental DNA sample using Bacteria- or Archaea-specific rDNA primers. Unique rDNA types were identified by restriction fragment length polymorphism (RFLP) analysis and representative sequences were determined. Family 10 glycoside hydrolase consensus PCR primers were used to explore the occurrence and diversity of xylanase genes in the hot pool environmental DNA sample. Partial sequences for three different xylanases were obtained and genomic walking PCR (GWPCR), in combination with nested primer pairs, was used to obtained a unique 1,741-bp nucleotide sequence. Analysis of this sequence identified a putative XynA protein encoded by the xynA open reading frame. The single module novel xylanase shared sequence similarity to the family 10 glycoside hydrolases. The purified recombinant enzyme, XynA expressed in E. coli exhibited optimum activity at 100 degrees C and pH 6.0, and was extremely thermostable at 90 degrees C. The enzyme showed high specificity toward different xylans and xylooligosaccharides.
Im Bereich der medizinischen Diagnostik spielen DNA-Chips eine immer wichtigere Rolle. Dabei werden Glas- oder Silikon-Oberflächen mit Tausenden von einzelsträngigen DNA-Fragmenten, sog. Sonden, bestückt, die mit den passenden DNA-Fragmenten in der zugefügten Patientenprobe verschmelzen. Die Auswertung solcher Messungen liefert die Diagnose für Krankheiten wie z.B. Krebs, Alzheimer oder für den Nachweis pathogener Erreger. Durch fortschreitende Miniaturisierung dieser Meßsysteme können bis zu 40.000 Genfragmente des Menschen in einer einzigen Messung analysiert werden. Neben den DNA-Fragmenten können Bio-Chips auch für andere biologische Komponenten wie Antikörper und Proteine eingesetzt werden, wobei bei letzteren neben der Bindung auch die Aktivität ein wichtiger Diagnoseparamter ist. Am Fraunhofer-Institut für medizinische Technik und am Lehrstuhl für Analytische Biochemie der Universität Potsdam wurden im Rahmen einer Doktorarbeit Methoden entwickelt, die es ermöglichen auf nukleinsäuremodifizierten Sensoroberflächen die Aktivität von Proteinen zu messen. Es wurden Nukleinsäuren auf Oberflächen optischer Sensoren verankert. Diese fungierten als Rezeptor für die Proteine sowie auch als Substrat für Restriktionsenzyme, die Nukleinsäuren schneiden und Polymerasen, die Nukleinsäuren synthetisieren und verlängern können. Seine Anwendung fand diese Messmethode in der Messung der Aktivität des Proteins Telomerase, das in 90% aller Tumore erhöhte Aktivität gegenüber gesunden Zellen aufweist. Die Vorteile dieses neuen Assays gegenüber älteren Methoden liegt im Verzicht auf radioaktiv-markierten Komponenten und einer deutlich verkürzten Analysezeit. Die Arbeit schliesst mit einem funktionsfähigen Nachweis der Telomeraseaktivität im Zellextrakt von gesunden und kranken Zellen. Der direkte Einfluß von Hemmstoffen auf die Aktivität konnte sichtbar gemacht werden, und steht daher bei der Entwicklung neuer Tumor-Diagnostika und Therapeutika zur Verfügung.
Electro-chemical signal transduction is the basis of communication between n eurons and their target cells. An important group of neuroactive substances that are released by action potentials from neurons are the biogenic amines. These a re small organic molecules that bind to specific receptors located in the target cell membrane. Once activated these receptors cause changes in the intracellula r concentration of second messengers, i.e. cyclic nucleotides, phosphoinositides , or Ca2+, leading to slow but long-lasting cellular responses. Biochemical, pha rmacological, physiological, and molecular biological approaches have unequivoca lly shown that biogenic amines are important regulators of cellular function in both vertebrates and invertebrates. In this review, we will concentrate on the p roperties of two biogenic amines and their receptors that were originally identi fied in invertebrates: tyramine and octopamine.
The development of fast and reliable biochemical tools for on-site screening in environmental analysis was the main target of the present work. Due to various hazardous effects such as endocrine disruption and toxicity phenolic compounds are key analytes in environmental analysis and thus were chosen as model analytes. Three different methods were developed: For the enzymatic detection of phenols in environmental samples an enzyme-based biosensor was developed. In contrast to reported work using tyrosinase or peroxidases, we developed a biosensor based on glucose dehydrogenase as biorecognition element. This biosensor was devoted for an application in a laboratory flow system as well as in a portable device for on-site measurements. This enzymatic detection is applicable only for a limited number of phenols due to substrate specificity of the enzyme. For other relevant compounds based on a phenolic structure (i.e. nitrophenol, alkylphenols and alkylphenol ethoxylates) immunological methods had to be developed. The electrochemical GDH-biosensor was used as the label detector in these immunoassays. Two heterogeneous immunoassays were developed where ßGal was used as the label. An electrochemical method for the determination of the marker enzyme activity was processed. The separation step was realized with protein A/G columns (laboratory flow system) or by direct immobilization of the antibodies in small disposable capillaries (on-site analysis). All methods were targeted on the contemporary analysis of small numbers of samples.
Dopamine is found in many invertebrate organisms, including insects, however, the mechanisms through which this amine operates remain unclear. We have expressed two dopamine receptors cloned from honey bee (AmDOP1 and AmDOP2) in insect cells (Spodoptera frugiperda), and compared their pharmacology directly using production of cAMP as a functional assay. In each assay, AmDOP1 receptors required lower concentrations of dopamine and 6,7-ADTN for maximal activation than AmDOP2 receptors. Conversely, butaclamol and cis(Z)-flupentixol were more potent at blocking the cAMP response mediated through AmDOP2 than AmDOP1 receptors. Expression of AmDOP1, but not AmDOP2, receptors significantly increased levels of cAMP even in the absence of ligand. This constitutive activity was blocked by cis(Z)-flupentixol. This work provides the first evidence of a constitutively activated dopamine receptor in invertebrates and suggests that although AmDOP1 and AmDOP2 share much less homology than their vertebrate counterparts, they display a number of functional parallels with the mammalian D1-like dopamine receptors.