Enzimspecifitás megváltoztatása in vitro evolúciós stratégiával = Alteration of enzyme specificity by a strategy based on in vitro evolution

A kutatás célja előző - hasonló című - pályázatunk, amelyben is vitro mesterséges evolúcióval előállítottunk egy megváltozott specifitású DNS-modifikációs metiláz enzimet, kiegészítése és lezárása volt. Elért eredményeink a következőkben foglalhatók össze: 1. Előállítottuk és részlegesen jellemeztük két újabb, szintén megváltozott specifitású mutánsát a korábban vizsgált M.SinI enzimnek. 2. Előállítottuk és részlegesen jellemeztük az M.SinI enzimnek és egyik megváltozott specifitású mutánsának az N-terminális régió első 65 aminosavától megfosztott, rövidített változatát, amelyek működőképesek voltak. 3. Újszerű, az irodalomban egyedülálló, kisérleti rendszert dolgoztunk ki az M.SinI enzim kívánt irányú specifitásváltozásának in vivo detektálására és jellemzésére. | This was the extension and continuation of the work done during our previous OTKA project (with the same title), in which we constructed, by using in vitro directed evolution techniques, a mutant DNA-modification enzyme with changed recognition specificity. The results of the present work could be summarized as follows: 1. By applying the same methods, we constructed two different new mutant variants of the previously studied M.SinI enzyme, with similarly changed recognition specificities, and partially characterized them. 2. We constructed and partially characterized truncated versions of the wild-type and one mutant variant of the M.SinI enzyme, without the N-terminal first 65 amino acids. These truncated enzymes preserved their enzymatic activity. 3. We developed an entirely new experimental system, for the in vivo detection and semiquantitative measurement of the changed recognition specificity of the studied M.SinI enzyme

Magnetic Cellular Nonlinear Network with Spin Wave Bus for Image Processing

We describe and analyze a cellular nonlinear network based on magnetic nanostructures for image processing. The network consists of magneto-electric cells integrated onto a common ferromagnetic film - spin wave bus. The magneto-electric cell is an artificial two-phase multiferroic structure comprising piezoelectric and ferromagnetic materials. A bit of information is assigned to the cell's magnetic polarization, which can be controlled by the applied voltage. The information exchange among the cells is via the spin waves propagating in the spin wave bus. Each cell changes its state as a combined effect of two: the magneto-electric coupling and the interaction with the spin waves. The distinct feature of the network with spin wave bus is the ability to control the inter-cell communication by an external global parameter - magnetic field. The latter makes possible to realize different image processing functions on the same template without rewiring or reconfiguration. We present the results of numerical simulations illustrating image filtering, erosion, dilation, horizontal and vertical line detection, inversion and edge detection accomplished on one template by the proper choice of the strength and direction of the external magnetic field. We also present numerical assets on the major network parameters such as cell density, power dissipation and functional throughput, and compare them with the parameters projected for other nano-architectures such as CMOL-CrossNet, Quantum Dot Cellular Automata, and Quantum Dot Image Processor. Potentially, the utilization of spin waves phenomena at the nanometer scale may provide a route to low-power consuming and functional logic circuits for special task data processing

Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway

In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a Kd of ca. 10−5 M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI's interaction with a partly-folded protein, and the first to analyze this folding catalyst's changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding – differential affinity, rapid ligand exchange and conformational flexibility

A Novel Cloning Template Designing Method by Using an Artificial Bee Colony Algorithm for Edge Detection of CNN Based Imaging Sensors

Cellular Neural Networks (CNNs) have been widely used recently in applications such as edge detection, noise reduction and object detection, which are among the main computer imaging processes. They can also be realized as hardware based imaging sensors. The fact that hardware CNN models produce robust and effective results has attracted the attention of researchers using these structures within image sensors. Realization of desired CNN behavior such as edge detection can be achieved by correctly setting a cloning template without changing the structure of the CNN. To achieve different behaviors effectively, designing a cloning template is one of the most important research topics in this field. In this study, the edge detecting process that is used as a preliminary process for segmentation, identification and coding applications is conducted by using CNN structures. In order to design the cloning template of goal-oriented CNN architecture, an Artificial Bee Colony (ABC) algorithm which is inspired from the foraging behavior of honeybees is used and the performance analysis of ABC for this application is examined with multiple runs. The CNN template generated by the ABC algorithm is tested by using artificial and real test images. The results are subjectively and quantitatively compared with well-known classical edge detection methods, and other CNN based edge detector cloning templates available in the imaging literature. The results show that the proposed method is more successful than other methods

Hepatocyte Nuclear Factor 4 Provokes Expression of Epithelial Marker Genes, Acting As a Morphogen in Dedifferentiated Hepatoma Cells

Abstract. We have recently shown that stable expression of an epitope-tagged cDNA of the hepatocyte- enriched transcription factor, hepatocyte nuclear factor (HNF)4, in dedifferentiated rat hepatoma H5 cells is sufficient to provoke reexpression of a set of hepatocyte marker genes. Here, we demonstrate that the effects of HNF4 expression extend to the reestablishment of differentiated epithelial cell morphology and simple epithelial polarity. The acquisition of epithelial morphology occurs in two steps. First, expression of HNF4 results in reexpression of cytokeratin proteins and partial reestablishment of E-cadherin production. Only the transfectants are competent to respond to the synthetic glucocorticoid dexamethasone, which induces the second step of morphogenesis, including formation of the junctional complex and expression of a polarized cell phenotype. Cell fusion experiments revealed that the transfectant cells, which show only partial restoration of E-cadherin expression, produce an extinguisher that is capable of acting in trans to downregulate the E-cadherin gene of well-differentiated hepatoma cells. Bypass of this repression by stable expression of E-cadherin in H5 cells is sufficient to establish some epithelial cell characteristics, implying that the morphogenic potential of HNF4 in hepatic cells acts via activation of the E-cadherin gene. Thus, HNF4 seems to integrate the genetic programs of liver-specific gene expression and epithelial morphogenesis

Protein disulphide isomerase-assisted functionalization of proteinaceous substrates

Protein disulphide isomerase (PDI) is an enzyme that catalyzes thiol-disulphide exchange reactions among a broad spectrum of substrates, including proteins and low-molecular thiols and disulphides. As the first protein-folding catalyst reported, the study of PDI has mainly involved the correct folding of several cysteine-containing proteins. Its application on the functionalization of protein-based materials has not been extensively reported. Herein, we review the applications of PDI on the modification of proteinaceous substrates and discuss its future potential. The mechanism involved in PDI functionalization of fibrous protein substrates is discussed in detail. These approaches allow innovative applications in textile dyeing and finishing, medical textiles, controlled drug delivery systems and hair or skin care products.We thank to FCT 'Fundacao para a Ciencia e Tecnologia' (scholarship SFRH/BD/38363/2007) for providing Margarida Fernandes the grant for PhD studies

Megismerhetők és megváltoztathatók-e génjeink? | Is It Possible to Know and Alter Our Genes?

A genetika divatban van. A világsajtó főcímekben tudósított az amerikai elnök és a brit miniszterelnök közös sajtóértekezletéről: Bill Clinton és Tony Blair 2000. június 26-án bejelentette a Humán Genom Program sikeres befejezését, azaz az emberi öröklési állomány teljes szerkezetének megismerését. Naponta értesülünk a hírekből egy-egy új betegségokozó gén megismeréséről, a génterápia sikereiről és kudarcairól vagy éppen a genetikailag módosított élelmiszerekkel kapcsolatos vitákról, viharokról. A tudományos fantasztikus filmeknek és regényeknek is kedvelt témája, hogyan próbálják a gonosz genetikusok manipulálni az embert és a világot. Elôadásom ezekről a kérdésekrôl fog szólni. Célom az, hogy segítsem a tájékozódást, az ítéletalkotást, és eloszlassak néhány közkeletű félreértést. | Pál Venetianer, biologist and member of HAS, presented genetics, one of the most popular branches of science. The history of genetics is the story of uninterrupted developments from the early days of the science of heredity through the discovery of the double helix, and to the newest developments of protein research, including a widening range of applications. A breakthrough was recently achieved when, as a result of the Human Genome Project, the nucleotide order of the DNA double helix was defined. This is the first step towards the understanding of this basic functionality of the human organism, as the functions – or meanings – of these sequences are yet hardly known. New prospects are opened for gene surgery, and, drawing on the experience gained in the world of plants and animals, new treatments may be developed to cure human illnesses. This is why gene technology is not heading toward a horrifying future – on the contrary: it opens up a range of opportunities which require the assertion of high responsibility on the part of the scientist