An NAD(P)(+)-dependent secondary-alcohol dehydrogenase of Alcaligenes eutrophus: Purification, characterization and its application for the production of chiral alcohols

A soil micro-organism identified as Alcaligenes eutrophus capable of utilizing nerolidol, a sesquiterpene alcohol as the sole source of carbon, contains an inducible NAD(P)(+)-linked secondary-alcohol dehydrogenase (SADH), The enzyme was purified 252-fold from crude cell-free extract by a combination of salt precipitation, ion-exchange and affinity-matrix chromatography, Native and SDS/PAGE PAGE of the purified enzyme showed a single protein band and the enzyme appears to be a homotetramer having an apparent molecular mass of 139 kDa comprising four identical subunits of 38.5 kDa, The isoelectric point (pi) of SADH was determined to be 6.2, Depending on pH of the reaction media, the enzyme carried out both oxidation and reductions of various terpenoids and steroids, At pH 5.5, the enzyme catalysed the stereospecific reduction of prochiral ketones to optically active (S)-alcohols and the oxidation reaction was predominated over the former at pH 9.5, NADP(+) and NADPH were respectively preferred over NAD(+) and NADH for oxidation and reduction reactions, The K-m values for testosterone, NADP(+) and NAD(+) were 11.8, 55.6, and 122 mu M respectively, Neither enzyme was significantly inhibited by metal-binding agents, but some thiol-blocking compounds inhibited it, SADH tolerates moderate concentrations of water-miscible organic solvents such as ethanol, methanol, acetone and dioxan, Some of the properties of this enzyme were found to be significantly different from those thus far described.

Interaction Signatures Stabilizing the NAD(P)-Binding Rossmann Fold: A Structure Network Approach

The fidelity of the folding pathways being encoded in the amino acid sequence is met with challenge in instances where proteins with no sequence homology, performing different functions and no apparent evolutionary linkage, adopt a similar fold. The problem stated otherwise is that a limited fold space is available to a repertoire of diverse sequences. The key question is what factors lead to the formation of a fold from diverse sequences. Here, with the NAD(P)-binding Rossmann fold domains as a case study and using the concepts of network theory, we have unveiled the consensus structural features that drive the formation of this fold. We have proposed a graph theoretic formalism to capture the structural details in terms of the conserved atomic interactions in global milieu, and hence extract the essential topological features from diverse sequences. A unified mathematical representation of the different structures together with a judicious concoction of several network parameters enabled us to probe into the structural features driving the adoption of the NAD(P)-binding Rossmann fold. The atomic interactions at key positions seem to be better conserved in proteins, as compared to the residues participating in these interactions. We propose a ``spatial motif'' and several ``fold specific hot spots'' that form the signature structural blueprints of the NAD(P)-binding Rossmann fold domain. Excellent agreement of our data with previous experimental and theoretical studies validates the robustness and validity of the approach. Additionally, comparison of our results with statistical coupling analysis (SCA) provides further support. The methodology proposed here is general and can be applied to similar problems of interest.

Conserved water-mediated recognition and dynamics of NAD(+) (carboxamide group) to hIMPDH enzyme: water mimic approach toward the design of isoform-selective inhibitor

Inosine monophosphate dehydrogenase (IMPDH) enzyme involves in GMP biosynthesis pathway. Type I hIMPDH is expressed at lower levels in all cells, whereas type II is especially observed in acute myelogenous leukemia, chronic myelogenous leukemia cancer cells, and 10 ns simulation of the IMP-NAD(+) complex structures (PDB ID. 1B3O and 1JCN) have revealed the presence of a few conserved hydrophilic centers near carboxamide group of NAD(+). Three conserved water molecules (W1, W, and W1 `) in di-nucleotide binding pocket of enzyme have played a significant role in the recognition of carboxamide group (of NAD(+)) to D274 and H93 residues. Based on H-bonding interaction of conserved hydrophilic (water molecular) centers within IMP-NAD(+)-enzyme complexes and their recognition to NAD(+), some covalent modification at carboxamide group of di-nucleotide (NAD(+)) has been made by substituting the -CONH(2)group by -CONHNH2 (carboxyl hydrazide group) using water mimic inhibitor design protocol. The modeled structure of modified ligand may, though, be useful for the development of antileukemic agent or it could be act as better inhibitor for hIMPDH-II.

Modulação das vias de sinalização intracelulares pelo sistema NAD(P)H oxidase em melanoma humano

Evidências têm mostrado que as espécies reativas de oxigênio (ROS) geradas pela NAD(P)H oxidase são importantes moduladores de diversas funções celulares como migração, crescimento, proliferação e sobrevivência. Estudos recentes demonstraram o envolvimento da atividade da NAD(P)H oxidase no crescimento e sobrevivência de células de melanoma. Neste trabalho, investigamos o efeito da inibição da NAD(P)H oxidase por difenileneiodônio (DPI) sobre o crescimento das células de melanoma humano MV3 e observamos que este composto reduziu o crescimento destas células em aproximadamente 50%. A inibição da NAD(P)H oxidase induziu mudanças no formato celular, com arredondamento, diminuição do espraiamento e descolamento celular. Esta redução foi acompanhada por um rearranjo do citoesqueleto de actina, diminuição da fosforilação no resíduo Tyr397 da quinase de adesão focal (FAK) e redução na associação de FAK com actina e com a tirosina quinase c-Src. Isto indica que a inibição da geração de ROS está modulando negativamente vias de sinalização ativadas por integrinas, o que freqüentemente conduz a um tipo particular de morte celular conhecida por anoikis. Comprovando a ocorrência deste fenômeno, observamos que a inibição da atividade da NAD(P)H oxidase aumentou a apoptose das células de melanoma e induziu a ativação da caspase-3. Nossos resultados mostram ainda que a inibição da viabilidade celular por DPI foi revertida com o pré-tratamento das células MV3 com um inibidor de tirosina fosfatases (ortovanadato de sódio). Em resumo, este estudo mostra que a geração de ROS por NAD(P)H oxidase está envolvida nos mecanismos de sobrevivência em células de melanoma, uma vez que afetam as vias de sinalização dependentes de FAK-Src, através da inibição da atividade de proteína tirosina fosfatases.

Przegląd dotychczasowych badań nad związkami cech osobowości nauczycieli i uczniów z procesem nauczania / uczenia się

Poniższy artykuł oparty jest na dwóch pozycjach: Psychologia dla nauczycieli Davida Fontana oraz Individual differences in second language learning Petera Skehana i stanowi podstawę teoretyczną dla jednego z tandemów badawczych, w projekcie dotyczącym autonomizacji procesu uczenia się / nauczania. Techniką wykorzystaną w doświadczeniu jest rozwinięta forma znanej w metodyce techniki role play do postaci role playing game. (gry fabularne). Celem doświadczenia jest sprawdzenie skuteczności tejże techniki w kształtowaniu autokontroli i autokorekty, jako elementów Indywidualnej Kompetencji Komunikacyjnej w powiązaniu z cechami osobowości.

Charakter i cele Polskiego Towarzystwa Badania Gier oraz perspektywy badań nad grami w Polsce

Wartość poznawcza zamieszczonych w tomie artykułów polega na oglądzie gier jako nowego zjawiska w kulturze i na staraniach o zrozumienie ich atrakcyjności oraz na wysiłku poszukiwania ich pozytywnej roli wobec użytkowników. Autorzy artykułów podejmują rozmaite tematy związane w pierwszym rzędzie z grami komputerowymi i fabularnymi. Przedstawiają ich nowe cechy, oświetlają je z różnych punktów widzenia: medialnego, socjologicznego, psychologicznego, etycznego. I co równie interesujące, dostrzegają wiele powiązań z dobrze zakorzenionymi w kulturze dziedzinami, na przykład z literaturą czy filozofią. Czytelnik zyskuje wgląd w dzisiejszy stan badań nad grami. Nie tylko otrzymuje obraz wstępnych refleksji, ale ma szansę zetknąć się z postawą badawczą w stosunku do gier. (z recenzji wydawniczej prof. dr hab. Maryli Hopfinger)

Jakość opieki nad dzieckiem w rodzinie a jego emocjonalno-społeczne funkcjonowanie

Wydział Studiów Edukacyjnych: Zakład Teoretycznych Podstaw Edukacji

Antyk i współczesność: wokół pragmatyki tekstu antycznego (1Tm 1, 17). Z badań nad tradycją antyczną na Górnym Śląsku

The article presents an interpretation of the Soli Deo honor et gloria inscription from the fronton of the metropolitan cathedral of Christ the King in Katowice. The interpretation of the text depends on whether the word soli is taken as solely attributive or also as predicative. Given both the ancient and the contemporary historical and cultural contexts the former appears more plausible.

Spór o wspólnotę w perspektywie badań nad miastem postindustrialnym

Wydział Nauk Społecznych: Instytut Kulturoznawstwa

W stronę uniwersytetu jako instytucji dobra wspólnego. Filozoficzne podstawy krytycznych badań nad szkolnictwem wyższym

Wydział Nauk Społecznych: Instytut Filozofii