Fusing two cytochromes b of Rhodobacter capsulatus cytochrome bc1 using various linkers defines a set of protein templates for asymmetric mutagenesis

Cytochrome bc1 (mitochondrial complex III), one of the key enzymes of biological energy conversion, is a functional homodimer in which each monomer contains three catalytic subunits: cytochrome c1, the iron–sulfur subunit and cytochrome b. The latter is composed of eight transmembrane α-helices which, in duplicate, form a hydrophobic core of a dimer. We show that two cytochromes b can be fused into one 16-helical subunit using a number of different peptide linkers that vary in length but all connect the C-terminus of one cytochrome with the N-terminus of the other. The fusion proteins replace two cytochromes b in the dimer defining a set of available protein templates for introducing mutations that allow breaking symmetry of a dimer. A more detailed comparison of the form with the shortest, 3 amino acid, linker to the form with 12 amino acid linker established that both forms display similar level of structural plasticity to accommodate several, but not all, asymmetric patterns of mutations that knock out individual segments of cofactor chains. While the system based on a fused gene does not allow for the assessments of the functionality of electron-transfer paths in vivo, the family of proteins with fused cytochrome b offers attractive model for detailed investigations of molecular mechanism of catalysis at in vitro/reconstitution level

Catalytically-relevant electron transfer between two hemes b_{L} in the hybrid cytochrome bc_{1}-like complex containing a fusion of Rhodobacter sphaeroides and capsulatus cytochromes b

AbstractTo address mechanistic questions about the functioning of dimeric cytochrome bc1 new genetic approaches have recently been developed. They were specifically designed to enable construction of asymmetrically-mutated variants suitable for functional studies. One approach exploited a fusion of two cytochromes b that replaced the separate subunits in the dimer. The fusion protein, built from two copies of the same cytochrome b of purple bacterium Rhodobacter capsulatus, served as a template to create a series of asymmetrically-mutated cytochrome bc1-like complexes (B–B) which, through kinetic studies, disclosed several important principles of dimer engineering. Here, we report on construction of another fusion protein complex that adds a new tool to investigate dimeric function of the enzyme through the asymmetrically mutated forms of the protein. This complex (BS–B) contains a hybrid protein that combines two different cytochromes b: one coming from R. capsulatus and the other — from a closely related species, R. sphaeroides. With this new fusion we addressed a still controversial issue of electron transfer between the two hemes bL in the core of dimer. Kinetic data obtained with a series of BS–B variants provided new evidence confirming the previously reported observations that electron transfer between those two hemes occurs on a millisecond timescale, thus is a catalytically-relevant event. Both types of the fusion complexes (B–B and BS–B) consistently implicate that the heme-bL–bL bridge forms an electronic connection available for inter-monomer electron transfer in cytochrome bc1

Najnowsze techniki biologii molekularnej w profilaktyce przewlekłych zakażeń wirusowych

Postęp medycyny i związany z tym postęp technik biologii molekularnej stworzyły daleko idące możliwości kontrolowania i zapobiegania powikłaniom w przebiegu przewlekłych chorób wirusowych, takich jak zakażenia wirusowe wątroby oraz wirusem ludzkiego niedoboru odporności (HIV) Jeszcze kilkanaście lat wstecz niemożliwe było dokładne kontrolowanie poziomu wiremii oraz serokonwersji wirusa HBV podczas zakażeń. Od kilku lat istnieją już w pełni wystandaryzowane i wykorzystywane w diagnostyce metody biologii molekularnej, potwierdzające zakażenia HCV, HBV oraz HIV nawet podczas nieobecności przeciwciał we krwi pacjenta, wykrywające bezpośrednio obecność genomu wirusa. Coraz częściej wykorzystuje się również techniki pozwalające monitorowanie stanu zakażenia oraz szybkie reagowanie na zmiany zachodzące w organizmie zakażonego. Do wyżej wymienionych technik należy przede wszystkim odmiana reakcji PCR, czyli ilościowy PCR w czasie rzeczywistym (Real-Time PCR) czy sekwencjonowanie kwasów nukleinowych. Każda z tych technik pozwala nie tylko na wykrycie zakażenia na poziomie dotychczas nieosiągalnym, lecz również na jego skuteczne obserwowanie i skuteczne leczenie. Przy pomocy reakcji Real-Time PCR możliwe jest określenie ilości cząstek wirusa w badanej jednostce objętości krwi. Technika sekwencjonowania pozwala na dokładne określenie typu wirusa, a co za tym idzie zjadliwości. Wszystkie te techniki wzięte razem pozwalają na dokładne określenie ilości wirusów we krwi oraz na skuteczną walkę z nimi.Progress either in medicine or molecular biology technics induces perspective possibilities of control and complication prevention in case of chronic viral diseases such as viral hepatitis and HIV infection. Accurate control of HBV viremia and seroconvertion level during infection was impossible several years ago. For the last couple of years molecular biology methods for HCV, HBV and HIV infection have been generally accepted and used for diagnosis, even without antibodies detectable in blood. Infection monitoring technics, such as Real-Time PCR, nucleic acid sequencing have been usefull and fl ow cytofl uorometry, have been useful in rapid reacting on changes in the infected patient state. Each of the mentioned technics provides either the infection detection on the level unavailable in the past, or efficient observation and, consequently, proper treatment of the infection. Real-Time PCR allows to defi ne the quantity of viral molecules in the volume of tested blood sample. Sequentioning technic provides viruses type ane virulence defi nition. The combination of all the other hand the cytofluorometry technic allows to defi ne precisely the quantity of viral proteins presented on the infected cells and detect other blood proteins expression. The combination of all those technics provides accurate defi nition of virus ammount and efficient treatment

Depressive and anxiety disorders in the cardiological conditions: psychological interventions

Cardiovascular disease (CVD) remains a leading cause of mortality worldwide, despite diagnostic and pharmacological advances. These adverse effects lead to the search for new etiological factors and effective ways to prevent, treat and rehabilitate CVD. The impact of psychological factors on the development of CVD has gained scientific recognition. Mental and mood disorders, such as depressive disorders, anxiety disorders, and post-traumatic stress disorder (PTSD), can contribute to CVD and significantly affect the quality of life and prognosis of a CVD patient, often contributing to premature death. Therefore, it is essential to study the socio-demographic and psychosocial factors that are most likely to support pro-health behaviour and identify effective psychotherapeutic interventions for CVD patients. These findings result in new interventions, such as meta-cognitive therapy and mindfulness-based training, which promote healthy behaviours and improve clinical conditions and prognosis in CVD patients

Enzymatic Activities of Isolated Cytochrome bc1-like Complexes Containing Fused Cytochrome b Subunits with Asymmetrically Inactivated Segments of Electron Transfer Chains

Homodimeric structure of cytochrome bc_1, a common component of biological energy conversion systems, builds in four catalytic quinone oxidation/reduction sites and four chains of cofactors (branches) that, connected by a centrally located bridge, form a symmetric H-shaped electron transfer system. The mechanism of operation of this complex system is under constant debate. Here, we report on isolation and enzymatic examination of cytochrome bc1-like complexes containing fused cytochrome b subunits in which asymmetrically introduced mutations inactivated individual branches in various combinations. The structural asymmetry of those forms was confirmed spectroscopically. All the asymmetric forms corresponding to cytochrome bc_1 with partial or full inactivation of one monomer retain high enzymatic activity but at the same time show a decrease in the maximum turnover rate by a factor close to 2. This strongly supports the model assuming independent operation of monomers. The cross-inactivated form corresponding to cytochrome bc_1 with disabled complementary parts of each monomer retains the enzymatic activity at the level that, for the first time on isolated from membranes and purified to homogeneity preparations, demonstrates that intermonomer electron transfer through the bridge effectively sustains the enzymatic turnover. The results fully support the concept that electrons freely distribute between the four catalytic sites of a dimer and that any path connecting the catalytic sites on the opposite sides of the membrane is enzymatically competent. The possibility to examine enzymatic properties of isolated forms of asymmetric complexes constructed using the cytochrome b fusion system extends the array of tools available for investigating the engineering of dimeric cytochrome bc1 from the mechanistic and physiological perspectives

Troska o zdrowie w aspekcie społecznym. Cz. 2

Praca recenzowana / peer-reviewed pape

Zagrożenia zdrowotne wśród dzieci i młodzieży. T. 3

Praca recenzowana / Peer-reviewed pape

Inter-Church Organizations in the Register of Churches and Denominations

Churches and other religious denominations can establish inter-church organisations that have a legal personality by virtue of the Freedom of Conscience and Confession Act of 17 May 1989. These inter-church organisations are subject to the rules of the Act regarding the registration of churches and other religious denominations. As of 15 February 2011, the B section of the Register of Churches and Denominations contains entries for five inter-church organisations: Polish Ecumenical Council, Polish Bible Society, Council of Evangelical Churches, Budhist Union of Poland and Evangelical Alliance in the Republic of Poland. There are two joint commissions that include representatives of churches that are associated in inter-church organisations and the government: Joint Commission of Polish Government Representatives and Polish Ecumenical Council as well as Joint Commission of Polish Government Representatives and Evangelical Alliance in the Republic of Poland

Business activity in religious cemeteries done by judicial persons of the Catholic Church

Prawodawstwo Rzeczypospolitej Polskiej zapewnia kościołom i innym związkom wyznaniowym prawo posiadania, zarządzania oraz zakładania i poszerzania cmentarzy grzebalnych przy zachowaniu przepisów dotyczących cmentarzy i chowania zmarłych. Miejscom tym, co do zasady, Państwo gwarantuje nienaruszalność. Jednakże z decyzji Prezesa Urzędu Ochrony Konkurencji i Konsumentów dotyczących kościelnych osób prawnych zarządzających cmentarzami, które to decyzje były potwierdzone przez sądownictwo antymonopolowe, wyraźnie wynika, że do cmentarzy wyznaniowych odnoszone są wszystkie obowiązujące przepisy prawa, w tym ustawy z dnia 16 lutego 2007 r. o ochronie konkurencji i konsumentów. Ze wspomnianą linią orzeczniczą nie zgadzają się przedstawiciele Kościoła Katolickiego, którzy poruszają tę sprawę m.in. na forum Komisji Wspólnej Przedstawicieli Rządu Rzeczypospolitej Polskiej i Konferencji Episkopatu Polski. Kwestia prowadzenia działalności gospodarczej na cmentarzach wyznaniowych wymaga szerszych wyjaśnień, szczególnie z uwagi na fakt różnego rozumienia definicji przedsiębiorcy na gruncie ustawy o ochronie konkurencji i konsumentów.Polish legislation gives churches and other religious associations the right of ownership, management and establishment of cemeteries in compliance with the relevant legal provisions on cemeteries and burial. In principle, the state guarantees inviolability of such sites. However, judging by the decisions of the President of the Office of Competition and Consumer Protection concerning judicial persons managing cemeteries, such decisions having been upheld by antitrust jurisdiction, it transpires that religious cemeteries are affected by all applicable laws, including the Act of 16 February 2007 on competition and consumer protection. This trend in the existing case-law has been opposed by the Catholic Church representatives who raise this issue, for example, in the Joint Commission of the Representatives of the Polish Government and the Polish Episcopal Conference. The issue of conducting business activity in religious burial grounds calls for a broader elucidation, especially given diverse interpretations of the definition of an entrepreneur under the Act on competition and consumer protection