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

Oribatid communities and heavy metal bioaccumulation in selected species associated with lichens in a heavily contaminated habitat

The study examines oribatid communities and heavy metal bioaccumulation in selected species associated with different microhabitats of a post-smelting dump, i.e. three lichen species of Cladonia with various growth forms and the slag substrate. The abundance of oribatids collected from the substrate was significantly lower than observed in lichen thalli. The morphology and chemical properties of lichens, and to some extent varying concentrations of heavy metals in thalli, are probably responsible for significant differences in oribatid communities inhabiting different Cladonia species. Some oribatids demonstrate the ability to accumulate zinc and cadmium with unusual efficiency, whereas lead is the most effectively regulated element by all species. A positive correlation was found between Zn content in all studied oribatids and their microhabitats. Oribatids exploring different food resources, i.e. fungivorous and non-fungivorous grazers, show considerable differences in bioconcentrations of certain elements

Degradation, Bioactivity, and Osteogenic Potential of Composites Made of PLGA and Two Different Sol–Gel Bioactive Glasses

We have developed poly(l-lactide-co-glycolide) (PLGA) based composites using sol–gel derived bioactive glasses (S-BG), previously described by our group, as composite components. Two different composite types were manufactured that contained either S2—high content silica S-BG, or A2—high content lime S-BG. The composites were evaluated in the form of sheets and 3D scaffolds. Sheets containing 12, 21, and 33 vol.% of each bioactive glass were characterized for mechanical properties, wettability, hydrolytic degradation, and surface bioactivity. Sheets containing A2 S-BG rapidly formed a hydroxyapatite surface layer after incubation in simulated body fluid. The incorporation of either S-BG increased the tensile strength and Young’s modulus of the composites and tailored their degradation rates compared to starting compounds. Sheets and 3D scaffolds were evaluated for their ability to support growth of human bone marrow cells (BMC) and MG-63 cells, respectively. Cells were grown in non-differentiating, osteogenic or osteoclast-inducing conditions. Osteogenesis was induced with either recombinant human BMP-2 or dexamethasone, and osteoclast formation with M-CSF. BMC viability was lower at higher S-BG content, though specific ALP/cell was significantly higher on PLGA/A2-33 composites. Composites containing S2 S-BG enhanced calcification of extracellular matrix by BMC, whereas incorporation of A2 S-BG in the composites promoted osteoclast formation from BMC. MG-63 osteoblast-like cells seeded in porous scaffolds containing S2 maintained viability and secreted collagen and calcium throughout the scaffolds. Overall, the presented data show functional versatility of the composites studied and indicate their potential to design a wide variety of implant materials differing in physico-chemical properties and biological applications. We propose these sol–gel derived bioactive glass–PLGA composites may prove excellent potential orthopedic and dental biomaterials supporting bone formation and remodeling

Photosynthetic growth despite a broken Q-cycle

Central in respiration or photosynthesis, the cytochrome bc1 and b6f complexes are regarded as functionally similar quinol oxidoreductases. They both catalyse a redox loop, the Q-cycle, which couples electron and proton transfer. This loop involves a bifurcated electron transfer step considered as being mechanistically mandatory, making the Q-cycle indispensable for growth. Attempts to falsify this paradigm in the case of cytochrome bc1 have failed. The rapid proteolytic degradation of b6f complexes bearing mutations aimed at hindering the Q-cycle has precluded so far the experimental assessment of this model in the photosynthetic chain. Here we combine mutations in Chlamydomonas that inactivate the redox loop but preserve high accumulation levels of b6f complexes. The oxidoreductase activity of these crippled complexes is sufficient to sustain photosynthetic growth, which demonstrates that the Q-cycle is dispensable for oxygenic photosynthesis

Cryo-EM structure of the spinach cytochrome b6 f complex at 3.6 Å resolution.

The cytochrome b6 f (cytb6 f ) complex has a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and converting solar energy into a transmembrane proton gradient for ATP synthesis1-3. Electron transfer within cytb6 f occurs via the quinol (Q) cycle, which catalyses the oxidation of plastoquinol (PQH2) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation2. In higher plants, cytb6 f also acts as a redox-sensing hub, pivotal to the regulation of light harvesting and cyclic electron transfer that protect against metabolic and environmental stresses3. Here we present a 3.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the dimeric cytb6 f complex from spinach, which reveals the structural basis for operation of the Q cycle and its redox-sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is located in one cytb6 f monomer near the PQ oxidation site (Qp) adjacent to haem bp and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Qp site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Qn) on the PQ1 side and committing the electron transfer network to turnover at the occupied Qn site in the neighbouring monomer. A conformational switch involving the haem cn propionate promotes two-electron, two-proton reduction at the Qn site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Qp and Qn sites in opposite monomers during the Q cycle. The spinach cytb6 f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis

Unveiling novel genes upregulated by both rhBMP2 and rhBMP7 during early osteoblastic transdifferentiation of C2C12 cells

<p>Abstract</p> <p>Findings</p> <p>We set out to analyse the gene expression profile of pre-osteoblastic C2C12 cells during osteodifferentiation induced by both rhBMP2 and rhBMP7 using DNA microarrays. Induced and repressed genes were intercepted, resulting in 1,318 induced genes and 704 repressed genes by both rhBMP2 and rhBMP7. We selected and validated, by RT-qPCR, 24 genes which were upregulated by rhBMP2 and rhBMP7; of these, 13 are related to transcription (<it>Runx2, Dlx1, Dlx2, Dlx5, Id1, Id2, Id3, Fkhr1, Osx, Hoxc8, Glis1, Glis3 </it>and <it>Cfdp1</it>), four are associated with cell signalling pathways (<it>Lrp6, Dvl1, Ecsit </it>and <it>PKCδ</it>) and seven are associated with the extracellular matrix (<it>Ltbp2, Grn, Postn, Plod1, BMP1, Htra1 </it>and <it>IGFBP-rP10</it>). The novel identified genes include: <it>Hoxc8, Glis1, Glis3, Ecsit, PKCδ, LrP6, Dvl1, Grn, BMP1, Ltbp2, Plod1, Htra1 </it>and <it>IGFBP-rP10</it>.</p> <p>Background</p> <p>BMPs (bone morphogenetic proteins) are members of the TGFβ (transforming growth factor-β) super-family of proteins, which regulate growth and differentiation of different cell types in various tissues, and play a critical role in the differentiation of mesenchymal cells into osteoblasts. In particular, rhBMP2 and rhBMP7 promote osteoinduction <it>in vitro </it>and <it>in vivo</it>, and both proteins are therapeutically applied in orthopaedics and dentistry.</p> <p>Conclusion</p> <p>Using DNA microarrays and RT-qPCR, we identified both previously known and novel genes which are upregulated by rhBMP2 and rhBMP7 during the onset of osteoblastic transdifferentiation of pre-myoblastic C2C12 cells. Subsequent studies of these genes in C2C12 and mesenchymal or pre-osteoblastic cells should reveal more details about their role during this type of cellular differentiation induced by BMP2 or BMP7. These studies are relevant to better understanding the molecular mechanisms underlying osteoblastic differentiation and bone repair.</p

Cladonia diversa [Cladoniaceae, lichenized Ascomycota] - overlooked lichen in Poland

This is the first report on Cladonia diversa Asperges in Poland. Till the present this species was overlooked and usually included under the name C. coccifera. The study is based on revision of over a thousand of specimens of the genus Cladonia section Cocciferae deposited in Polish herbaria. The morphology, chemistry, habitat requirements and known distribution of the lichen in Poland are discussed. A taxonomical remarks concerning the species are also provided

Cladonia metacorallifera, nowy porost dla Karpat Wschodnich

A rare lichen Cladonia metacorallifera was found in the Bieszczady Mts. and this is the first report of the species from the Eastern Carpathians. In Poland, its occurrence is probably limited to small populations scattered in high mountain situations. The data of distribution of the species in Poland, ecological characterization of the new locality and some taxonomical remarks concerning the recorded specimen, as well as a photograph of its habit are provided.Cladonia metacorallifera Asahina jest kieliszkowato zakończonym porostem należącym do sekcji Cocciferae (Delise) A. Evans. Duża zmienność morfologiczna taksonów z tej sekcji wciąż stwarza problemy taksonomiczne i utrudnia prawidłową identyfikację gatunków (por. Stenroos 1989a; Osyczka 2009). Cechą charakterystyczną C. metacorallifera są liczne łuseczki na ogół gęsto rozmieszczone na całej długości podecjów oraz czerniejące fragmenty plechy widoczne w miejscach pozbawionych kory i łuseczek. Cladonia metacorallifera wytwarza kwas usninowy, didymowy oraz skwamatowy (lub tamnoliowy). Ta właściwość chemiczna jest ważną cechą diagnostyczną gatunku. Istotne cechy taksonomiczne odróżniające C. metacorallifera od morfologicznie podobnych taksonów z sekcji Cocciferae (np. C. coccifera (L.) Willd., C. diversa Asperges lub C. borealis S. Stenroos) zawarte zostały w kilku publikacjach (np. Stenroos 1989b; zobacz również: Kowalewska, Kukwa 2004; Osyczka et al. 2006). W Polsce po raz pierwszy C. metacorallifera znaleziono na jednym stanowisku w masywie Śnieżki w Karkonoszach (Kowalewska, Kukwa 2004), następnie podano go z dwóch stanowisk w polskiej części Tatr Wysokich (Osyczka et al. 2006). W wyniku rewizji materiałów zielnikowych porostów należących do C. coccifera s.l., zdeponowanych w zielniku lichenologicznym Uniwersytetu Pedagogicznego w Krakowie (KRAP-L), stwierdzono występowanie C. metacorallifera również w polskiej części Bieszczadów. Jest to pierwsze doniesienie o występowaniu tego gatunku w Karpatach Wschodnich. Nowe stanowisko C. metacorallifera znajduje się w granicach Bieszczadzkiego Parku Narodowego, na zachodnim zboczu masywu Rozsypańca, na wysokości 1153 m n.p.m., ponad górną granicą lasu, w obrębie rumoszu skalnego. Stanowisko to charakteryzuje się wysokim nasłonecznieniem i ekspozycją południowo-zachodnią. Porost ten rósł wśród mchu na humusie (pH = 4.65). Występowanie C. metacorallifera w Polsce prawdopodobnie ograniczone jest do niewielkich populacji rozproszonych w obszarach wysokogórskich wzdłuż południowej granicy kraju