High-resolution cryo-EM structures of plant cytochrome b6fb_{6}f at work

Plants use solar energy to power cellular metabolism. The oxidation of plastoquinol and reduction of plastocyanin by cytochrome $b_{6}f$ (Cyt $b_{6}f$) is known as one of the key steps of photosynthesis, but the catalytic mechanism in the plastoquinone oxidation site ($Q_{p}$) remains elusive. Here, we describe two high-resolution cryo-EM structures of the spinach Cyt $b_{6}f$ homodimer with endogenous plastoquinones and in complex with plastocyanin. Three plastoquinones are visible and line up one after another head to tail near $Q_{p}$ in both monomers, indicating the existence of a channel in each monomer. Therefore, quinones appear to flow through Cyt $b_{6}f$ in one direction, transiently exposing the redox-active ring of quinone during catalysis. Our work proposes an unprecedented one-way traffic model that explains efficient quinol oxidation during photosynthesis and respiration. Structures of cytochrome $b_{6}f$ with and without plastocyanin imply a one-way traffic of quinones for efficient photosynthesis

How do xanthophylls protect lipid membranes from oxidative damage?

Here, we address the problem of the antioxidant activity of carotenoids in biomembranes. The activity of lutein and zeaxanthin in the quenching of singlet oxygen generated by photosensitization was monitored in lipid vesicles using a singlet oxygen-sensitive fluorescent probe and with the application of fluorescence lifetime imaging microscopy. The antioxidant activity of xanthophylls was interpreted on the basis of electron paramagnetic resonance oximetry results showing that xanthophylls constitute a barrier to the penetration of molecular oxygen into lipid membranes: to a greater extent in the 13-cis configuration than in all-trans. These results are discussed in relation to the trans-cis photoisomerization of xanthophylls observed in the human retina. It can be concluded that photoisomerization of xanthophylls is a regulatory mechanism that is important for both the modulation of light filtration through the macula and photoprotection by quenching singlet oxygen and creating a barrier to oxygen permeation to membranes

Codzienna praktyka kliniczna w ostrych zespołach wieńcowych bez uniesienia odcinka ST w szpitalach rejonowych - rejestr w Małopolsce

Wstęp: Ostre zespoły wieńcowe bez uniesień odcinka ST (NSTE ACS) rozpoznaje się u ponad połowy pacjentów przyjmowanych do szpitali z powodu ostrego zespołu wieńcowego (ACS). Ze względu na całodobową dostępność pracowni hemodynamicznych w Małopolsce każdy pacjent z NSTE ACS wysokiego ryzyka przyjęty do szpitala rejonowego może zostać przetransportowany do ośrodka kardiologii interwencyjnej. Celem badania była ocena częstości kierowania pacjentów z rozpoznaniem NSTE ACS na badanie diagnostyczne tętnic wieńcowych do ośrodków kardiologii interwencyjnej oraz charakterystyka demograficzna i zastosowana farmakoterapia w tej grupie. Materiał i metody: Korzystając z ankiet, zebrano dane dotyczące 2382 kolejnych pacjentów z rozpoznaniem ACS, przyjętych do szpitali rejonowych w Małopolsce w okresie od kwietnia 2002 do lutego 2003 r. U 1396 chorych potwierdzono ostatecznie przy wypisie rozpoznanie ostrego zespołu wieńcowego bez uniesienia odcinka ST. Wyniki: U 42% (n = 582) chorych z końcowym rozpoznaniem NSTE ACS stwierdzono podwyższone stężenie markerów martwicy mięśnia sercowego, takich jak troponina T/I lub CK-MB (CM+), natomiast u 58% (n = 814) pacjentów nie odnotowano podwyższonego stężenia markerów (CM-). W grupie CM+ zaobserwowano większą śmiertelność wewnątrzszpitalną niż w grupie CM- (3,3% vs. 0,4%; p = 0,0002). Tylko 17,7% pacjentów z całej grupy CM+ skierowano w trakcie hospitalizacji do ośrodka kardiologii interwencyjnej w celu wykonania koronarografii i ewentualnego leczenia inwazyjnego. Wraz ze wzrostem ryzyka określanego w skali TIMI Risk Score zwiększał się odsetek osób kierowanych do pracowni hemodynamiki (TIMI Risk Score 0-2 pkt: 14%; 3-4 pkt: 15%; 5-7 pkt: 22%; p = 0,02 dla 3-4 vs. 5-7 oraz p = 0,01 dla 0&#8211;2 vs. 5&#8211;7). Jednocześnie obserwowano wzrost śmiertelności wewnątrzszpitalnej w grupie pacjentów nieskierowanych na leczenie inwazyjne 0,8% vs. 1,9% vs. 3,5% (p = 0,02 dla 0-2 vs. 5-7) odpowiednio dla grup z ryzykiem wynoszącym w skali TIMI Risk Score 0-2 vs. 3-4 vs. 5-7. Pacjenci kierowani na leczenie inwazyjne znamiennie częściej otrzymywali tienopirydyny (68,3% vs. 44,5%; p < 0,0001), blokery receptora IIb/IIIa (1,5% vs. 0,3%; p = 0,04), heparyny (92,7% vs. 85%; p = 0,003) oraz ß-blokery (88,3% vs. 78,8%; p = 0,002). Wnioski: Pomimo 24-godzinnego dostępu do pracowni hemodynamiki tylko niewielki odsetek pacjentów z NSTE ACS jest kierowanych ze szpitali rejonowych. U pacjentów z grup wysokiego ryzyka według klasyfikacji TIMI (TIMI Risk Score 5&#8211;7 pkt, w tym z podwyższonymi stężeniami markerów martwicy mięśnia sercowego), niekierowanych na leczenie inwazyjne, pomimo stosowanej terapii farmakologicznej rokowanie nadal jest złe. (Folia Cardiol. 2005; 12: 21&#8211;31

Novel AlkB Dioxygenases—Alternative Models for In Silico and In Vivo Studies

Background: ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a direct, single-protein repair system, protecting cellular DNA and RNA against the cytotoxic and mutagenic activity of alkylating agents, chemicals significantly contributing to tumor formation and used in cancer therapy. In silico analysis and in vivo studies have shown the existence of AlkB homologs in almost all organisms. Nine AlkB homologs (ALKBH1–8 and FTO) have been identified in humans. High ALKBH levels have been found to encourage tumor development, questioning the use of alkylating agents in chemotherapy. The aim of this work was to assign biological significance to multiple AlkB homologs by characterizing their activity in the repair of nucleic acids in prokaryotes and their subcellular localization in eukaryotes. Methodology and Findings: Bioinformatic analysis of protein sequence databases identified 1943 AlkB sequences with eight new AlkB subfamilies. Since Cyanobacteria and Arabidopsis thaliana contain multiple AlkB homologs, they were selected as model organisms for in vivo research. Using E. coli alkB2 mutant and plasmids expressing cyanobacterial AlkBs, we studied the repair of methyl methanesulfonate (MMS) and chloroacetaldehyde (CAA) induced lesions in ssDNA, ssRNA, and genomic DNA. On the basis of GFP fusions, we investigated the subcellular localization of ALKBHs in A. thaliana and established its mostly nucleo-cytoplasmic distribution. Some of the ALKBH proteins were found to change their localization upon MMS treatment. Conclusions: Our in vivo studies showed highly specific activity of cyanobacterial AlkB proteins towards lesions and nucleic acid type. Subcellular localization and translocation of ALKBHs in A. thaliana indicates a possible role for these proteins in the repair of alkyl lesions. We hypothesize that the multiplicity of ALKBHs is due to their involvement in the metabolism of nucleo-protein complexes; we find their repair by ALKBH proteins to be economical and effective alternative to degradation and de novo synthesis

Analogs of Cinnamic Acid Benzyl Amide As Nonclassical Inhibitors of Activated JAK2 Kinase.

Scaffold-based analogs of cinnamic acid benzyl amide (CABA) exhibit pleiotropic effects in cancer cells, and their exact molecular mechanism of action is under investigation. The present study is part of our systemic analysis of interactions of CABA analogs with their molecular targets. These compounds were shown to inhibit Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) and JAK2/signal transducer and activator of transcription 5 (STAT5) signaling and thus are attractive scaffolds for anticancer drug design. To identify the potential mechanisms of action of this class of compounds, direct interactions of the selected CABA analogs with JAK2 kinase were examined. Inhibition of JAK2 enzymatic activity was assessed, and molecular modeling studies of selected compounds-(E)-2-cyano-N-[(S)-1-phenylethyl]-3-(pyridin-2-yl)acrylamide (WP1065), (E)-2-cyano-N-[(S)-1-phenylbutyl]- 3-(3-bromopyridin-2-yl)acrylamide (WP1130), and (E)-2-cyano-N-[(S)-1,4-diphenylbutyl]-3-(3-bromopyridin-2-yl)acrylamide (WP1702)-in the JAK2 kinase domain were used to support interpretation of the experimental data. Our results indicated that the tested CABA analogs are nonclassical inhibitors of activated (phosphorylated) JAK2, although markedly weaker than clinically tested ATP-competitive JAK2 inhibitors. Relatively small structural changes in the studied compounds affected interactions with JAK2, and their mode of action ranged from allosteric-noncompetitive to bisubstrate-competitive. These results demonstrated that direct inhibition of JAK2 enzymatic activity by the WP1065 (half-maximal inhibitory concentration [IC50] = 14.8 µM), WP1130 (IC50 = 3.8 µM), and WP1702 (IC50 = 2.9 µM) potentially contributes, albeit minimally, to suppression of the JAK2/STAT signaling pathways in cancer cells and that additional specific structural modifications may amplify JAK2-inhibitory effects

Immobilization of His-tagged kinase JAK2 onto the surface of a plasmon resonance gold disc modified with different copper (II) complexes.

New surface plasmon resonance (SPR) sensing platforms which consists of copper (II) complexes of a pentetic acid thiol ligand (DPTA-Cu(II)) and of a thiol derivative of dipyrromethene (DPM-Cu(II) created on the surface of gold SPR disc were applied to oriented immobilization of His-tagged Janus kinase 2 (GST-His6-JAK2). This method is based on the covalent bond formation between histidine from a His-tag chain of a protein and Cu(II) centres from the complexes. The kinetic and thermodynamic parameters of the oriented immobilization of GST-His6-JAK2 protein to DPTA-Cu(II) and DPM-Cu(II) complexes attached to the Au surface of a SPR disc were discussed

Pentetic acid (DPTA) Cu(II) monolayer deposited on gold electrode—The base of biosensors for electrochemical screening of kinase JAK2 and potential inhibitor interactions

Here, the new biosensor destined for screening of interactions between kinase JAK2 and compounds which may act as inhibitors was presented. The Cu(II) complex of pentetic acid thiol ligand was applied for immobilization of kinase JAK2 on the gold electrode surface through his-tagged chemistry. The base of the biosensor response was the change of the electrochemical properties of the Cu(II) redox centres upon formation of the kinase JAK2–potential inhibitor complex. The increasing inhibitor concentration caused the decrease of reduction/oxidation Cu(II) current observed with Osteryoung square wave voltammetry. The biosensor usability was checked using known inhibitors, berberines–isoquinolone alkaloids, as well as caffeic acid—a control compound which has no affinity to kinase JAK2. The possible parameters suitable for estimation of the strength of the interactions between kinase JAK2 covalently attached to the Cu(II) complex of pentetic acid thiol ligand deposited on the gold electrode surface and potential inhibitors present in the solution were presented

Structure&ndash;Activity Relationship of the Dimeric and Oligomeric Forms of a Cytotoxic Biotherapeutic Based on Diphtheria Toxin

Protein aggregation is a well-recognized problem in industrial preparation, including biotherapeutics. These low-energy states constantly compete with a native-like conformation, which is more pronounced in the case of macromolecules of low stability in the solution. A better understanding of the structure and function of such aggregates is generally required for the more rational development of therapeutic proteins, including single-chain fusion cytotoxins to target specific receptors on cancer cells. Here, we identified and purified such particles as side products of the renaturation process of the single-chain fusion cytotoxin, composed of two diphtheria toxin (DT) domains and interleukin 13 (IL-13), and applied various experimental techniques to comprehensively understand their molecular architecture and function. Importantly, we distinguished soluble purified dimeric and fractionated oligomeric particles from aggregates. The oligomers are polydisperse and multimodal, with a distribution favoring lower and even stoichiometries, suggesting they are composed of dimeric building units. Importantly, all these oligomeric particles and the monomer are cystine-dependent as their innate disulfide bonds have structural and functional roles. Their reduction triggers aggregation. Presumably the dimer and lower oligomers represent the metastable state, retaining the native disulfide bond. Although significantly reduced in contrast to the monomer, they preserve some fraction of bioactivity, manifested by their IL-13RA2 receptor affinity and selective cytotoxic potency towards the U-251 glioblastoma cell line. These molecular assemblies probably preserve structural integrity and native-like fold, at least to some extent. As our study demonstrated, the dimeric and oligomeric cytotoxin may be an exciting model protein, introducing a new understanding of its monomeric counterpart&rsquo;s molecular characteristics

The <i>Cyanobacteria</i> strains with indicated <i>alkB</i> homologs (GI numbers according to NCBI database).

<p>The <i>Cyanobacteria</i> strains with indicated <i>alkB</i> homologs (GI numbers according to NCBI database).</p

CLANS clustering of 1943 ALKBH proteins.

<p>Particular ALKBH groups (A) or taxons (B) are color coded. The groups numbered 9–16 are novel ALKBH family members described in this paper. Color codes are explained in the legend in the upper right corner of (B).</p