Detection of Free Radicals and Cholesterol Hydroperoxides in Blood Taken from the Coronary Sinus of Man During Percutaneous Transluminal Coronary Angioplasty

Patients undergoing percutaneous transluminal coronary angioplasty (PTCA) were investigated for the production of free radicals and cholesterol hydroperoxides during reperfusion. Fifteen patients were studied. Ischaemia during balloon inflation was assessed by serial coronary sinus lactate analysis (mean maximal increase in anterior descending artery diltation was 130%), and by the demonstration of reperfusion hyperaemia (mean increase of coronary sinus oxygen saturation 74%).

Free radicals were detected by electron spin resonance (ESR) spin trapping using the spin trap PBN (N-t-Butyl-α-phenylnitrone). Radical adducts were detected in up to 50% of samples taken during reperfusion after anterior descending lesion angioplasty. No radicals were detected in control samples or during the ischaemic phase. Radical detection was positively correlated with the change in coronary sinus lactate (p<0.025).

Coronary sinus cholesterol hydroperoxide analysis did not show a significant increase over control during reperfusion, due in part to unexpectedly high pre angioplasty levels.

This study provides clear evidence for the production of a burst of free radicals and evidence for lipid peroxidation in the minutes following myocardial reperfusion during angioplasty. A relationship between the severity of the ischaemic insult and the detection of radical adducts has also been found.     

Ascorbate in thylakoid lumen as an endogenous electron donor to Photosystem II: Protection of thylakoids from photoinhibition and regeneration of ascorbate in stroma by dehydroascorbate reductase

Photoinhibition of the electron transport activity from tyrosine Z (Y_Z) in PS II to NADP⁺in Tris-treated thylakoids was suppressed by electron donation with either diphenylcarbazide or ascorbate (AsA) during the photoinhibition treatment. This suggests that AsA prevents donor side-induced photoinhibition in vivo as an endogenous donor. AsA in the lumen is photooxidized to monodehydroascorbate (MDA) in Tris-treated thylakoids, as detected by electron spin resonance spectrometry, but not in oxygenic thylakoids. Redox analysis of pyridine nucleotide in the presence of either MDA reductase or dehydroascorbate (DHA) reductase showed that the MDA photoproduced in the lumen is disproportionated to AsA and DHA, and the DHA leaking into the stroma is reduced to AsA by DHA reductase. No leakage of MDA through the thylakoid membrane was observed. Thus, the DHA-reducing enzyme system is indispensable in maintaining AsA concentrations in chloroplasts.     

A glycosylated form of the human cardiac hormone pro B-type natriuretic peptide is an intrinsically unstructured monomeric protein

The N-terminal fragment of pro B-type natriuretic peptide (NT-proBNP) and proBNP are used as gold standard clinical markers of myocardial dysfunction such as cardiac hypertrophy and left ventricle heart failure. The actual circulating molecular forms of these peptides have been the subject of intense investigation particularly since these analytes are measured in clinical assays. Conflicting data has been reported and no firm consensus on the exact nature of the molecular species exists. Because these clinical assays are immunoassay-based, specific epitopes are detected. It is conceivable then that certain epitopes may be masked and therefore unavailable for antibody binding, thus the importance of determining the nature of the circulating molecular forms of these analytes. This situation is an unavoidable Achilles’ heel of immunoassays in general.A recombinant O-linked glycosylated form of proBNP has been show to mimic some of the properties of extracted plasma from a heart failure patient. In particular the recombinant and native material co-migrated as diffuse Western-immunostained bands on SDS-PAGE and each band collapsed to an apparent homogeneous band following deglycosylation. Thus, glycosylated-proBNP may be one such circulating form. Here we provide extensive physiochemical characterization for this O-linked protein and compare these results to other described circulating species, non-glycosylated-proBNP and NT-proBNP. It will be shown that glycosylation has no influence on the secondary and quaternary structure of proBNP. In fact, at moderate concentration in benign physiological neutral pH buffer, all three likely circulating species are essentially devoid of major secondary structure, i.e., are intrinsically unstructured proteins (IUPs). Furthermore, all three proteins exist as monomers in solution. These results may have important implications in the design of NT-proBNP/BNP immunoassays.     

Backbone dynamics of alamethicin bound to lipid membranes: spin-echo electron paramagnetic resonance of TOAC-spin labels

Alamethicin F50/5 is a hydrophobic peptide that is devoid of charged residues and that induces voltage-dependent ion channels in lipid membranes. The peptide backbone is likely to be involved in the ion conduction pathway. Electron spin-echo spectroscopy of alamethicin F50/5 analogs in which a selected Aib residue (at position n = 1, 8, or 16) is replaced by the TOAC amino-acid spin label was used to study torsional dynamics of the peptide backbone in association with phosphatidylcholine bilayer membranes. Rapid librational motions of limited angular amplitude were observed at each of the three TOAC sites by recording echo-detected spectra as a function of echo delay time, 2τ. Simulation of the time-resolved spectra, combined with conventional EPR measurements of the librational amplitude, shows that torsional fluctuations of the peptide backbone take place on the subnanosecond to nanosecond timescale, with little temperature dependence. Associated fluctuations in polar fields from the peptide could facilitate ion permeation.     

Agonist-induced conformational changes in bovine rhodopsin: insight into activation of G-protein-coupled receptors

Activation of G-protein-coupled receptors (GPCRs) is initiated by conformational changes in the transmembrane (TM) helices and the intra- and extracellular loops induced by ligand binding. Understanding the conformational changes in GPCRs leading to activation is imperative in deciphering the role of these receptors in the pathology of diseases. Since the crystal structures of activated GPCRs are not yet available, computational methods and biophysical techniques have been used to predict the structures of GPCR active states. We have recently applied the computational method LITiCon to understand the ligand-induced conformational changes in β₂-adrenergic receptor by ligands of varied efficacies. Here we report a study of the conformational changes associated with the activation of bovine rhodopsin for which the crystal structure of the inactive state is known. Starting from the inactive (dark) state, we have predicted the TM conformational changes that are induced by the isomerization of 11-cis retinal to all-trans retinal leading to the fully activated state, metarhodopsin II. The predicted active state of rhodopsin satisfies all of the 30 known experimental distance constraints. The predicted model also correlates well with the experimentally observed conformational switches in rhodopsin and other class A GPCRs, namely, the breaking of the ionic lock between R135^3.50 at the intracellular end of TM3 (part of the DRY motif) and E247^6.30 on TM6, and the rotamer toggle switch on W265^6.48 on TM6. We observe that the toggling of the W265^6.48 rotamer modulates the bend angle of TM6 around the conserved proline. The rotamer toggling is facilitated by the formation of a water wire connecting S298^7.45, W265^6.48 and H211^5.46. As a result, the intracellular ends of TMs 5 and 6 move outward from the protein core, causing large conformational changes at the cytoplasmic interface. The predicted outward movements of TM5 and TM6 are in agreement with the recently published crystal structure of opsin, which is proposed to be close to the active-state structure. In the predicted active state, several residues in the intracellular loops, such as R69, V139^3.54, T229, Q237, Q239, S240, T243 and V250^6.33, become more water exposed compared to the inactive state. These residues may be involved in mediating the conformational signal from the receptor to the G protein. From mutagenesis studies, some of these residues, such as V139^3.54, T229 and V250^6.33, are already implicated in G-protein activation. The predicted active state also leads to the formation of new stabilizing interhelical hydrogen-bond contacts, such as those between W265^6.48 and H211^5.46 and E122^3.37 and C167^4.56. These hydrogen-bond contacts serve as potential conformational switches offering new opportunities for future experimental investigations. The calculated retinal binding energy surface shows that binding of an agonist makes the receptor dynamic and flexible and accessible to many conformations, while binding of an inverse agonist traps the receptor in the inactive state and makes the other conformations inaccessible.     

Ocellatin‐PT antimicrobial peptides: High‐resolution microscopy studies in antileishmania models and interactions with mimetic membrane systems

Although the mechanism of action of antimicrobial peptides (AMPs) is not clear, they can interact electrostatically with the cell membranes of microorganisms. New ocellatin‐PT peptides were recently isolated from the skin secretion of Leptodactylus pustulatus. The secondary structure of these AMPs and their effect on Leishmania infantum cells, and on different lipid surface models was characterized in this work. The results showed that all ocellatin‐PT peptides have an α‐helix structure and five of them (PT3, PT4, PT6 to PT8) have leishmanicidal activity; PT1 and PT2 affected the cellular morphology of the parasites and showed greater affinity for leishmania and bacteria‐mimicking lipid membranes than for those of mammals. The results show selectivity of ocellatin‐PTs to the membranes of microorganisms and the applicability of biophysical methods to clarify the interaction of AMPs with cell membranes.     

Regularity and Synchrony in Motor Proteins

We investigate the origin of the regularity and synchrony which have been observed in numerical experiments of two realistic models of molecular motors, namely Fisher–Kolomeisky’s model of myosin V for vesicle transport in cells and Duke’s model of myosin II for sarcomere shortening in muscle contraction. We show that there is a generic organizing principle behind the emergence of regular gait for a motor pulling a large cargo and synchrony of action of many motors pulling a single cargo. These results are surprising in that the models used are inherently stochastic, and yet they display regular behaviors in the parameter range relevant to experiments. Our results also show that these behaviors are not tied to the particular models used in these experiments, but rather are generic to a wide class of motor protein models.     

顺磁物质对土壤低场核磁共振信号特征及含水量测定的影响

核磁共振(NMR)技术由于具有高效快速、不破坏土壤结构且对人体无害等优点,逐渐被应用到土壤学相关领域研究中。然而,土壤中顺磁物质的存在对核磁共振信号特征的影响仍不明确。本研究旨在揭示顺磁物质对不同类型土壤低场核磁共振(LF-NMR)信号特征和土壤含水量测定的影响。结果表明:土壤水的LF-NMR信号量最高可达150左右,土壤矿物、有机质和微生物等固相物质的LF-NMR信号量基本不超过0.3,相对可以忽略。质地和顺磁物质对土壤水的LF-NMR信号量测量有更大影响。LF-NMR仪器存在弛豫时间监测盲区,信号量损失主要是由于顺磁物质加速了水中氢质子的弛豫过程,导致小孔隙中水分反馈的极快的LF-NMR信号不能被监测设备捕获。对于顺磁物质含量较少的壤性潮土(1.2%)和黏壤性黑土(1.3%),LF-NMR信号量损失不大,其与土壤含水量呈线性关系;但对于黏粒含量(45.3%)和顺磁物质含量(4.0%)较高的黏性红壤,测定中会损失一部分LF-NMR信号量,监测到的LF-NMR信号量与土壤含水量不再呈线性关系。此外,外源添加顺磁物质(3.0 g·L^-1的MnCl₂溶液)也会降低黑土和红壤中可被监测的LF-NMR信号量,黑土和红壤的信号量最大损失率分别为41.0%和46.7%,极大地改变其与土壤含水量之间的定量关系。因此,在利用LF-NMR测量富含顺磁物质(>1.3%)或有外源顺磁物质进入的黏性土壤的含水量时,应先通过校正降低顺磁物质等对LF-NMR信号量的影响。研究结果对利用低场核磁共振技术准确分析土壤水分分布及土壤孔隙结构具有重要意义。     

Conservation of Dynamics Associated with Biological Function in an Enzyme Superfamily

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Association of the eukaryotic V1VO ATPase subunits a with d and d with A

Owing to the complex nature of V₁V_O ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V₁ headpiece and the V_O-domain of the yeast V₁V_O ATPase via subunit A and d as well as the V_O subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200 nM have been determined for the a-d and d-A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A₃B₃ hexamer with V_O.

Structured summary

MINT-7012054: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by fluorescence correlation spectroscopy (MI:0052)MINT-7012041: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by surface plasmon resonance (MI:0107)MINT-7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107)