EPR spectroscopic investigation of membrane protein structure and folding on light harvesting complex LHCIIb

Structure and folding of membrane proteins are important issues in molecular and cell biology. In this work new approaches are developed to characterize the structure of folded, unfolded and partially folded membrane proteins. These approaches combine site-directed spin labeling and pulse EPR techniques. The major plant light harvesting complex LHCIIb was used as a model system. Measurements of longitudinal and transversal relaxation times of electron spins and of hyperfine couplings to neighboring nuclei by electron spin echo envelope modulation(ESEEM) provide complementary information about the local environment of a single spin label. By double electron electron resonance (DEER) distances in the nanometer range between two spin labels can be determined. The results are analyzed in terms of relative water accessibilities of different sites in LHCIIb and its geometry. They reveal conformational changes as a function of micelle composition. This arsenal of methods is used to study protein folding during the LHCIIb self assembly and a spatially and temporally resolved folding model is proposed. The approaches developed here are potentially applicable for studying structure and folding of any protein or other self-assembling structure if site-directed spin labeling is feasible and the time scale of folding is accessible to freeze-quench techniques.

Ribosome Biogenesis and cell cycle regulation: Effect of RNA Polymerase III inhibition

In cycling cells positive stimuli like nutrient, growth factors and mitogens increase ribosome biogenesis rate and protein synthesis to ensure both growth and proliferation. In contrast, under stress situation, proliferating cells negatively modulate ribosome production to reduce protein synthesis and block cell cycle progression. The main strategy used by cycling cell to coordinate cell proliferation and ribosome biogenesis is to share regulatory elements, which participate directly in ribosome production and in cell cycle regulation. In fact, there is evidence that stimulation or inhibition of cell proliferation exerts direct effect on activity of the RNA polymerases controlling the ribosome biogenesis, while several alterations in normal ribosome biogenesis cause changes of the expression and the activity of the tumor suppressor p53, the main effector of cell cycle progression inhibition. The available data on the cross-talk between ribosome biogenesis and cell proliferation have been until now obtained in experimental model in which changes in ribosome biogenesis were obtained either by reducing the activity of the RNA polymerase I or by down-regulating the expression of the ribosomal proteins. The molecular pathways involved in the relationship between the effect of the inhibition of RNA polymerase III (Pol III) activity and cell cycle progression have been not yet investigated. In eukaryotes, RNA Polymerase III is responsible for transcription of factors involved both in ribosome assembly (5S rRNA) and rRNA processing (RNAse P and MRP).Thus, the aim of this study is characterize the effects of the down-regulation of RNA Polymerase III activity, or the specific depletion of 5S rRNA. The results that will be obtained might lead to a deeper understanding of the molecular pathway that controls the coordination between ribosome biogenesis and cell cycle, and might give useful information about the possibility to target RNA Polymerase III for cancer treatment.

Neural network activity in the neonatal acute slice, slice culture and cell culture

Information processing and storage in the brain may be presented by the oscillations and cell assemblies. Here we address the question of how individual neurons associate together to assemble neural networks and present spontaneous electrical activity. Therefore, we dissected the neonatal brain at three different levels: acute 1-mm thick brain slice, cultured organotypic 350-µm thick brain slice and dissociated neuronal cultures. The spatio-temporal properties of neural activity were investigated by using a 60-channel Micro-electrode arrays (MEA), and the cell assemblies were studied by using a template-matching method. We find local on-propagating as well as large- scale propagating spontaneous oscillatory activity in acute slices, spontaneous network activity characterized by synchronized burst discharges in organotypic cultured slices, and autonomous bursting behaviour in dissociated neuronal cultures. Furthermore, repetitive spike patterns emerge after one week of dissociated neuronal culture and dramatically increase their numbers as well as their complexity and occurrence in the second week. Our data indicate that neurons can self-organize themselves, assembly to a neural network, present spontaneous oscillations, and emerge spatio-temporal activation patterns. The spontaneous oscillations and repetitive spike patterns may serve fundamental functions for information processing and storage in the brain.

Der enzymatische und anti-oxidative Mechanismus des anti-atherogenen Enzyms Paraoxonase-2

Das humane Enzym PON2 ist in eine Vielzahl pathophysiologischer Prozesse involviert und ist durch zwei Funktionen gekennzeichnet - eine enzymatische Laktonase-Aktivität und eine anti-oxidative Aktivität. Durch die Laktonase-Aktivität hydrolysiert PON2 vorwiegend das bakterielle Signalmolekül 3oxoC12. PON2 ist als Bestandteil des angeborenen Immunsystems anzusehen und trägt wahrscheinlich zur Immunabwehr gegen Infektionen mit den human-pathogenen Pseudomonas aeruginosa Bakterien bei. Durch die anti-oxidative Aktivität vermindert PON2 oxidative Schäden und verringert redox-abhängige pro-apoptotische Stimulation. Diese einzigartige Funktion von PON2 ist jedoch ambivalent zu betrachten, da hohe PON2-Spiegel zwar Arteriosklerose reduzieren können, aber im Verdacht stehen Tumorzellen zu stabilisieren.rnIn dieser Arbeit wurden die noch unbekannten Mechanismen und der Zusammenhang der enzymatischen und der anti-oxidativen Aktivität analysiert. In diesem Rahmen wurde gezeigt, dass PON2 spezifisch die Superoxidfreisetzung an Komplex I und III der Atmungskette in der inneren Mitochondrienmembran reduzieren kann. PON2 veränderte dabei weder die Aktivitäten der Superoxiddismutasen noch die Cytochrom C-Expression. Weiterhin konnte in dieser Arbeit erstmals gezeigt werden, dass PON2 O2- nicht direkt abbaut, sondern vielmehr dessen Bildung verhindert. Diese Erkenntnisse implizieren, dass PON2 die anti-oxidative Aktivität über eine Beeinflussung des Quinon-Pools vermittelt. Anhand von verschiedenen Punktmutationen konnte gezeigt werden, dass die Histidinreste-114 und -133 für die Laktonase-Aktivität essentiell sind. Weiterhin wurden die Glykosylierungsstellen von PON2 identifiziert und gezeigt, dass die Glykosylierung, nicht aber der natürliche Polymorphismus Ser/Cys311 für die Laktonase-Aktivität von Bedeutung ist. Von besonderer Bedeutung ist, dass keine dieser Mutationen die anti-oxidative Aktivität beeinflusste, wodurch erstmals die Unabhängigkeit der beiden Funktionen von PON2 gezeigt werden konnte. rnEs war bekannt, dass PON2 gegen intrinsische und ER-Stress-induzierte Apoptose schützt. Die Spezifität der anti-oxidativen / anti-apoptotischen Wirkung wurde hier an einem weiteren pathophysiologischen Modell untersucht. 7-Ketocholesterol (7-KC) ist der Hauptbestandteil des pro-arteriosklerotischen oxLDL und verursacht in Zellen des Gefäßsystems ER-Stress, oxidativen Stress und Apoptose. Unerwarteterweise konnte PON2 Endothelzellen nicht gegen den 7-KC-induzierten Zelltod schützen. Mehrere unabhängige experimentelle Ansätze belegen, dass 7-KC in Endothelzellen im Gegensatz zu Gefäßmuskelzellen den Zelltod über Autophagie und nicht über ER-Stress oder intrinsische Apoptose bewirkt. Weiterhin führt 7-KC, wie auch 3oxoC12 und Thapsigargin zu einem Abbau der PON2-mRNA, die über die 5’UTR der PON2-mRNA vermittelt wird. Diese Arbeit vermittelt detaillierte mechanistische Einsichten in die Funktionen von PON2, die für ihre Rolle bei Arteriosklerose, in der körpereigenen Immunabwehr und bei Krebs entscheidend sind.rn

Designing an antimicrobial and cell-adhesive multilayer via plasma polymerization

In dieser Arbeit wurden Oberflächenmodifizierungen entwickelt, die sowohl rnzelladhäsive als auch antimikrobielle Eigenschaften tragen. Rasche Zelladhäsion rnund Wundheilung ist gewünscht für Biomaterialien, da sonst das Material als rnFremdkörper erkannt werden würde und Infektionskeime in die Kavität zwischen rnMaterial und Gewebe eindringen könnten. Plasmapolymerisation dient hierbei als rnBeschichtungsverfahren, da es ein breites Spektrum an Materialien beschichten rnkann unabhängig von dessen Beschaffenheit. Als zelladhäsive Schicht wurde rnplasmapolymerisiertes Allylamin gewählt, da es zellfreundlich ist und dabei rnweitere nasschemische Modifikationen, wie die Anbindung von Fibronektin, rnzulässt. Dabei dient es zugleich als Barriereschicht für darunterliegende zink- und silberhaltige Filme, die der Beschichtung durch Freisetzung von Silber und Zink antimikrobielle Eigenschaften verleihen. Die Schichtsysteme wurden rnspektroskopisch und mikroskopisch untersucht sowie zelladhäsive und rnantimikrobielle Wirkung mit verschiedenen Zell- und Bakterientypen getestet.

Control of Steroid 21-oic Acid Synthesis by Peroxisome Proliferator-activated Receptor and Role of the Hypothalamic-Pituitary-Adrenal Axis

A previous study identified the peroxisome proliferator-activated receptor alpha (PPARalpha) activation biomarkers 21-steroid carboxylic acids 11beta-hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11beta,20-dihydroxy-3-oxo-pregn-4-en-21-oic acid (DHOPA). In the present study, the molecular mechanism and the metabolic pathway of their production were determined. The PPARalpha-specific time-dependent increases in HDOPA and 20alpha-DHOPA paralleled the development of adrenal cortex hyperplasia, hypercortisolism, and spleen atrophy, which was attenuated in adrenalectomized mice. Wy-14,643 activation of PPARalpha induced hepatic FGF21, which caused increased neuropeptide Y and agouti-related protein mRNAs in the hypothalamus, stimulation of the agouti-related protein/neuropeptide Y neurons, and activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased adrenal cortex hyperplasia and corticosterone production, revealing a link between PPARalpha and the HPA axis in controlling energy homeostasis and immune regulation. Corticosterone was demonstrated as the precursor of 21-carboxylic acids both in vivo and in vitro. Under PPARalpha activation, the classic reductive metabolic pathway of corticosterone was suppressed, whereas an alternative oxidative pathway was uncovered that leads to the sequential oxidation on carbon 21 resulting in HDOPA. The latter was then reduced to the end product 20alpha-DHOPA. Hepatic cytochromes P450, aldehyde dehydrogenase (ALDH3A2), and 21-hydroxysteroid dehydrogenase (AKR1C18) were found to be involved in this pathway. Activation of PPARalpha resulted in the induction of Aldh3a2 and Akr1c18, both of which were confirmed as target genes through introduction of promoter luciferase reporter constructs into mouse livers in vivo. This study underscores the power of mass spectrometry-based metabolomics combined with genomic and physiologic analyses in identifying downstream metabolic biomarkers and the corresponding upstream molecular mechanisms.

Peroxisome proliferator-activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}) promotes skeletal muscle lipid refueling in vivo by activating de novo lipogenesis and the pentose phosphate pathway

Exercise induces a pleiotropic adaptive response in skeletal muscle, largely through peroxisome proliferator-activated receptor coactivator 1 (PGC-1 ). PGC-1 enhances lipid oxidation and thereby provides energy for sustained muscle contraction. Its potential implication in promoting muscle refueling remains unresolved, however. Here, we investigated a possible role of elevated PGC-1 levels in skeletal muscle lipogenesis in vivo and the molecular mechanisms that underlie PGC-1 -mediated de novo lipogenesis. To this end, we studied transgenic mice with physiological overexpression of PGC-1 and human muscle biopsies pre- and post-exercise. We demonstrate that PGC-1 enhances lipogenesis in skeletal muscle through liver X receptor -dependent activation of the fatty acid synthase (FAS) promoter and by increasing FAS activity. Using chromatin immunoprecipitation, we establish a direct interaction between PGC-1 and the liver X receptor-responsive element in the FAS promoter. Moreover, we show for the first time that increased glucose uptake and activation of the pentose phosphate pathway provide substrates for RNA synthesis and cofactors for de novo lipogenesis. Similarly, we observed increased lipogenesis and lipid levels in human muscle biopsies that were obtained post-exercise. Our findings suggest that PGC-1 coordinates lipogenesis, intramyocellular lipid accumulation, and substrate oxidation in exercised skeletal muscle in vivo.

Lamellar body ultrastructure revisited: high-pressure freezing and cryo-electron microscopy of vitreous sections

Lamellar bodies are the storage sites for lung surfactant within type II alveolar epithelial cells. The structure-function models of lamellar bodies are based on microscopic analyses of chemically fixed tissue. Despite available alternative fixation methods that are less prone to artifacts, such as cryofixation by high-pressure freezing, the nature of the lung, being mostly air filled, makes it difficult to take advantage of these improved methods. In this paper, we propose a new approach and show for the first time the ultrastructure of intracellular lamellar bodies based on cryo-electron microscopy of vitreous sections in the range of nanometer resolution. Thus, unspoiled by chemical fixation, dehydration and contrasting agents, a close to native structure is revealed. Our approach uses perfluorocarbon to substitute the air in the alveoli. Lung tissue was subsequently high-pressure frozen, cryosectioned and observed in a cryo-electron microscope. The lamellar bodies clearly show a tight lamellar morphology. The periodicity of these lamellae was 7.3 nm. Lamellar bifurcations were observed in our cryosections. The technical approach described in this paper allows the examination of the native cellular ultrastructure of the surfactant system under near in vivo conditions, and therefore opens up prospectives for scrutinizing various theories of lamellar body biogenesis, exocytosis and recycling.

Diversity of structure and function of alpha1alpha6beta3delta GABAA receptors: comparison with alpha1beta3delta and alpha6beta3delta receptors

delta subunit-containing gamma-aminobutyric acid, type A (GABA(A))receptors are expressed extrasynaptically and mediate tonic inhibition. In cerebellar granule cells, they often form receptors together with alpha(1) and/or alpha(6) subunits. We were interested in determining the architecture of receptors containing both subunits. We predefined the subunit arrangement of several different GABA(A) receptor pentamers by concatenation. These receptors composed of alpha(1), alpha(6), beta(3), and delta subunits were expressed in Xenopus oocytes. Currents elicited in response to GABA were determined in the presence and absence of 3alpha,21-dihydroxy-5alpha-pregnan-20-one (THDOC) or ethanol, or currents were elicited by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP). Several subunit configurations formed active channels. We therefore conclude that delta can assume multiple positions in a receptor pentamer made up of alpha(1), alpha(6), beta(3), and delta subunits. The different receptors differ in their functional properties. Functional expression of one receptor type was only evident in the combined presence of the neurosteroid THDOC with the channel agonist GABA. Most, but not all, receptors active with GABA/THDOC responded to THIP. None of the receptors was modulated by ethanol concentrations up to 30 mm. Several observations point to a preferred position of delta subunits between two alpha subunits in alpha(1)alpha(6)beta(3)delta receptors. This property is shared by alpha(1)beta(3)delta and alpha(6)beta(3)delta receptors, but there are differences in the additionally expressed isoforms.

Tailored protection against plasmalemmal injury by annexins with different Ca2+ sensitivities

The annexins, a family of Ca(2+)- and lipid-binding proteins, are involved in a range of intracellular processes. Recent findings have implicated annexin A1 in the resealing of plasmalemmal injuries. Here, we demonstrate that another member of the annexin protein family, annexin A6, is also involved in the repair of plasmalemmal lesions induced by a bacterial pore-forming toxin, streptolysin O. An injury-induced elevation in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) triggers plasmalemmal repair. The highly Ca(2+)-sensitive annexin A6 responds faster than annexin A1 to [Ca(2+)](i) elevation. Correspondingly, a limited plasmalemmal injury can be promptly countered by annexin A6 even without the participation of annexin A1. However, its high Ca(2+) sensitivity makes annexin A6 highly amenable to an unproductive binding to the uninjured plasmalemma; during an extensive injury accompanied by a massive elevation in [Ca(2+)](i), its active pool is severely depleted. In contrast, annexin A1 with a much lower Ca(2+) sensitivity is ineffective at the early stages of injury; however, it remains available for the repair even at high [Ca(2+)](i). Our findings highlight the role of the annexins in the process of plasmalemmal repair; a number of annexins with different Ca(2+)-sensitivities provide a cell with the means to react promptly to a limited injury in its early stages and, at the same time, to withstand a sustained injury accompanied by the continuous formation of plasmalemmal lesions.

Purification, cDNA structure and biological significance of a single insulin-like growth factor-binding domain protein (SIBD-1) identified in the hemocytes of the spider Cupiennius salei

Cupiennius salei single insulin-like growth factor-binding domain protein (SIBD-1), which exhibits an IGFBP N-terminal domain-like profile, was identified in the hemocytes of the spider C. salei. SIBD-1 was purified by RP-HPLC and the sequence determined by a combination of Edman degradation and 5'-3'- RACE PCR. The peptide (8676.08 Da) is composed of 78 amino acids, contains six intrachain disulphide bridges and carries a modified Thr residue at position 2. SIBD-1 mRNA expression was detected by quantitative real-time PCR mainly in hemocytes, but also in the subesophageal nerve mass and muscle. After infection, the SIBD-1 content in the hemocytes decreases and, simultaneously, the temporal SIBD-1 expression seems to be down-regulated. Two further peptides, SIBD-2 and IGFBP-rP1, also exhibiting IGFBP N-terminal domain variants with unknown functions, were identified on cDNA level in spider hemocytes and venom glands. We conclude that SIBD-1 may play an important role in the immune system of spiders.

Metabolomics reveals attenuation of the SLC6A20 kidney transporter in nonhuman primate and mouse models of type 2 diabetes mellitus

To enhance understanding of the metabolic indicators of type 2 diabetes mellitus (T2DM) disease pathogenesis and progression, the urinary metabolomes of well characterized rhesus macaques (normal or spontaneously and naturally diabetic) were examined. High-resolution ultra-performance liquid chromatography coupled with the accurate mass determination of time-of-flight mass spectrometry was used to analyze spot urine samples from normal (n = 10) and T2DM (n = 11) male monkeys. The machine-learning algorithm random forests classified urine samples as either from normal or T2DM monkeys. The metabolites important for developing the classifier were further examined for their biological significance. Random forests models had a misclassification error of less than 5%. Metabolites were identified based on accurate masses (<10 ppm) and confirmed by tandem mass spectrometry of authentic compounds. Urinary compounds significantly increased (p < 0.05) in the T2DM when compared with the normal group included glycine betaine (9-fold), citric acid (2.8-fold), kynurenic acid (1.8-fold), glucose (68-fold), and pipecolic acid (6.5-fold). When compared with the conventional definition of T2DM, the metabolites were also useful in defining the T2DM condition, and the urinary elevations in glycine betaine and pipecolic acid (as well as proline) indicated defective re-absorption in the kidney proximal tubules by SLC6A20, a Na(+)-dependent transporter. The mRNA levels of SLC6A20 were significantly reduced in the kidneys of monkeys with T2DM. These observations were validated in the db/db mouse model of T2DM. This study provides convincing evidence of the power of metabolomics for identifying functional changes at many levels in the omics pipeline.

Plant volatiles: production, function and pharmacology

Plant volatiles typically occur as a complex mixture of low-molecular weight lipophilic compounds derived from different biosynthetic pathways, and are seemingly produced as part of a defense strategy against biotic and abiotic stress, as well as contributing to various physiological functions of the producer organism. The biochemistry and molecular biology of plant volatiles is complex, and involves the interplay of several biochemical pathways and hundreds of genes. All plants are able to store and emit volatile organic compounds (VOCs), but the process shows remarkable genotypic variation and phenotypic plasticity. From a physiological standpoint, plant volatiles are involved in three critical processes, namely plant–plant interaction, the signaling between symbiotic organisms, and the attraction of pollinating insects. Their role in these ‘‘housekeeping’’ activities underlies agricultural applications that range from the search for sustainable methods for pest control to the production of flavors and fragrances. On the other hand, there is also growing evidence that VOCs are endowed with a range of biological activities in mammals, and that they represent a substantially under-exploited and still largely untapped source of novel drugs and drug leads. This review summarizes recent major developments in the study of biosynthesis, ecological functions and medicinal applications of plant VOCs.