The structural effects of divalent cations and insulin on phospholipid model membranes /

It is well accepted that structural studies with model membranes are of considerable value in understanding the structure of biological membranes. Many studies with models of pure phospholipids have been done; but the effects of divalent cations and protein on these models would make these studies more applicable to intact membrane. The present study, performed with above view, is a structural analysis of divalent io~cardio1ipin complexes using the technique of x-ray diffraction. Cardiolipin, precipitated from dilute solution by divalent ionscalcium, magnesium and barium, contains little water and the structure formed is similar to the structure of pure cardiolipin with low water content. The calcium-cardiolipin complex forms a pure hexagonal type II phase that exists from 40 to 400 C. The molar ratio of calcium and cardiolipin in the complex is 1 : 1. Cardiolipin, precipitated with magnesium and barium forms two co-existing phases, lamellar and hexagonal, the relative quantity of the two phases being dependent on temperature. The hexagonal phase type II consisting of water filled channels formed by adding calcium to cardiolipin may have a remarkable permeability property in intact membrane. Pure cardiolipin and insulin at pH 3.0 and 4.0 precipitate but form no organised structure. Lecithin/cardiolipin and insulin precipitated at pH 3.0 give a pure lamellar phase. As the lecithin/cardiolipin molar ratio changes from 93/7 to SO/50, (a) the repeat distance of the lamellar changes from 72.8 X to 68.2 A; (b) the amount of protein bound increases in such a way that cardiolipin/insulin molar ratio in the complex reaches a maximum constant value at lecithin/cardiolipin molar ratio 70/30. A structural model based on these data shows that the molecular arrangement of lipid and protein is a lipid bilayer coated with protein molecules. The lipid-protein interaction is chiefly electrostatic and little, if any, hydrophobic bonding occurs in this particular system. So, the proposed model is essentially the same as Davson-Daniellifs model of biological membrane.

Mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in RNA binding by using surface plasmon resonance

Phosphorylation of the coronavirus nucleoprotein (N protein) has been predicted to play a role in RNA binding. To investigate this hypothesis, we examined the kinetics of RNA binding between nonphosphorylated and phosphorylated infectious bronchitis virus N protein with nonviral and viral RNA by surface plasmon resonance (Biacore). Mass spectroscopic analysis of N protein identified phosphorylation sites that were proximal to RNA binding domains. Kinetic analysis, by surface plasmon resonance, indicated that nonphospborylated N protein bound with the same affinity to viral RNA as phosphorylated N protein. However, phosphorylated N protein bound to viral RNA with a higher binding affinity than nonviral RNA, suggesting that phosphorylation of N protein determined the recognition of virus RNA. The data also indicated that a known N protein binding site (involved in transcriptional regulation) consisting of a conserved core sequence present near the 5' end of the genome (in the leader sequence) functioned by promoting high association rates of N protein binding. Further analysis of the leader sequence indicated that the core element was not the only binding site for N protein and that other regions functioned to promote high-affinity binding.

Loss of 3-chlorotyrosine by inflammatory oxidants: Implications for the use of 3-chlorotyrosine as a bio-marker in vivo

Activated neutrophils generate the potent oxidant hypochlorous acid (HOCl) from the enzyme myeloperoxidase (MPO). A proposed bio-marker for MPO-derived HOCl in vivo is 3-chlorotyrosine, elevated levels of which have been measured in several human inflammatory pathologies. However, it is unlikely that HOCl is produced as the sole oxidant at sites of chronic inflammation as other reactive species are also produced during the inflammatory response. The work presented shows that free and protein bound 3-chlorotyrosine is lost upon addition of the pro-inflammatory oxidants, HOCl, peroxynitrite, and acidified nitrite. Furthermore, incubation of 3-chlorotyrosine with activated RAW264.7 macrophages or neutrophil-like HL-60 cells resulted in significant loss of 3-chlorotyrosine. Therefore, at sites of chronic inflammation where there is concomitant ONOO- and HOCl formation, it is possible measurement of 3-chlorotyrosine may represent an underestimate of the true extent of tyrosine chlorination. This finding could account for some of the discrepancies reported between 3-chlorotyrosine levels in tissues in the literature. (c) 2008 Elsevier Inc. All rights reserved.

Glutathione and related thiol compounds. I. Glutathione and related thiol compounds in flour

The high pressure liquid chromatography method for determination of glutathione in free and protein-bound forms was re-established and has successfully been developed to measure glutathione related thiol compounds, i.e. L-cysteine, gamma-L-glutamyl-L-cysteine and L-cysteinyl-L-glycine, in both free and protein-bound forms. The natural levels of those compounds in typical strong, weak flours, and flours from 36 wheat varieties grown in the UK were investigated. The total free and protein-bound glutathione compounds found in the 36 UK varieties was 358 +/- 51 and 190 +/- 17 nmol/g, respectively. Multiple correlation analysis did not show a clear-cut relationship between the natural level of glutathione and any related thiol compound in either free or protein-bound forms and flour quality attributes, including rheological properties, baking performance, protein content and SDS sedimentation test values. Therefore, it can be suggested that glutathione and related thiol compounds at natural levels do not lead to significant differences in the rheological properties of dough and the baking performance of flour. (C) 2003 Elsevier Ltd. All rights reserved.

The effect of dietary nitrate on salivary, plasma, and urinary nitrate metabolism in humans

Dietary nitrate is metabolized to nitrite by bacterial flora on the posterior surface of the tongue leading to increased salivary nitrite concentrations. In the acidic environment of the stomach, nitrite forms nitrous acid, a potent nitrating/nitrosating agent. The aim of this study was to examine the pharmacokinetics of dietary nitrate in relation to the formation of salivary, plasma, and urinary nitrite and nitrate in healthy subjects. A secondary aim was to determine whether dietary nitrate increases the formation of protein-bound 3-nitrotyrosine in plasma, and if dietary nitrate improves platelet function. The pharmacokinetic profile of urinary nitrate excretion indicates total clearance of consumed nitrate in a 24 h period. While urinary, salivary, and plasma nitrate concentrations increased between 4- and 7-fold, a significant increase in nitrite was only detected in saliva (7-fold). High dietary nitrate consumption does not cause a significant acute change in plasma concentrations of 3-nitrotyrosine or in platelet function.

Monitoring the penetration enhancer dimethyl sulfoxide in human stratum corneum in vivo by confocal raman spectroscopy

The stratum corneum (SC) barrier typically consists of layers of corneocytes embedded in a lipid continuum that regulates barrier function. The lipid domain containing ceramides, cholesterol, and free fatty acids provides the major pathway for most drugs permeating across SC. Penetration enhancers diminish the SC barrier function. The classic enhancer is dimethyl sulfoxide (DMSO). Its mechanisms of action remain unclear, although DMSO disrupts lipid organisation and may displace protein-bound water. Here we use confocal Raman spectroscopy to probe molecular interactions between a finite (depleting) dose of DMSO and SC, as functions of depth and time, providing novel information about residence time and location of DMSO in human SC in vivo

An isotope dilution model for partitioning phenylalanine and tyrosine uptake by the mammary gland of lactating dairy cows

An isotope dilution model for partitioning phenylalanine and tyrosine uptake by the mammary gland of the lactating dairy cow is constructed and solved in the steady state. The model contains four intracellular and four extracellular pools and conservation of mass principles are applied to generate the fundamental equations describing the behaviour of the system. The experimental measurements required for model solution are milk secretion and plasma flow rate across the gland in combination with phenylalanine and tyrosine concentrations and plateau isotopic enrichments in arterial and venous plasma and free and protein bound milk during a constant infusion of [1-(13)C]phenylalanine and [2,3,5,6-(2)H]tyrosine tracer. If assumptions are made, model solution enables determination of steady state flows for phenylalanine and tyrosine inflow to the gland, outflow from it and bypass, and flows representing the synthesis and degradation of constitutive protein and hydroxylation. The model is effective in providing information about the fates of phenylalanine and tyrosine in the mammary gland and could be used as part of a more complex system describing amino acid metabolism in the whole ruminant.

Regulation of Catalase-Peroxidase KatG Is OxyR Dependent and Fur Independent in Caulobacter crescentus

Most organisms that grow in the presence of oxygen possess catalases and/or peroxidases, which are necessary for scavenging the H(2)O(2) produced by aerobic metabolism. In this work we investigate the pathways that regulate the Caulobacter crescentus katG gene, encoding the only enzyme with catalase-peroxidase function in this bacterium. The transcriptional start site of the katG gene was determined, showing a short 5` untranslated region. The katG regulatory region was mapped by serial deletions, and the results indicate that there is a single promoter, which is responsible for induction at stationary phase. An oxyR mutant strain was constructed; it showed decreased katG expression, and no KatG protein or catalase-peroxidase activity was detected in stationary-phase cell extracts, implying that OxyR is the main positive regulator of the C. crescentus katG gene. Purified OxyR protein bound to the katG regulatory region between nucleotides -42 and -91 from the transcription start site, as determined by a DNase I footprinting assay, and a canonical OxyR binding site was found in this region. Moreover, OxyR binding was shown to be redox dependent, given that only oxidized proteins bound adjacent to the -35 sequence of the promoter and the katG P1 promoter was activated by OxyR in an H(2)O(2)-dependent manner. On the other hand, this work showed that the iron-responsive regulator Fur does not regulate C. crescentus katG, since a fur mutant strain presented wild-type levels of katG transcription and catalase-peroxidase production and activity, and the purified Fur protein was not able to bind to the katG regulatory region.

Dietary supplementation of lysine and/or methionine on performance, nitrogen retention and excretion in pacu Piaractus mesopotamicus reared in cages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Study of the binding of Eu3+ and Tb3+ to L-phenylalanine and L-tryptophan

Trivalent europium and terbium ions have ionic radii similar to that of Ca2+. So they are employed as probes of calcium binding sites in biological molecules. These ions exhibit very useful spectroscopic characteristics, chiefly a pronounced luminescence. In protein bound lanthanide, visible light emission from the lanthanide excited states can be observed when UV light is absorbed by aromatic amino acids. Subsequently, the energy is transferred to the lanthanide ion. The present work was carried out to define the binding sites of Eu3+ and Tb3+ in complexes with the aromatic amino acids L-phenylalanine and L-tryptophan. The techniques utilized were infrared and C nuclear magnetic resonance spectroscopies. It was found that trivalent europium and terbium interact with the carboxylate group of both amino acids. With L-tryptophan, the imino group of the indole ring is also involved representing another coordination site.

Estudo comparativo entre heparina sódica e heparina de baixo peso molecular (nadroparina cálcica) em cães (Canis familiaris) submetidos à hemódialise

Pós-graduação em Medicina Veterinária - FMVZ

Digestibilidade de nutrientes em dietas com diferentes fontes e níveis para juvenis de tilápia-do-nilo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Biomimetisches Modell eines Photosystems aus dem pflanzlichen Photosyntheseapparat

In dieser Arbeit wurde ein biomimetisches Modell für ein pflanzliches Photosystem bestehend aus dem rekombinanten Hauptlichtsammlerkomplex (LHCII) als Absorptions- und Energietransfereinheit und einem N-terminal an das Protein gebundenen Farbstoff als Energieakzeptor hergestellt. Mehrere LHCII-Farbstoff-Konstrukte wurden getestet, die höchste Energietransfereffizienz von komplexgebundenem Chlorophyll-a zum Energieakzeptor konnte an einem LHCII-Benzoylterrylendicarboximid-Konstrukt gemessen werden. Bei Raumtemperatur wurde hier 70% der Chlorophyll-a-Anregungsenergie auf den Farbstoff übertragen, bei 77 K sogar 85%. LHCII-Farbstoffkonstrukte können helfen, strukturelle und funktionelle Eigenschaften des LHCII näher zu beleuchten. So konnte bereits in dieser Arbeit gezeigt werden, daß der N-Terminus des Komplexes im zeitlichen Mittel in eine größere Annäherung zum pigmentierten Teil des LHCII kommen muß, sonst sind Energietransfereffizienzen obiger Größenordnung nicht möglich. Weitere Erkenntnisse werden von einzelmolekülspektroskopischen Untersuchungen erwartet. Voraussetzung hierfür ist jedoch eine orientierte Immobilisierung des LHCII auf einer Glasoberfläche. Es gelang, den Komplex über eine auf molekularer Ebene eingeführte Aminosäuresequenz aus sechs Histidinen an die Nickelchelatgruppe einer auf Glas immobilisierten Meerrettich-Peroxidase zu binden. Einzelmolekülspektroskopisch konnte eine LHCII-Immobilisation senkrecht zur Proteinsymmetrieachse nachgewiesen werden. Mittelfristig wird angestrebt, LHCII-Farbstoffkonstrukte auch für photovoltaische Anwendungen nutzbar zu machen. Ein erster Meilenstein wurde in dieser Arbeit erreicht, indem es gelang, LHCII an Titandioxid, Halbleiter der sog. Grätzelzelle, zu binden.

LrhA als Regulator der Flagellen, Motilität, Chemotaxis und Typ 1 Fimbrien in Escherichia coli

Das lrhA-Gen von E. coli kodiert für einen Transkriptionsregulator der LysR-Familie. Die Funktion von LrhA war ungeklärt und sollte durch Vergleich der Gesamt-mRNA aus einem E. coli-Wildtyp und einer isogenen lrhA-Mutante mit Hilfe von Genomanalysen untersucht werden. In der lrhA-Mutante war der mRNA-Gehalt vieler Gene um den Faktor 3 bis 80 erhöht. Es handelt sich um Flagellen-, Motilitäts- und Chemotaxisgene, bzw. um Gene der Typ 1 Fimbrien. Diese Ergebnisse wurden in Expressionsmessungen bestätigt. LrhA war in der Lage an den Promotor von flhDC zu binden, aber nicht an die Promotoren der übrigen Gene für Motilität und Chemotaxis. FlhDC kodiert für den übergeordneten Regulator FlhD2C2 der Fagellensynthese.LrhA war außerdem in der Lage an die Promotoren der Gene für Typ 1 Fimbrien fimA und fimE zu binden. Typ 1 Fimbrien stellen in E. coli Virulenzfaktoren dar. Eine Regulation weiterer Virulenzfaktoren durch LrhA konnte in DNA-Pathoarrays ausgeschlossen werden.LrhA ist damit ein wichtiger Transkriptionsregulator, der die Expression der Gene für Flagellen, Motilität, Chemotaxis und Typ 1 Fimbrien reguliert. FlhDC, fimA und fimE stellen dabei direkte Zielgene von LrhA dar. Außerdem konnte eine positive Autoregulation von LrhA nachgewiesen werden.

Zeitaufgelöste Messung der Faltung und Pigmentbindung des Lichtsammlerproteins LHCIIb anhand verschiedener spektroskopischer Monitore

Über die Biogenese des Lichtsammelkomplexes des Photosystems II höherer Pflanzen (LHCII) in der Thylakoidmembran der Chloroplasten existieren wenige Daten. Deswegen soll die Aufklärung des Faltungsmechanismus in vitro anhand von zeitaufgelösten Messungen der Rückfaltung des Komplexes Rückschlüsse auf die Situation in vivo ermöglichen.Zur Beobachtung der Rückfaltung wurden Methoden der Fluoreszenz- und CD-Spektroskopie verwendet. Die Pigmentbindung und die Ausbildung von α-helikaler Sekundärstruktur erfolgt in einem schnelleren und einem langsameren apparenten Schritt (Sekunden und Minuten); beide Vorgänge sind eng gekoppelt und limitiert durch die Bindung der Carotinoide. In der schnelleren Phase ist die Bindung von Chl a und Lutein ausreichend für die Zunahme an α-helikaler Struktur. Ein thermodynamisch stabiler Komplex erfordert die Bindung von Chl b und Carotinoiden. In der schnellen Phase bindet Chl a vor Chl b und Lutein mindestens so schnell wie Chl b; beide Pigmente limitieren die Bindung von Chl b. Chl b ist notwendig für die Ereignisse der langsameren Phase.Bzgl. der Situation in vivo deuten die Daten auf (1) eine aktive Rolle der Pigmentbindung für die Membraninsertion des Proteins, (2) einen Schutz vor Photooxidation der Chlorophylle durch die obligatorische Carotinoidbindung und (3) die Möglichkeit der Umsetzung von LHCII-gebundem Chl a zu Chl b.