Structure of the haptoglobin-haemoglobin complex

Red cell haemoglobin is the fundamental oxygen-transporting molecule in blood, but also a potentially tissue-damaging compound owing to its highly reactive haem groups. During intravascular haemolysis, such as in malaria and haemoglobinopathies(1), haemoglobin is released into the plasma, where it is captured by the protective acute-phase protein haptoglobin. This leads to formation of the haptoglobin-haemoglobin complex, which represents a virtually irreversible non-covalent protein-protein interaction(2). Here we present the crystal structure of the dimeric porcine haptoglobin-haemoglobin complex determined at 2.9 angstrom resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected beta-strand swap between two complement control protein (CCP) domains, defining a new fusion CCP domain structure. The haptoglobin serine protease domain forms extensive interactions with both the alpha- and beta-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the alpha beta dimer is highly overlapping with the interface between the two alpha beta dimers that constitute the native haemoglobin tetramer. Several haemoglobin residues prone to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin-haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin-haemoglobin to the macrophage scavenger receptor CD163 (ref. 3) protrudes from the surface of the distal end of the complex, adjacent to the associated haemoglobin alpha-subunit. Small-angle X-ray scattering measurements of human haptoglobin-haemoglobin bound to the ligand-binding fragment of CD163 confirm receptor binding in this area, and show that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of multimeric haptoglobin-haemoglobin for CD163 (ref. 4).

Cu(GlyGlyHis) effects on MCF7 cells: Copper uptake, reactive oxygen species generation and membrane topography changes

Elevated levels of copper have been detected in various types of human cancer cells, such as breast cancer cells, and a number of mechanisms have been proposed to explain the action and influence of copper on tumor progress. In this work, we found that stimulating the proliferation of mammary epithelial MCF7 cells with the high-redox-potential copper complex Cu (GlyGlyHis) is associated with the copper-induced intracellular generation of reactive oxygen species (ROS) that induces lipid peroxidation and causes increased roughness of external cell membranes, which leads to the formation of larger cell domes. The results presented herein provide new insights into the molecular link between copper and the proliferation of breast cancer cells and, consequently, into the mechanism by which changes in redox balance and ROS accumulation regulates cell membrane roughness. (C) 2012 Elsevier Inc. All rights reserved.

The role of reactive oxygen species in the modulation of the contraction induced by angiotensin II in carotid artery from diabetic rat

The modulation played by reactive oxygen species on the angiotensin II-induced contraction in type I-diabetic rat carotid was investigated. Concentration-response curves for angiotensin II were obtained in endothelium-intact or endothelium-denuded carotid from control or streptozotocin-induced diabetic rats, pre-treated with tiron (superoxide scavenger), PEG-catalase (hydrogen peroxide scavenger), dimethylthiourea (hydroxyl scavenger), apocynin [NAD(P) H oxidase inhibitor], SC560 (cyclooxygenase-1 inhibitor), SC236 (cyclooxygenase-2 inhibitor) or Y-27632 (Rho-kinase inhibitor). Reactive oxygen species were measured by flow cytometry in dihydroethidium (DHE)-loaded endothelial cells. Cyclooxygenase and AT1-receptor expression was assessed by immunohistochemistry. Diabetes increased the angiotensin II-induced contraction but reduced the agonist potency in rat carotid. Endothelium removal, tiron or apocynin restored the angiotensin II-induced contraction in diabetic rat carotid to control levels. PEG-catalase, DMTU or SC560 reduced the angiotensin II-induced contraction in diabetic rat carotid at the same extent. SC236 restored the angiotensin II potency in diabetic rat carotid. Y-27632 reduced the angiotensin II-induced contraction in endothelium-intact or -denuded diabetic rat carotid. Diabetes increased the DHE-fluorescence of carotid endothelial cells. Apocynin reduced the DHE-fluorescence of endothelial cells from diabetic rat carotid to control levels. Diabetes increased the muscular cyclooxygenase-2 expression but reduced the muscular AT1-receptor expression in rat carotid. In summary, hydroxyl radical, hydrogen peroxide and superoxide anion-derived from endothelial NAD(P) H oxidase mediate the hyperreactivity to angiotensin II in type I-diabetic rat carotid, involving the participation of cyclooxygenase-1 and Rho-kinase. Moreover, increased muscular cyclooxygenase-2 expression in type I-diabetic rat carotid seems to be related to the local reduced AT1-receptor expression and the reduced angiotensin II potency. (C) 2011 Elsevier B. V. All rights reserved.

Do blood components affect the production of reactive oxygen species (ROS) by equine synovial cells in vitro?

Brossi P.M., Baccarin R.Y.A. & Massoco C.O. 2012 Do blood components affect the production of reactive oxygen species (ROS) by equine synovial cells in vitro? Pesquisa Veterinaria Brasileira 32(12):1355-1360. Departamento de Clinica Medica, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, Butanta, Sao Paulo, SP 5508-210, Brazil. E-mail: baccarin@ usp.br Blood-derived products are commonly administered to horses and humans to treat many musculoskeletal diseases, due to their potential antioxidant and anti-inflammatory effects. Nevertheless, antioxidant effects have never been shown upon horse synovial fluid cells in vitro. If proved, this could give a new perspective to justify the clinical application of blood-derived products. The aim of the present study was to investigate the antioxidant effects of two blood-derived products - plasma (unconditioned blood product - UBP) and a commercial blood preparation (conditioned blood product - CBP)(4) - upon stimulated equine synovial fluid cells. Healthy tarsocrural joints (60) were tapped to obtain synovial fluid cells; these cells were pooled, processed, stimulated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), and evaluated by flow cytometry for the production of reactive oxygen species (ROS). Upon addition of any blood-derived product here used - UBP and CBP - there was a significant decrease in the oxidative burst of synovial fluid cells (P<0.05). There was no difference between UBP and CBP effects. In conclusion, treatment of stimulated equine synovial cells with either UBP or CBP efficiently restored their redox equilibrium.

Annealing effects on nanostructured gold-polymethylmethacrylate composites: Small-angle x-ray scattering analysis

Composites formed of a polymer-embedded layer of sub-10 nm gold nanoclusters were fabricated by very low energy (49 eV) gold ion implantation into polymethylmethacrylate. We used small angle x-ray scattering to investigate the structural properties of these metal-polymer composite layers that were fabricated at three different ion doses, both in their original form (as-implanted) and after annealing for 6 h well above the polymer glass transition temperature (150 degrees C). We show that annealing provides a simple means for modification of the structure of the composite by coarsening mechanisms, and thereby changes its properties. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4720464]

Gaussian deconvolution: a useful method for a form-free modeling of scattering data from mono- and multilayered planar systems

A new method for analysis of scattering data from lamellar bilayer systems is presented. The method employs a form-free description of the cross-section structure of the bilayer and the fit is performed directly to the scattering data, introducing also a structure factor when required. The cross-section structure (electron density profile in the case of X-ray scattering) is described by a set of Gaussian functions and the technique is termed Gaussian deconvolution. The coefficients of the Gaussians are optimized using a constrained least-squares routine that induces smoothness of the electron density profile. The optimization is coupled with the point-of-inflection method for determining the optimal weight of the smoothness. With the new approach, it is possible to optimize simultaneously the form factor, structure factor and several other parameters in the model. The applicability of this method is demonstrated by using it in a study of a multilamellar system composed of lecithin bilayers, where the form factor and structure factor are obtained simultaneously, and the obtained results provided new insight into this very well known system.

Procalcitonin (PCT) and C-reactive Protein (CRP) as severe systemic infection markers in febrile neutropenic adults

Abstract Background Procalcitonin (PCT) is an inflammatory marker that has been used as indicator of severe bacterial infection. We evaluated the concentrations of PCT as a marker for systemic infection compared to C-reactive protein (CRP) in patients neutropenic febrile. Methods 52 adult patients were enrolled in the study. Blood sample was collected in order to determine the serum concentrations of PCT, CRP and other hematological parameters at the onset of fever. The patients were divided into 2 groups, one with severe infection (n = 26) and the other in which the patients did not present such an infection (n = 26). Then PCT and CRP concentrations at the fever onset were compared between groups using non parametric statistical tests, ROC curve, sensitivity, specificity, likelihood ratio, and Spearman's correlation coefficient. Results The mean of PCT was significantly higher in the group with severe infection (6.7 ng/mL versus 0.6 ng/mL – p = 0.0075) comparing with CRP. Serum concentrations of 0.245 ng/mL of PCT displayed 100% de sensitivity and 69.2% specificity. PCT concentrations of 2,145 ng/mL presented a likelihood ratio of 13, which was not observed for any concentration of CRP. Conclusion PCT seems to be an useful marker for the diagnosis of systemic infection in febrile neutropenic patients, probably better than CRP.

Diet-Sensitive Sources of Reactive Oxygen Species in Liver Mitochondria: Role of Very Long Chain Acyl-CoA Dehydrogenases

High fat diets and accompanying hepatic steatosis are highly prevalent conditions. Previous work has shown that steatosis is accompanied by enhanced generation of reactive oxygen species (ROS), which may mediate further liver damage. Here we investigated mechanisms leading to enhanced ROS generation following high fat diets (HFD). We found that mitochondria from HFD livers present no differences in maximal respiratory rates and coupling, but generate more ROS specifically when fatty acids are used as substrates. Indeed, many acyl-CoA dehydrogenase isoforms were found to be more highly expressed in HFD livers, although only the very long chain acyl-CoA dehydrogenase (VLCAD) was more functionally active. Studies conducted with permeabilized mitochondria and different chain length acyl-CoA derivatives suggest that VLCAD is also a source of ROS production in mitochondria of HFD animals. This production is stimulated by the lack of NAD+. Overall, our studies uncover VLCAD as a novel, diet-sensitive, source of mitochondrial ROS.

Chain extension of poly (ethylene terephthalate) by reactive extrusion with secondary stabilizer

Poly(ethylene tereftalate) (PET) is a polymer highly susceptible to the hydrolytic reactions that occur during applications and mainly in thermomechanical processing. These reactions lead to the decrease of molecular weight of the polymer, limiting the recycling number of the material. The reactive extrusion of the PET in presence of chain extenders is an alternative to recover mechanical and rheological properties that were depreciated by the polymer degradation. In this study, PET wastes from nonwoven fabrics production were extruded in presence of the secondary stabilizer Irgafos 126 (IRG) on variable concentrations. The results showed that Irgafos 126 increased molecular weight, decreased crystallinity and changed processing behavior of the PET, similarly to the effects produced by the well-known chain extender pyromellitic dianhydride (PMDA), showing that the secondary stabilizer Irgafos 126 can also act as a chain extender for the PET.

Loudness scattering due to vibro-acoustic model variability

The use of numerical simulation in the design and evaluation of products performance is ever increasing. To a greater extent, such estimates are needed in a early design stage, when physical prototypes are not available. When dealing with vibro-acoustic models, known to be computationally expensive, a question remains, which is related to the accuracy of such models in view of the well-know variability inherent to the mass manufacturing production techniques. In addition, both academia and industry have recently realized the importance of actually listening to a products sound, either by measurements or by virtual sound synthesis, in order to assess its performance. In this work, the scatter of significant parameter variations on a simplified vehicle vibro-acoustic model is calculated on loudness metrics using Monte Carlo analysis. The mapping from the system parameters to sound quality metric is performed by a fully-coupled vibro-acoustic finite element model. Different loudness metrics are used, including overall sound pressure level expressed in dB and Specific Loudness in Sones. Sound quality equivalent sources are used to excite this model and the sound pressure level at the driver's head position is acquired to be evaluated according to sound quality metrics. No significant variation has been perceived when evaluating the system using regular sound pressure level expressed in in dB and dB(A). This happens because of the third-octave filters that averages the results under some frequency bands. On the other hand, Zwicker Loudness presents important variations, arguably, due to the masking effects.

Variability in light absorption and scattering of phytoplankton in Patagonian waters: Role of community size structure and pigment composition

Intense phytoplankton blooms were observed along the Patagonian shelf-break with satellite ocean color data, but few in situ optical observations were made in that region. We examine the variability of phytoplankton absorption and particulate scattering coefficients during such blooms on the basis of field data. The chlorophyll-a concentration, [Chla], ranged from 0.1 to 22.3 mg m−3 in surface waters. The size fractionation of [Chla] showed that 80% of samples were dominated by nanophytoplankton (N-group) and 20% by microphytoplankton (M-group). Chlorophyll-specific phytoplankton absorption coefficients at 440 and 676 nm, a*ph(440) and a*ph(676), and particulate scattering coefficient at 660 nm, b*p(660), ranged from 0.018 to 0.173, 0.009 to 0.046, and 0.031 to 2.37 m2 (mg Chla)−1, respectively. Both a*ph(440) and a*ph(676) were statistically higher for the N-group than M-group and also considerably higher than expected from global trends as a function of [Chla]. This result suggests that size of phytoplankton cells in Patagonian waters tends to be smaller than in other regions at similar [Chla]. The phytoplankton cell size parameter, Sf, derived from phytoplankton absorption spectra, proved to be useful for interpreting the variability in the data around the general inverse dependence of a*ph(440), a*ph(676), and b*p(660) on [Chla]. Sf also showed a pattern along the increasing trend of a*ph(440) and a*ph(676) as a function of the ratios of some accessory pigments to [Chla]. Our results suggest that the variability in phytoplankton absorption and scattering coefficients in Patagonian waters is caused primarily by changes in the dominant phytoplankton cell size accompanied by covariation in the concentrations of accessory pigments.

Low energy elastic and electronically inelastic electron scattering from biomolecules.

Reactions initiated by collisions with low-energy secondary electrons has been found to be the prominent mechanism toward the radiation damage on living tissues through DNA strand breaks. Now it is widely accepted that during the interaction with these secondary species the selective breaking of chemical bonds is triggered by dissociative electron attachment (DEA), that is, the capture of the incident electron and the formation of temporary negative ion states [1,2,3]. One of the approaches largely used toward a deeper understanding of the radiation damage to DNA is through modeling of DEA with its basic constituents (nucleotide bases, sugar and other subunits). We have tried to simplify this approach and attempt to make it comprehensible at a more fundamental level by looking at even simple molecules. Studies involving organic systems such as carboxylic acids, alcohols and simple ¯ve-membered heterocyclic compounds are taken as starting points for these understanding. In the present study we investigate the role played by elastic scattering and electronic excitation of molecules on electron-driven chemical processes. Special attention is focused on the analysis of the in°uence of polarization and multichannel coupling e®ects on the magnitude of elastic and electronically inelastic cross-sections. Our aim is also to investigate the existence of resonances in the elastic and electronically inelastic channels as well as to characterize them with respect to its type (shape, core-excited or Feshbach), symmetry and position. The relevance of these issues is evaluated within the context of possible applications for the modeling of discharge environments and implications in the understanding of mutagenic rupture of DNA chains. The scattering calculations were carried out with the Schwinger multichannel method (SMC) [4] and its implementation with pseudopotentials (SMCPP) [5] at di®erent levels of approximation for impact energies ranging from 0.5 eV to 30 eV. References [1] B. Boudai®a, P. Cloutier, D. Hunting, M. A. Huels and L. Sanche, Science 287, 1658 (2000). [2] X. Pan, P. Cloutier, D. Hunting and L. Sanche, Phys. Rev. Lett. 90, 208102 (2003). [3] F. Martin, P. D. Burrow, Z. Cai, P. Cloutier, D. Hunting and L. Sanche, Phys. Rev. Lett. 93, 068101 (2004). [4] K. Takatsuka and V. McKoy, Phys. Rev. A 24, 2437 (1981); ibid. Phys. Rev. A 30, 1734 (1984). [5] M. H. F. Bettega, L. G. Ferreira and M. A. P. Lima, Phys. Rev. A 47, 1111 (1993).

Reactive sputter magnetron reactor for preparation of thin films and simultaneous in-situ structural study by X-ray diffraction.

Reactive Sputter Magnetron (RSM) is a widely used technique to thin films growing of compounds both, in research laboratories and in industrial processes. The nature of the deposited compound will depend then on the nature of the magnetron target and the nature of the ions generated in the plasma. One important aspect of the problem is the knowledge of the evolution of the film during the process of growing itself. In this work, we present the design, construction of a chamber to be installed in the Huber goniometer in the XRD2 line of LNLS in Campinas, which allows in situ growing kinetic studies of thin films.