Convergencia de vías de señalización en un modelo de apoptosis

La sepsis es un evento inflamatorio generalizado del organismo inducido por un daño causado generalmente por un agente infeccioso. El patógeno más frecuentemente asociado con esta entidad es el Staphylococcus aureus, responsable de la inducción de apoptosis en células endoteliales debida a la producción de ceramida. Se ha descrito el efecto protector de la proteína C activada (PCA) en sepsis y su relación con la disminución de la apoptosis de las células endoteliales. En este trabajo se analizó la activación de las quinasas AKT, ASK1, SAPK/JNK y p38 en un modelo de apoptosis endotelial usando las técnicas de Western Blotting y ELISA. Las células endoteliales (EA.hy926), se trataron con C2-ceramida (130μM) en presencia de inhibidores químicos de cada una de estas quinasas y PCA. La supervivencia de las células en presencia de inhibidores químicos y PCA fue evaluada por medio de ensayos de activación de las caspasas 3, 7 y 9, que verificaban la muerte celular por apoptosis. Los resultados evidencian que la ceramida reduce la activación de AKT y aumenta la activación de las quinasas ASK, SAPK/JNK y p38, en tanto que PCA ejerce el efecto contrario. Adicionalmente se encontró que la tiorredoxina incrementa la activación/fosforilación de AKT, mientras que la quinasa p38 induce la defosforilación de AKT.

L´onconasa com a model per l´estudi de les bases moleculars de la citotoxicitat de les ribonucleases pancreàtiques. Aplicacions a la construcció de ribonucleases amb activitat antimicrobiana

La primera part d'aquest treball s´ha centrat en la caracterització i optimització del procés d'activació de l´onconasa recombinant per tal d'obtenir l´enzim igual a la forma nativa. Per això, les reaccions d'eliminació de la Met-1 i la ciclació de la Glu1, necessàries per generar el piroglutamic han estat seguides per MALDI-TOF MS. La segona part d´aquest treball s´ha centrat en l´estudi de la contribució del pont disulfur 30-75 de l´onconasa a les seves propietats biològiques. Els resultats suggereixen que el potencial redox del citosol cel·lular podria reduir el pont disulfur 30-75 de l´onconasa salvatge afectant la unió onconasa -inhibidor proteic de ribonucleases. La tercera part ha consistit en la construcció de variants de l´HP-RNasa i onconasa amb activitat bactericida. Per això, s´ha introduït el determinant bactericida (YRWR) descrit per la proteïna catiònica d'eosinòfils en els dos enzims. Els resultats obtinguts han evidenciat que les dues ribonucleases amb el determinant bactericida presenten activitat citotòxica contra bacteris gram-negatius preferentment.

Effect of combined heat and high-pressure treatments on the texture of chicken breast muscle (Pectoralis fundus)

Commercially supplied chicken breast muscle was subjected to simultaneous heat and pressure treatments. Treatment conditions ranged from ambient temperature to 70 °C and from 0.1 to 800 MPa, respectively, in various combinations. Texture profile analysis (TPA) of the treated samples was performed to determine changes in muscle hardness. At treatment temperatures up to and including 50 °C, heat and pressure acted synergistically to increase muscle hardness. However, at 60 and 70 °C, hardness decreased following treatments in excess of 200 MPa. TPA was performed on extracted myofibrillar protein gels that after treatment under similar conditions revealed similar effects of heat and pressure. Differential scanning calorimetry analysis of whole muscle samples revealed that at ambient pressure the unfolding of myosin was completed at 60 °C, unlike actin, which completely denatured only above 70 °C. With simultaneous pressure treatment at >200 MPa, myosin and actin unfolded at 20 °C. Unfolding of myosin and actin could be induced in extracted myofibrillar protein with simultaneous treatment at 200 MPa and 40 °C. Electrophoretic analysis indicated high pressure/temperature regimens induced disulfide bonding between myosin chains.

Contribution of redox status to hepatitis C virus E2 envelope protein function and antigenicity

Disulfide bonding contributes to the function and antigenicity of many viral envelope glycoproteins. We assessed here its significance for the hepatitis C virus E2 envelope protein and a counterpart deleted for hypervariable region-1 (HVR1). All 18 cysteine residues of the antigens were involved in disulfides. Chemical reduction of up to half of these disulfides was compatible with anti-E2 monoclonal antibody reaction, CD81 receptor binding, and viral entry, whereas complete reduction abrogated these properties. The addition of 5,5'-dithiobis-2-nitrobenzoic acid had no effect on viral entry. Thus, E2 function is only weakly dependent on its redox status, and cell entry does not require redox catalysts, in contrast to a number of enveloped viruses. Because E2 is a major neutralizing antibody target, we examined the effect of disulfide bonding on E2 antigenicity. We show that reduction of three disulfides, as well as deletion of HVR1, improved antibody binding for half of the patient sera tested, whereas it had no effect on the remainder. Small scale immunization of mice with reduced E2 antigens greatly improved serum reactivity with reduced forms of E2 when compared with immunization using native E2, whereas deletion of HVR1 only marginally affected the ability of the serum to bind the redox intermediates. Immunization with reduced E2 also showed an improved neutralizing antibody response, suggesting that potential epitopes are masked on the disulfide-bonded antigen and that mild reduction may increase the breadth of the antibody response. Although E2 function is surprisingly independent of its redox status, its disulfide bonds mask antigenic domains. E2 redox manipulation may contribute to improved vaccine design.

Cell entry by enveloped viruses: redox considerations for HIV and SARS-coronavirus

For enveloped viruses, genome entry into the target cell involves two major steps: virion binding to the cell-surface receptor and fusion of the virion and cell membranes. Virus-cell membrane fusion is mediated by the virus envelope complex, and its fusogenicity is the result of an active virus-cell interaction process that induces conformation changes within the envelope. For some viruses, such as influenza, exposure to an acidic milieu within the cell during the early steps of infection triggers the necessary structural changes. However, for other pathogens which are not exposed to such environmental stress, activation of fusogenicity can result from precise thiol/disulfide rearrangements mediated by either an endogenous redox autocatalytic isomerase or a cell-associated oxidoreductase. Study of the activation of HIV envelope fusogenicity has revealed new knowledge about how redox changes within a viral envelope trigger fusion. We discuss these findings and their implication for anti-HIV therapy. In addition, to compare and contrast the situation outlined for HIV with an enveloped virus that can fuse with the cell plasma membrane independent of the redox status of its envelope protein, we review parallel data obtained on SARS coronavirus entry.

In vivo selection for metastasis promoting genes in the mouse

Here, we report the identification of a metastasis promoting factor by a forward genetic screen in mice. A retroviral cDNA library was introduced into the nonmetastatic cancer cell line 168FARN, which was then orthotopically transplanted into mouse mammary fat pads, followed by selection for cells that metastasize to the lung. The genes encoding the disulfide isomerase ERp5 and beta-catenin were found to promote breast cancer invasion and metastasis. Disulfide isomerases (thiol isomerases), which catalyze disulfide bond formation, reduction, and isomerization, have not previously been implicated in cancer cell signaling and tumor metastasis. Overexpression of ERp5 promotes both in vitro migration and invasion and in vivo metastasis of breast cancer cells. These effects were shown to involve activation of ErbB2 and phosphoinositicle 3-kinase (PI3K) pathways through dimerization of ErbB2. Activation of ErbB2 and PI3K subsequently stimulates RhoA and beta-catenin, which mediate the migration and invasion of tumor cells. Inhibition of ErbB2 and PI3K reverses the phenotypes induced by ERp5. Finally, ERp5 was shown to be up-regulated in human surgical samples of invasive breast cancers. These data identify a link between disulfide isomerases and tumor development, and provide a mechanism that modulates ErbB2 and PI3K signaling in the promotion of cancer progression.

Significant redox insensitivity of the functions of the SARS-CoV spike glycoprotein - Comparison with HIV envelope

The capacity of the surface glycoproteins of enveloped viruses to mediate virus/cell binding and membrane fusion requires a proper thiol/disulfide balance. Chemical manipulation of their redox state using reducing agents or free sulfhydryl reagents affects virus/cell interaction. Conversely, natural thiol/disulfide rearrangements often occur during the cell interaction to trigger fusogenicity, hence the virus entry. We examined the relationship between the redox state of the 20 cysteine residues of the SARS-CoV (severe acute respiratory syndrome coronavirus) Spike glycoprotein S1 subdomain and its functional properties. Mature S1 exhibited similar to 4 unpaired cysteines, and chemically reduced S1 displaying up to similar to 6 additional unpaired cysteines still bound ACE2 and enabled fusion. In addition, virus/cell membrane fusion occurred in the presence of sulfhydryl-blocking reagents and oxidoreductase inhibitors. Thus, in contrast to various viruses including HIV (human immunodeficiency virus) examined in parallel, the functions of the SARS-CoV Spike glycoprotein exhibit a significant and surprising independence of redox state, which may contribute to the wide host range of the virus. These data suggest clues for molecularly engineering vaccine immunogens.

A role for the thiol isomerase. protein ERP5 in platelet function

Formation and rearrangement of disulfide bonds during the correct folding of nascent proteins is modulated by a family of enzymes known as thiol isomerases, which include protein disulfide isomerase (PDI), endoplasmic reticulum protein 5 (ERP5), and ERP57. Recent evidence supports an alternative role for this family of proteins on the surface of cells, where they are involved in receptor 'remodeling and recognition. In platelets, blocking PDI with inhibitory antibodies inhibits a number of platelet activation pathways, including aggregation, secretion, and fibrinogen binding. Analysis of human platelet membrane fractions identified the presence of the thiol isomerase protein ERP5. Further study showed that ERP5 is resident mainly on platelet intracellular membranes, although it is rapidly recruited to the cell, surface in response to a range of platelet agonists. Blocking cell-surface ERP5 using inhibitory antibodies leads to a decrease in platelet aggregation in response to agonists, and a decrease in fibrinogen binding and P-selectin exposure. It is Possible that this is based on the disruption of integrin function, as we observed that ERP5 becomes physically associated with the integrin beta(3) subunit during platelet stimulation. These results provide new insights into the involvement of thiol isomerases and regulation of platelet activation. (C) 2005 by The American Society of Hematology.

Alternative syntheses of linear polyurethanes using masked isocyanate monomers

A range of linear polyurethanes featuring aliphatic, aromatic and ether residues have been prepared by co-polymerisation of novel 'masked' isocyanate A(2)-type monomers and diols. The reactive isocyanate monomers were generated in situ via the triphenylphosphine mediated decomposition of the heterocyclic disulfide, 1,2,4-dithiazolidine-3,5-dione. Two different synthetic approaches were examined and assessed for the construction of the novel A(2)-type monomers, which involved either coupling two 1,2,4-dithiazolidine-3,5-diones together through a spacer group or construction of 1,2,4-dithiazolidine-3,5-diones directly from diamines. The resulting polyurethanes were purified via solvent extraction and analysed using GPC, NMR and IR spectroscopic analyses. Molecular weight data were obtained and compared from both GPC and H-1 NMR (via end-group analysis) spectroscopic analysis. The thermal properties of the polyurethanes were determined using DSC and their solubility in common aprotic organic solvents was also assessed and related to their structural composition. (c) 2006 Elsevier B.V. All rights reserved.

The component polypeptide chains of bovine insulin nucleate or inhibit aggregation of the parent protein in a conformation-dependent manner

Amyloid fibrils are typically rigid, unbrariched structures with diameters of similar to 10 nm and lengths up to several micrometres, and are associated with more than 20 diseases including Alzheimer's disease and type II diabetes. Insulin is a small, predominantly alpha-helical protein consisting of 51 residues in two disulfide-linked polypeptide chains that readily assembles into amyloid fibrils under conditions of low PH and elevated temperature. We demonstrate here that both the A-chain and the B-chain of insulin are capable of forming amyloid fibrils in isolation under similar conditions, with fibrillar morphologies that differ from those composed of intact insulin. Both the A-chain and B-chain fibrils were found to be able to cross-seed the fibrillization of the parent protein, although these reactions were substantially less efficient than self-seeding with fibrils composed of full-length insulin. In both cases, the cross-seeded fibrils were morphologically distinct from the seeding, material, but shared common characteristics with typical insulin fibrils, including a very similar helical repeat. The broader distribution of heights of the cross-seeded fibrils compared to typical insulin fibrils, however, indicates that their underling protofilament hierarchy may be subtly different. In addition, and remarkably in view of this seeding behavior, the soluble forms of the A-chain and B-chain peptides were found to be capable of inhibiting insulin fibril formation. Studies using mass spectrometry suggest that this behavior might be attributable to complex formation between insulin and the A-chain and B-chain peptides. The finding that the same chemical form of a polypeptide chain in different physical states can either stimulate or inhibit the conversion of a protein into amyloid fibrils sheds new light on the mechanisms underlying fibril formation, fibril strain propagation and amyloid disease initiation and progression. (c) 2006 Elsevier Ltd. All rights reserved.

Two new mu-(1,3-azido)-bridged polymers: alternating single and double bridges in a 1D nickel(II) complex and uniform bridge in a 2D copper(II) complex: Syntheses, single-crystal structures and magnetic studies

Two polymeric azido bridged complexes [Ni2L2(N-3)(3)](n)(ClO4). (1) and [Cu(bpdS)(2)(N-3)],(ClO4),(H2O)(2.5n) (2) [L = Schiff base, obtained from the condensation of pyridine-2-aldehyde with N,N,2,2-tetramethyl-1,3-propanediamine; bpds = 4,4'-bipyridyl disulfide] have been synthesized and their crystal structures have been determined. Complex 1, C26H42ClN15Ni2O4, crystallizes in a triclinic system, space group P1 with a 8.089(13), b = 9.392(14), c = 12.267(18) angstrom, a = 107.28(l), b 95.95(1), gamma = 96.92(1)degrees and Z = 2; complex 2, C20H21ClCuN7O6.5S4, crystallizes in an orthorhombic system, space group Pnna with a = 10.839(14), b = 13.208(17), c = 19.75(2) angstrom and Z = 4. The crystal structure of I consists of 1D polymers of nickel(L) units, alternatively connected by single and double bridging mu-(1,3-N-3) ligand with isolated perchlorate anions. Variable temperature magnetic susceptibility data of the complex have been measured and the fitting,of magnetic data was carried out applying the Borris-Almenar formula for such types of alternating one-dimensional S = 1 systems, based on the Hamiltonian H = -J Sigma(S2iS2i-1 + aS(2i)S(2i+1)). The best-fit parameters obtained are J = -106.7 +/- 2 cm(-1); a = 0.82 +/- 0.02; g = 2.21 +/- 0.02. Complex 2 is a 2D network of 4,4 topology with the nodes occupied by the Cu-II ions, and the edges formed by single azide and double bpds connectors. The perchlorate anions are located between pairs of bpds. The magnetic data have been fitted considering the complex as a pseudo-one-dimensional system, with all copper((II)) atoms linked by [mu(1,3-azido) bridging ligands at axial positions (long Cu...N-3 distances) since the coupling through long bpds is almost nil. The best-fit parameters obtained with this model are J = -1.21 +/- 0.2 cm(-1), g 2.14 +/- 0.02. (c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005).

The effects of diet, breed and age of animal at slaughter on the aroma compounds of grilled beef

The aroma volatiles of grilled beef, from animals fed either grass silage or cereal concentrates, were compared. Aberdeen Angus and Holstein-Friesian cross-breed steers, slaughtered at 14 or 24 months, were studied. Compounds formed from linoleic acid, in particular 2-pentylfuran, 1-octen-3-ol, (Z)-2-octen-1-ol, and hexanal were at higher levels in the meat from the animals fed concentrates. Phytenes and compounds formed from α-linolenic acid, in particular 1-penten-3-ol and (Z)-2-penten-1-ol, were at higher levels in the meat of animals fed silage. Differences due to breed were small and not consistent with slaughter age. Dimethyl disulfide, dimethyl disulfide and phenol were at higher levels in the meat of animals slaughtered at 24 months and may contribute to grilled beef aroma.

Alignment of a model amyloid peptide fragment in bulk and at a solid surface

The alignment of model amyloid peptide YYKLVFFC is investigated in bulk and at a solid surface using a range of spectroscopic methods employing polarized radiation. The peptide is based on a core sequence of the amyloid beta (A beta) peptide, KLVFF. The attached tyrosine and cysteine units are exploited to yield information on alignment and possible formation of disulfide or dityrosine links. Polarized Raman spectroscopy on aligned stalks provides information on tyrosine orientation, which complements data from linear dichroism (LD) on aqueous solutions subjected to shear in a Couette cell. LD provides a detailed picture of alignment of peptide strands and aromatic residues and was also used to probe the kinetics of self-assembly. This suggests initial association of phenylalanine residues, followed by subsequent registry of strands and orientation of tyrosine residues. X-ray diffraction (XRD) data from aligned stalks is used to extract orientational order parameters from the 0.48 nm reflection in the cross-beta pattern, from which an orientational distribution function is obtained. X-ray diffraction on solutions subject to capillary flow confirmed orientation in situ at the level of the cross-beta pattern. The information on fibril and tyrosine orientation from polarized Raman spectroscopy is compared with results from NEXAFS experiments on samples prepared as films on silicon. This indicates fibrils are aligned parallel to the surface, with phenyl ring normals perpendicular to the surface. Possible disulfide bridging leading to peptide dimer formation was excluded by Raman spectroscopy, whereas dityrosine formation was probed by fluorescence experiments and was found not to occur except under alkaline conditions. Congo red binding was found not to influence the cross-beta XRD pattern.

Thiolated mucoadhesive and PEGylated nonmucoadhesive organosilica nanoparticles from 3-Mercaptopropyltrimethoxysilane

A novel approach has been developed to synthesize thiolated sub-100 nm organosilica nanoparticles from 3-mercaptopropyltrimethoxysilane (MPTS) through its self-condensation in dimethylsulfoxide in contact with atmospheric oxygen. The formation of MPTS nanoparticles proceeds through the condensation of methoxysilane groups and simultaneous disulfide bridging caused by partial oxidation of thiol groups. These nanoparticles showed excellent colloidal stability in dilute aqueous dispersions but underwent further self-assembly into chains and necklaces at higher concentrations. They exhibited very good ability to adhere to ocular mucosal surfaces, which can find applications in drug delivery. The thiolated nanoparticles could also be easily modified through PEGylation resulting in a loss of their mucoadhesive properties.

Staphylococcal extracellular adherence protein induces platelet activation by stimulation of thiol isomerases

OBJECTIVE: Staphylococcus aureus can induce platelet aggregation. The rapidity and degree of this correlates with the severity of disseminated intravascular coagulation, and depends on platelet peptidoglycans. Surface-located thiol isomerases play an important role in platelet activation. The staphylococcal extracellular adherence protein (Eap) functions as an adhesin for host plasma proteins. Therefore we tested the effect of Eap on platelets. METHODS AND RESULTS: We found a strong stimulation of the platelet-surface thiol isomerases protein disulfide isomerase, endoplasmic reticulum stress proteins 57 and 72 by Eap. Eap induced thiol isomerase-dependent glycoprotein IIb/IIIa activation, granule secretion, and platelet aggregation. Treatment of platelets with thiol blockers, bacitracin, and anti-protein disulfide isomerase antibody inhibited Eap-induced platelet activation. The effect of Eap on platelets and protein disulfide isomerase activity was completely blocked by glycosaminoglycans. Inhibition by the hydrophobic probe bis(1-anilinonaphthalene 8-sulfonate) suggested the involvement of hydrophobic sites in protein disulfide isomerase and platelet activation by Eap. CONCLUSIONS: In the present study, we found an additional and yet unknown mechanism of platelet activation by a bacterial adhesin, involving stimulation of thiol isomerases. The thiol isomerase stimulatory and prothrombotic features of a microbial secreted protein are probably not restricted to S aureus and Eap. Because many microorganisms are coated with amyloidogenic proteins, it is likely that the observed mechanism is a more general one.