A ditryptophan cross-link is responsible for the covalent dimerization of human superoxide dismutase 1 during its bicarbonate-dependent peroxidase activity

Unlike intermolecular disulfide bonds, other protein cross-links arising from oxidative modifications cannot be reversed and are presumably more toxic to cells because they may accumulate and induce protein aggregation. However, most of these irreversible protein cross-links remain poorly characterized. For instance, the antioxidant enzyme human superoxide dismutase 1 (hSod1) has been reported to undergo non-disulfide covalent dimerization and further oligomerization during its bicarbonate-dependent peroxidase activity. The dimerization was shown to be dependent on the oxidation of the single, solvent-exposed TrP(32) residue of hSod1, but the covalent dimer was not isolated nor was its structure determined. In this work, the hSod1 covalent dimer was isolated, digested with trypsin in H(2)O and H(2)(18)O, and analyzed by UV-Vis spectroscopy and mass spectrometry (MS). The results demonstrate that the covalent dimer consists of two hSod1 subunits cross-linked by a ditryptophan, which contains a bond between C3 and N1 of the respective Trp(32) residues. We further demonstrate that the cross-link cleaves under usual MS/MS conditions leading to apparently unmodified Trp(32), partially hinders proteolysis, and provides a mechanism to explain the formation of hSod1 covalent trimers and tetramers. This characterization of the covalent hSod1 dimer identifies a novel oxidative modification of protein Trp residues and provides clues for studying its occurrence in vivo. (C) 2010 Elsevier Inc. All rights reserved.

Effect of chemical treatment on the mechanical properties, water vapour permeability and sorption isotherms of gelatin-based films

Proteins contain hydrophilic groups, which can bind to water molecules through hydrogen bridges, resulting in water vapour adsorption. An increase in the degree of cross-linking can be a method to improve the cohesiveness force and functional properties of protein-based films. Thus, the objective of this work was to evaluate the effect of chemical treatment of gelatin with formaldehyde and glyoxal on the mechanical properties, water vapour permeability (WVP) and water vapour sorption characteristics of gelatin-based films. Films were produced using gelatin, with and without chemical treatment. The formaldehyde treatments caused a significant increase in the tensile strength and a reduction in the WVP of films. The Guggenheim-Anderson-De Boer and Halsey models could be used to model the sorption isotherms of films. It was observed that an increase in temperature produced a decrease in water sorption, and the chemical modifications did not affect the monolayer moisture content. Copyright (c) 2007 John Wiley & Sons, Ltd.

Recovery and upgrading bovine rumen protein by extrusion: effect of lipid content on protein disulphide cross-linking, solubility and molecular weight

Bovine rumen protein with two levels of residual lipids (1.9 per cent or 3.8 per cent) was subjected to thermoplastic extrusion under different temperatures and moisture contents. Protein solubility in different buffers, disulphide cross-linking and molecular weight distribution were determined on the extrudates. After extrusion, samples with 1.9 per cent residual lipids content had a higher concentration of protein insoluble by undetermined forces, irrespective of feed moisture and processing temperature used. Lipid content of 3.8 per cent in the feed material resulted in more protein participating in the extrudate network through non-covalent interactions (hydrophobic and electrostatic) and disulphide bonds. A small dependency of the extrusion process on moisture and temperature and a marked dependency on lipid content, especially phospholipid, was observed, Electrophoresis under non-reducing conditions showed that protein extrusion with low feed moisture promoted high molecular breakdown inside the barrel, probably due to intense shear force, and further protein aggregation at the die end

Recovery and upgrading bovine rumen protein by extrusion: Effect of lipid content on protein disulphide cross-linking, solubility and molecular weight

Bovine rumen protein with two levels of residual lipids (1.9% or 3.8%) was subjected to thermoplastic extrusion under different temperatures and moisture contents. Protein Solubility in different buffers, disulphide cross-linking and molecular weight distribution were determined on the extrudates. After extrusion, samples with 1.9% residual lipids content had a higher concentration of protein insoluble by undetermined forces, irrespective of feed moisture and processing temperature used. Lipid content of 3.8% in the feed material resulted in more protein participating in the extrudate network through non-covalent interactions (hydrophobic and electrostatic) and disulphide bonds. A small dependency of the extrusion process on moisture and temperature and a marked dependency on lipid content, especially phospholipid, was observed, Electrophoresis under non-reducing conditions showed that protein extrusion with low feed moisture promoted high molecular breakdown inside the barrel, probably due to intense shear force, and further protein aggregation at the die end. (C) 2009 Elsevier Ltd. All rights reserved.

Development of films based on blends of gelatin and poly(vinyl alcohol) cross linked with glutaraldehyde

Both gelatin and poly(vinyl alcohol) (PVA) can be cross linked with glutaraldehyde (GLU). In the case of gelatin, the GLU reacts with each e-NH2 functional group of adjacent lysine residues, while for PVA, the GLU reacts with two adjacent hydroxyl groups, forming acetal bridges. Thus it can be considered possible to cross link adjacent macromolecules of gelatin and PVA using GLU. In this context, the aims of this work were the development of biodegradable films based on blends of gelatin and poly(vinyl alcohol) cross linked with GLU, and the characterization of some of their main physical and functional properties. All the films were produced from film-forming solutions (FFS) containing 2 g macromolecules (PVA + gelatin)/100 g FFS, 25 g glycerol/100 g macromolecules, and 4 g GLU (25% solution)/100 g FFS. The FFS were prepared with two concentrations of PVA (20 or 50 g PVA/100 g macromolecules) and two reaction temperatures: 90 or 55 degrees C, applied for 30 min. The films were obtained after drying (30 degrees C/24 h) and conditioning at 25 degrees C and 58% of relative humidity for 7 days, and were then characterized. The results for the color parameters, mechanical properties, phase transitions and infrared spectra showed that some chemical modifications occurred, principally for the gelatin. However, in general, all the characteristics of the films were either typical of films based on blends of these macromolecules without cross linking, or slightly higher. A greater improvement in the properties of this material was probably not observed due to the crystallinity of the PVA, which has a melting point above 90 degrees C. The presence of microcrystals in the polymer chain probably reduced macromolecular mobility, hindering the reaction. Thus more research is necessary to produce biodegradable films with improved properties. (C) 2011 Elsevier Ltd. All rights reserved.

Thermal and mechanical characteristics of polyurethane/curaua fiber composites

Polyurethane composites reinforced with curaua fiber at 5, 10 and 20% mass/mass proportions were prepared by using the conventional melt-mixing method. The influence of curaua fibers on the thermal behavior and polymer cohesiveness in polyurethane matrix was evaluated by dynamic mechanical thermal analysis (DMTA) and by differential scanning calorimetry (DSC). This specific interaction between the fibers and the hard segment domain was influenced by the behavior of the storage modulus E` and the loss modulus EaEuro(3) curves. The polyurethane PU80 is much stiffer and resistant than the other composites at low temperatures up to 70A degrees C. All samples were thermoplastic and presented a rubbery plateau over a wide temperature range above the glass transition temperature and a thermoplastic flow around 170A degrees C.

Influence of curing rate on softening in ethanol, degree of conversion, and wear of resin composite

Purpose: To investigate the effect of curing rate on softening in ethanol, degree of conversion, and wear of resin composites. Methods: With a given energy density and for each of two different light-curing units (QTH or LED), the curing rate was reduced by modulating the curing mode. Thus, the irradiation of resin composite specimens (Filtek Z250, Tetric Ceram, Esthet-X) was performed in a continuous curing mode and in a pulse-delay curing mode. Wallace hardness was used to determine the softening of resin composite after storage in ethanol. Degree of conversion was determined by infrared spectroscopy (FTIR). Wear was assessed by a three-body test. Data were submitted to Levene`s test, one and three-way ANOVA, and Tukey HSD test (alpha= 0.05). Results: Immersion in ethanol, curing mode, and material all had significant effects on Wallace hardness. After ethanol storage, resin composites exposed to the pulse-delay curing mode were softer than resin composites exposed to continuous cure (P< 0.0001). Tetric Ceram was the softest material followed by Esthet-X and Filtek Z250 (P< 0.001). Only the restorative material had a significant effect on degree of conversion (P< 0.001): Esthet-X had the lowest degree of conversion followed by Filtek Z250 and Tetric Ceram. Curing mode (P= 0.007) and material (P< 0.001) had significant effect on wear. Higher wear resulted from the pulse-delay curing mode when compared to continuous curing, and Filtek Z250 showed the lowest wear followed by Esthet-X and Tetric Ceram. (Am J Dent 2011;24:115-118).

State of the art etch-and-rinse adhesives

Objectives: The aim of this study was to explore the therapeutic opportunities of each step of 3-step etch-and-rinse adhesives. Methods: Etch-and-rinse adhesive systems are the oldest of the multi-generation evolution of resin bonding systems. In the 3-step version, they involve acid-etching, priming and application of a separate adhesive. Each step can accomplish multiple goals. Acid-etching, using 32-37% phosphoric acid (pH 0.1-0.4) not only simultaneously etches enamel and dentin, but the low pH kills many residual bacteria. Results: Some etchants include anti-microbial compounds such as benzalkonium chloride that also inhibits matrix metalloproteinases (MMPs) in dentin. Primers are usually water and HEMA-rich solutions that ensure complete expansion of the collagen fibril meshwork and wet the collagen with hydrophilic monomers. However, water alone can re-expand dried dentin and can also serve as a vehicle for protease inhibitors or protein cross-linking agents that may increase the durability of resin-dentin bonds. In the future, ethanol or other water-free solvents may serve as dehydrating primers that may also contain antibacterial quaternary ammonium methacrylates to inhibit dentin MMPs and increase the durability of resin-dentin bonds. The complete evaporation of solvents is nearly impossible. Significance: Manufacturers may need to optimize solvent concentrations. Solvent-free adhesives can seal resin-dentin interfaces with hydrophobic resins that may also contain fluoride and antimicrobial compounds. Etch-and-rinse adhesives produce higher resin-dentin bonds that are more durable than most 1 and 2-step adhesives. Incorporation of protease inhibitors in etchants and/or cross-linking agents in primers may increase the durability of resin-dentin bonds. The therapeutic potential of etch-and-rinse adhesives has yet to be fully exploited. (C) 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Microhardness of resin cements in the intraradicular environment: Effects of water storage and softening treament

Objectives. To analyze the microhardness of four dual-cure resin cements used for cementing fiber-reinforced posts under the following conditions: after 7 days of storage in water, after additional 24 h of immersion in 75% ethanol, and after 3 months of storage in water. Hardness measurements were taken at the cervical, middle and apical thirds along the cement line. Methods. Root canals of 40 bovine incisors were prepared for post space. Fibrekor (R) glass fiber-reinforced posts (Jeneric/Pentron) of 1 mm in diameter were cemented using Panavia F 2.0 (Kuraray), Variolink (Ivoclar-Vivadent), Rely X Unicem (3M ESPE) or Duolink (Bisco) (N = 10). After 7 days of water storage at 37 degrees C, half the sample (N = 5) was longitudinally sectioned and the initial microhardness measured along the cement line from cervical to apex. These same samples were further immersed in 75% ethanol for 24 h and reassessed. The remaining half (N = 5) was kept unsectioned in deionized water at 37 degrees C for 3 months, followed by sectioning and measuring. Data were analyzed by a series of two-way ANOVA and Tukey tests at alpha = 5%. Results. Statistically significant differences were identified among the cements, thirds and conditions. Significant interactions were also observed between cements and thirds and between cements and conditions. Panavia F exhibited significantly higher initial microhardness than the other three cements, which showed no statistical difference among themselves. Variolink and Duolink showed significantly higher microhardness values in the cervical third, without significant difference among the thirds for the other cements. Immersion in ethanol significantly reduced the hardness values for all cements, regardless of the thirds. Storage in water for 3 months had no influence on the hardness of most of the cements, with the exception of Unicem that showed a significant increase in the hardness values after this period. Results showed heterogeneity in the microhardness of the cements inside the canal. All cements presented some degree of softening after ethanol treatment, which suggests instability of the polymer. The quality of curing of resin cements in the root canal environment seems unpredictable and highly material dependent. (C) 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Influence of curing protocol on selected properties of light-curing polymers: Degree of conversion, volume contraction, elastic modulus, and glass transition temperature

Objectives. The purpose of this study was to investigate the effect of light-curing protocol on degree of conversion (DC), volume contraction (C), elastic modulus (E), and glass transition temperature (T(g)) as measured on a model polymer. It was a further aim to correlate the measured values with each other. Methods. Different light-curing protocols were used in order to investigate the influence of energy density (ED), power density (PD), and mode of cure on the properties. The modes of cure were continuous, pulse-delay, and stepped irradiation. DC was measured by Raman micro-spectroscopy. C was determined by pycnometry and a density column. E was measured by a dynamic mechanical analyzer (DMA), and T(g) was measured by differential scanning calorimetry (DSC). Data were submitted to two-and three-way ANOVA, and linear regression analyses. Results. ED, PD, and mode of cure influenced DC, C, E, and T(g) of the polymer. A significant positive correlation was found between ED and DC (r = 0.58), ED and E (r = 0.51), and ED and T(g) (r = 0.44). Taken together, ED and PD were significantly related to DC and E. The regression coefficient was positive for ED and negative for PD. Significant positive correlations were detected between DC and C (r = 0.54), DC and E (r = 0.61), and DC and T(g) (r = 0.53). Comparisons between continuous and pulse-delay modes of cure showed significant influence of mode of cure: pulse-delay curing resulted in decreased DC, decreased C, and decreased T(g). Influence of mode of cure, when comparing continuous and step modes of cure, was more ambiguous. A complex relationship exists between curing protocol, microstructure of the resin and the investigated properties. The overall performance of a composite is thus indirectly affected by the curing protocol adopted, and the desired reduction of C may be in fact a consequence of the decrease in DC. (C) 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Effect of the anti-neoplastic drug doxorubicin on XPD-mutated DNA repair-deficient human cells

Doxorubicin (DOX), a member of the anthracycline group, is a widely used drug in cancer therapy. The mechanisms of DOX action include topoisomerase II-poisoning, free radical release, DNA adducts and interstrand cross-link (ICL) formation. Nucleotide excision repair(NER) is involved in the removal of helix-distorting lesions and chemical adducts, however, little is known about the response of NER-deficient cell lines to anti-tumoral drugs like DOX. Wild type and XPD-mutated cells, harbouring mutations in different regions of this gene and leading to XP-D, XP/CS or TTD diseases, were treated with this drug and analyzed for cell cycle arrest and DNA damage by comet assay. The formation of DSBs was also investigated by determination of gamma H2AX foci. Our results indicate that all three NER-deficient cell lines tested are more sensitive to DOX treatment, when compared to wild type cells or XP cells complemented by the wild type XPD cDNA, suggesting that NER is involved in the removal of DOX-induced lesions. The cell cycle analysis showed the characteristic G2 arrest in repair-proficient MRC5 cell line after DOX treatment, whereas the repair-deficient cell lines presented significant increase in sub-G1 fraction. The NER-deficient cell lines do not show different patterns of DNA damage formation as assayed by comet assay and phosphorylated H2AX foci formation. Knock-down of topoisomerase II alpha with siRNA leads to increased survival in both MRC5 and XP cells, however, XP cell line still remained significantly more sensitive to the treatment by DOX. Our study suggests that the enhanced sensitivity is due to DOX-induced DNA damage that is subject to NER, as we observed decreased unscheduled DNA synthesis in XP-deficient cells upon DOX treatment. Furthermore, the complementation of the XPD-function abolished the observed sensitivity at lower DOX concentrations, suggesting that the XPD helicase activity is involved in the repair of DOX-induced lesions. (C) 2009 Elsevier B.V. All rights reserved.

Structural parameters of polyacrylamide hydrogels obtained by the Equilibrium Swelling Theory

In this paper, the synthesis and structural characterization of a series of polyacrylamide hydrogels with different degrees of reticulation are reported. Although the Equilibrium Swelling Theory was recognized as a simple and reliable tool for the determination of structural hydrogels network parameters like equilibrium degree of swelling, cross-link ratio and mesh size, this is the first application of this methodology for polyacrylamide hydrogels. By changing the total monomer content in the synthesis solution (%T) from 5 to 30%, at a fixed value of cross-linker content in the total monomer amount (%C) of 5%, the final parameter obtained, the mesh size, can be tuned from 2 to 0.3 nm. It was also possible to change the mesh size (0.19-0.35) by varying %C from 5 to 12% (at %T = 20%). Scanning Electron Microscopy images for the most different formulations are shown and corroborate data obtained from the theory. (c) 2008 Elsevier Ltd. All rights reserved.

Sensitization Prevalence, Antibody Cross-Reactivity and Immunogenic Peptide Profile of Api g 2, the Non-Specific Lipid Transfer Protein 1 of Celery

Background: Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model. Methodology: 786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs. Results: IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides. Conclusions: Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.

Cross talk Initiated by Endothelial Cells Enhances Migration and Inhibits Anoikis of Squamous Cell Carcinoma Cells through STAT3/Akt/ERK Signaling

It is well known that cancer cells secrete angiogenic factors to recruit and sustain tumor vascular networks. However, little is known about the effect of endothelial cell-secreted factors on the phenotype and behavior of tumor cells. The hypothesis underlying this study is that endothelial cells initiate signaling pathways that enhance tumor cell survival and migration. Here, we observed that soluble mediators from primary human dermal microvascular endothelial cells induce phosphorylation of signal transducer and activator of transcription 3 (STAT3), Akt, and extracellular signal-regulated kinase (ERK) in a panel of head and neck squamous cell carcinoma (HNSCC) cells (OSCC-3, UM-SCC-1, UM-SCC-17B, UM-SCC-74A). Gene expression analysis demonstrated that interleukin-6 (IL-6), interleukin-8 (CXCL8), and epidermal growth factor (EGF) are upregulated in endothelial cells cocultured with HNSCC. Blockade of endothelial cell-derived IL-6, CXCL8, or EGF by gene silencing or neutralizing antibodies inhibited phosphorylation of STAT3, Akt, and ERK in tumor cells, respectively. Notably, activation of STAT3, Akt, and ERK by endothelial cells enhanced migration and inhibited anoikis of tumor cells. We have previously demonstrated that Bcl-2 is upregulated in tumor microvessels in patients with HNSCC. Here, we observed that Bcl-2 signaling induces expression of IL-6, CXCL8, and EGF, providing a mechanism for the upregulation of these cytokines in tumor-associated endothelial cells. This study expands the contribution of endothelial cells to the pathobiology of tumor cells. It unveils a new mechanism in which endothelial cells function as initiators of molecular crosstalks that enhance survival and migration of tumor cells.

Cross-Talk Between Mitochondria and NADPH Oxidase: Effects of Mild Mitochondrial Dysfunction on Angiotensin II-Mediated Increase in Nox Isoform Expression and Activity in Vascular Smooth Muscle Cells

Mitochondria and NADPH oxidase activation are concomitantly involved in pathogenesis of many vascular diseases. However, possible cross-talk between those ROS-generating systems is unclear. We induced mild mitochondrial dysfunction due to mitochondrial DNA damage after 24 h incubation of rabbit aortic smooth muscle (VSMC) with 250 ng/mL ethidium bromide (EtBr). VSMC remained viable and had 29% less oxygen consumption, 16% greater baseline hydrogen peroxide, and unchanged glutathione levels. Serum-stimulated proliferation was unaltered at 24 h. Although PCR amplification of several mtDNA sequences was preserved, D-Loop mtDNA region showed distinct amplification of shorter products after EtBr. Such evidence for DNA damage was further enhanced after angiotensin-II (AngII) incubation. Remarkably, the normally observed increase in VSMC membrane fraction NADPH oxidase activity after AngII was completely abrogated after EtBr, together with failure to upregulate Nox1 mRNA expression. Conversely, basal Nox4 mRNA expression increased 1.6-fold, while being unresponsive to AngII. Similar loss in AngII redox response occurred after 24 h antimycin-A incubation. Enhanced Nox4 expression was unassociated with endoplasmic reticulum stress markers. Protein disulfide isomerase, an NADPH oxidase regulator, exhibited increased expression and inverted pattern of migration to membrane fraction after EtBr. These results unravel functionally relevant cross-talk between mitochondria and NADPH oxidase, which markedly affects redox responses to AngII. Antioxid Redox Signal 11, 1265-1278.