Avaliação das características físico-químicas de biscoitos caninos naturais e a influência da adição de farinha de casca de uva na validade comercial / Physical and chemical properties analysis of natural canine treats and the influence of the addition of grape flour on shelf life

A indústria de alimentos pet está em constante expansão e vêm seguindo as tendências da alimentação humana, com o objetivo de promover benefícios para a saúde dos animais e satisfação aos tutores. A introdução de compostos naturais na formulação de rações permite a redução da ingestão de aditivos químicos, transgênicos e a possibilidade de ofertar ao animal uma dieta equilibrada e funcional. Desta forma, o presente estudo teve como intuito realizar análises: bromatológica, físico-química e a influência da adição da farinha de uva na validade comercial de um biscoito canino natural. Os resultados foram considerados satisfatórios conforme a legislação vigente. Em virtude disso, a adição da farinha de uva promoveu retardo da oxidação lipídica, contribuindo para o aumento de nove dias da validade comercial.

Conjugation with L,L-diphenylalanine Self-Assemblies Enhances In Vitro Antitumor Activity of Phthalocyanine Photosensitizer.

We present the synthesis and characterization of new peptide conjugates obtained by hierarchical co-assembly of L,L-diphenylalanine (FF) and zinc phthalocyanine complexes (ZnPc) in water. Self-assembly capabilities under defined conditions were investigated by scanning electron microscopy, and photophysical properties were evaluated using UV-Vis and fluorescence spectroscopy. AFM observations demonstrated that these ZnPcs form different highly ordered arrays on the crystalline faces of the FF microplates and that surface roughness significantly changes with the presence of differently substituted phthalocyanine units. XRD assays showed that the overall molecular packing of the conjugates is organized according to a hexagonal symmetry, with ZnPcs hosted in the interstices of the peptide phase. In vitro photodynamic studies were conducted on human breast cancer MCF-7 cells to investigate both cellular uptake and cytotoxicity. It was shown that FF self-assemblies are not toxicity and enhance accumulation of ZnPc in MCF-7 cells, improving apoptotic cell death upon irradiation. Our findings demonstrate enhancement of ZnPc antitumor efficiency by FF conjugates and a proof-of-concept for new photosensitizer carriers based on peptide conjugates.

Structure and Catalysis of Fe(III) and Cu(II) Microperoxidase-11 Interacting with the Positively Charged Interfaces of Lipids.

Numerous applications have been described for microperoxidases (MPs) such as in photoreceptors, sensing, drugs, and hydrogen evolution. The last application was obtained by replacing Fe(III), the native central metal, by cobalt ion and inspired part of the present study. Here, the Fe(III) of MP-11 was replaced by Cu(II) that is also a stable redox state in aerated medium, and the structure and activity of both MPs were modulated by the interaction with the positively charged interfaces of lipids. Comparative spectroscopic characterization of Fe(III) and Cu(II)MP-11 in the studied media demonstrated the presence of high and low spin species with axial distortion. The association of the Fe(III)MP-11 with CTAB and Cu(II)MP-11 with DODAB affected the colloidal stability of the surfactants that was recovered by heating. This result is consistent with hydrophobic interactions of MPs with DODAB vesicles and CTAB micelles. The hydrophobic interactions decreased the heme accessibility to substrates and the Fe(III) MP-11catalytic efficiency. Cu(II)MP-11 challenged by peroxides exhibited a cyclic Cu(II)/Cu(I) interconversion mechanism that is suggestive of a mimetic Cu/ZnSOD (superoxide dismutase) activity against peroxides. Hydrogen peroxide-activated Cu(II)MP-11 converted Amplex Red® to dihydroresofurin. This study opens more possibilities for technological applications of MPs.

Nanostructures for peroxidases.

Peroxidases are enzymes catalyzing redox reactions that cleave peroxides. Their active redox centers have heme, cysteine thiols, selenium, manganese, and other chemical moieties. Peroxidases and their mimetic systems have several technological and biomedical applications such as environment protection, energy production, bioremediation, sensors and immunoassays design, and drug delivery devices. The combination of peroxidases or systems with peroxidase-like activity with nanostructures such as nanoparticles, nanotubes, thin films, liposomes, micelles, nanoflowers, nanorods and others is often an efficient strategy to improve catalytic activity, targeting, and reusability.

Protective Effect of Plantago major Extract against t-BOOH-Induced Mitochondrial Oxidative Damage and Cytotoxicity.

Plantago major L. produces several chemical substances with anti-inflammatory and analgesic activities and its use in the treatment of oral and throat inflammation in popular medicine is well described. In this study, the antioxidant potential of the Plantago major hydroethanolic extract was screened and its protective action was evaluated against t-BOOH-induced oxidative stress. The extract was obtained by fractionated percolation using 50% ethanolic solution and, after drying, suspended in dimethyl sulfoxide. The chromatographic profile of crude extract was obtained with the identification of some phytochemical markers and the total phenols and flavonoids were quantified. The scavenger activity against DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals was determined and the antioxidant activity in biological systems was evaluated in isolated rat liver mitochondria and HepG2 cells. The extract exhibited a significant free radical scavenger activity at 0.1 mg/mL, and decreased the ROS (reactive oxygen species) generation in succinate-energized mitochondria. Such an effect was associated with the preservation of the intrinsic antioxidant defenses (reduced glutathione and NAD(P)H) against the oxidation by t-BOOH, and also to the protection of membranes from lipid oxidation. The cytoprotective effect of PmHE against t-BOOH induced cell death was also shown. These findings contribute to the understanding of the health benefits attributed to P. major.

Intermediate Tyrosyl Radical and Amyloid Structure in Peroxide-Activated Cytoglobin.

We characterized the peroxidase mechanism of recombinant rat brain cytoglobin (Cygb) challenged by hydrogen peroxide, tert-butylhydroperoxide and by cumene hydroperoxide. The peroxidase mechanism of Cygb is similar to that of myoglobin. Cygb challenged by hydrogen peroxide is converted to a Fe4+ oxoferryl π cation, which is converted to Fe4+ oxoferryl and tyrosyl radical detected by direct continuous wave-electron paramagnetic resonance and by 3,5-dibromo-4-nitrosobenzene sulfonate spin trapping. When organic peroxides are used as substrates at initial reaction times, and given an excess of peroxide present, the EPR signals of the corresponding peroxyl radicals precede those of the direct tyrosyl radical. This result is consistent with the use of peroxide as a reducing agent for the recycling of Cygb high-valence species. Furthermore, we found that the Cygb oxidation by peroxides leads to the formation of amyloid fibrils. This result suggests that Cygb possibly participates in the development of degenerative diseases; our findings also support the possible biological role of Cygb related to peroxidase activity.

Thiosemicarbazone p-Substituted Acetophenone Derivatives Promote the Loss of Mitochondrial Δψ, GSH Depletion, and Death in K562 Cells.

A series of thiosemicarbazone (TSC) p-substituted acetophenone derivatives were synthesized and chemically characterized. The p-substituents appended to the phenyl group of the TSC structures were hydrogen, fluor, chlorine, methyl, and nitro, producing compounds named TSC-H, TSC-F, TSC-Cl, TSC-Me, and TSC-NO2, respectively. The TSC compounds were evaluated for their capacity to induce mitochondrial permeability, to deplete mitochondrial thiol content, and to promote cell death in the K562 cell lineage using flow cytometry and fluorescence microscopy. TSC-H, TSC-F, and TSC-Cl exhibited a bell-shaped dose-response curve for the induction of apoptosis in K562 cells due to the change from apoptosis to necrosis as the principal mechanism of cell death at the highest tested doses. TSC-Me and TSC-NO2 exhibited a typical dose-response profile, with a half maximal effective concentration of approximately 10 µM for cell death. Cell death was also evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which revealed lower toxicity of these compounds for peripheral blood mononuclear cells than for K562 cells. The possible mechanisms leading to cell death are discussed based on the observed effects of the new TSC compounds on the cellular thiol content and on mitochondrial bioenergetics.

Effects of trichlorotelluro-dypnones on mitochondrial bioenergetics and their relationship to the reactivity with protein thiols.

The effect of four trichlorotelluro-dypnones, named compounds 1, 2, 3, and 4, on the bioenergetics of isolated rat liver mitochondria (RLM) and cells was investigated. In a dose-dependent manner, the studied organotelluranes promoted Ca(2+)-dependent mitochondrial swelling inhibited by cyclosporine A and were associated with a decrease of the total mitochondrial protein thiol content. These effects characterize the opening of the classical mitochondrial permeability transition pore. Despite the reactivity with mitochondrial protein thiol groups, these compounds did not promote significant glutathione depletion. In the absence of Ca(2+), the organotelluranes promoted mitochondrial loss of ΔΨ in RLM concomitant with respiratory control decrease due to an increase of the state 4 respiration rate. In these conditions, mitochondrial swelling was absent, and thiol content was higher than that in the presence of Ca(2+). The differentiated effects observed in the presence and absence of Ca(2+) are probably related to the effects of that ion on membrane structure, with repercussions for the exposure of specific reactive protein thiol groups. In smooth muscle cells, these compounds promoted the loss of mitochondrial ΔΨ and apoptosis. The loss of ΔΨ was not preceded by a decrease of cell viability that is consistent with mitochondria as the primary targets for the action of these organotelluranes.

Hydroxyl scavenging activity accounts for differential antioxidant protection of Plantago major against oxidative toxicity in isolated rat liver mitochondria.

OBJECTIVES: The aim of this work was to study the effects of P. major against the oxidative damage of isolated rat liver mitochondria. METHODS: The extracts were obtained using methanol (MeOH), ethyl acetate (EAc), dichloromethane (DCM), and hexane (Hex) as solvents. KEY FINDINGS: Hex, DCM, and EAc totally, and MeOH partially, inhibited ROS generation and lipid peroxidation of membranes induced by Fe(2+) or t-BOOH. However, only MeOH was able to prevent the t-BOOH-induced glutathione and NAD(P)H oxidation. All extracts chelated Fe(2+) and reduced DPP Hradicals. EPR analysis revealed that P. major exhibited potent scavenger activity for hydroxyl radicals. CONCLUSIONS: The potent antioxidant activity exhibited by P. major was able to prevent oxidative mitochondrial damage, contributing to the understanding of its hepatoprotective action against ROS-mediated toxicity.

Towards the mechanisms involved in the antioxidant action of MnIII [meso-tetrakis(4-N-methyl pyridinium) porphyrin] in mitochondria.

Aerobic organisms are afforded with an antioxidant enzymatic apparatus that more recently has been recognized to include cytochrome c, as it is able to prevent hydrogen peroxide generation by returning electrons from the superoxide ion back to the respiratory chain. The present study investigated the glutathione peroxidase (GPx), superoxide dismutase (SOD) and cytochrome c-like antioxidant activities of para Mn(III)TMPyP in isolated rat liver mitochondria (RLM) and mitoplasts. In RLM, Mn(III)TMPyP decreased the lipid-peroxide content associated with glutathione (GSH) depletion consistent with the use of GSH as a reducing agent for high valence states of Mn(III)TMPyP. SOD and cytochrome c antioxidant activities were also investigated. Mn(II)TMPyP was able to reduce ferric cytochrome c, indicating the potential to remove a superoxide ion by returning electrons back to the respiratory chain. In antimicyn A-poisoned mitoplasts, Mn(III)TMPyP efficiently decreased the EPR signal of DMPO-OH adduct concomitant with GSH depletion. The present results are consistent with SOD and GPx activities for Mn(III)TMPyP and do not exclude cytochrome c-like activity. However, considering that para Mn(III)TMPyP more efficiently reduces, rather than oxidizes, superoxide ion; electron transfer from the Mn(II)TMPyP to the respiratory chain might not significantly contribute to the superoxide ion removal, since most of Mn(II)TMPyP is expected to be produced at the expense of NADPH/GSH oxidation. The present results suggest GPx-like activity to be the principal antioxidant mechanism of Mn(III)TMPyP, whose efficiency is dependent on the NADPH/GSH content in cells.

Superoxide radical protects liposome-contained cytochrome c against oxidative damage promoted by peroxynitrite and free radicals.

The effects of nitrosative species on cyt c structure and peroxidase activity were investigated here in the presence of O(2)(*-) and anionic and zwitterionic vesicles. Nitrosative species were generated by 3-morpholinesydnonymine (SIN1) decomposition, using cyt c heme iron and/or molecular oxygen as electron acceptor. Far- and near-UV CD spectra of SIN1-treated cyt c revealed respectively a slight decrease of alpha-helix content (from 39 to 34%) and changes in the tryptophan structure accompanied by increased fluorescence. The Soret CD spectra displayed a significant decrease of the positive signal at 403 nm. EPR spectra revealed the presence of a low-spin cyt c form (S=1/2) with g(1)=2.736, g(2)=2.465, and g(3)=2.058 after incubation with SIN1. These data suggest that the concomitant presence of NO(*) and O(2)(*-) generated from dissolved oxygen, in a system containing cyt c and liposomes, promotes chemical and conformational modifications in cyt c, resulting in a hypothetical bis-histidine hexacoordinated heme iron. We also show that, paradoxically, O(2)(*-) prevents not only membrane lipoperoxidation by peroxide-derived radicals but also oxidation of cyt c itself due to the ability of O(2)(*-) to reduce heme iron. Finally, lipoperoxidation measurements showed that, although it is a more efficient peroxidase, SIN1-treated cyt c is not more effective than native cyt c in promoting damage to anionic liposomes in the presence of tert-ButylOOH, probably due to loss of affinity with negatively charged lipids.

Peroxidase catalytic cycle of MCM-41-entrapped microperoxidase-11 as a mechanism for phenol oxidation.

The encapsulation of microperoxidases (MPs) into molecular sieves with controlled pore size, such as the mesoporous silica MCM-41, represents a nanotechnology strategy to control the catalytic properties of MPs and mimic the enzymatic activity of hemoproteins. In this work, the ferric microperoxidase-11 (MP-11), obtained from trypsin-catalyzed hydrolysis of horse-heart cytochrome c, was entrapped in MCM-41, thus resulting in a catalyst (Fe(III)MP11MCM41) with catalase and monooxygenase properties. The entrapment of MP-11 inside MCM-41 was confirmed by elemental analysis and UV-visible spectrum, with a red shift in the Soret band indicating that the heme group was in a hydrophobic microenvironment. Similarly to catalase, the catalyst Fe(III)MP11MCM41 exhibited specificity for hydrogen peroxide to be converted to a high-valence oxidized intermediate, Compound II. Also mimicking catalase, the cleavage of hydrogen peroxide by MP11MCM41 resulted in O2 production detected by a Clark electrode. Phenol was able to act as reducing agent of MP11MCM41 Compound II leading to the completion of a peroxidase cycle, as confirmed by UV-visible spectrometry and EPR measurements. The analysis of the reaction products by high performance liquid chromatogram coupled to tandem mass spectrometry (HPLC/MS) revealed 2,4-dihydroxyphenol as the product of phenol oxidation by MP11MCM41. Therefore, in addition to catalase activity, the catalyst MP11MCM41 also displayed monooxygenase properties, which was possible because the MP-11 heme iron promotes homolytic cleavage of the hydrogen peroxide generating hydroxyl radicals. With such characteristics, MCM-41-entrapped MP-11 is a promising catalyst for nanobiotechnological devices.

Reaction route control by microperoxidase-9/CTAB micelle ratios.

Microperoxidases (MP) as water-soluble models attract interest to studying the reaction mechanism of peroxidases because these heme peptides are able to form the same enzyme intermediates during the reaction with peroxides. In this work we have demonstrated that the association of Fe(III)MP-9 and Fe(III)MP-11 with CTAB micelles (MP-9/CTAB and MP11/CTAB) provides a microenvironment with an alkaline interface and a hydrophobic core that exhibits peroxidase behavior. This microenvironment shifts positively the redox potential of microperoxidases by approximately 100 mV. tert-Butylhydroperoxide (t-BuOOH) when added to the medium, converted Fe(III)MP-9/CTAB to MP-9/CTAB Compound II, a high valence oxidized intermediate of the heme peptide. Subsequent addition of diphenylacetaldehyde (DPAA) to MP-9/CTAB Compound II regenerated the native form of the enzyme, Fe(III)MP-9/CTAB, what characterizes the occurrence of a peroxidase cycle. Fe(III)MP-9/CTAB regenerated during the peroxidase cycle reacted with residual DPAA in the medium to form Fe(II)MP-9/CTAB, which indicates that both Fe(III)MP-9/CTAB and its oxyferryl form can use aldehydes as reducing agents. According to the determined reduction potential, Fe(III)MP-9 and Fe(III)MP-9/CTAB should be able to oxidize DPAA (reduction potential -630 mV). The reaction of MP-9/CTAB with DPAA produced benzophenone as final product, detected by infrared spectroscopy and mass spectrometry. Interestingly, a significant difference was observed in the benzophenone yield according to the micelle/MP-9 molar ratio.

Low spin states of microperoxidases produced by inter- and intra-peptide chain sixth ligands: effect of pH and the oligopeptide type.

The low spin states of microperoxidases (MP)-8, -9 and -9 N-acetylated (N-Ac) were characterized using UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopies over the 6.0-12.0 pH range. The first MP-8 alkaline transition (pK(a)=8.53) produced hemepeptide aggregates in the low spin state in which a water molecule was replaced by the peptide chain N-terminal group of a neighboring MP-8 molecule. Higher pH led to the deprotonation of the MP-8 histidine imidazole ring (pK(a)=10.37) at the fifth coordination position. This MP-8 species was in equilibrium with a high spin state aggregate in which OH(-) replaced histidinate, the histidinate becoming the heme iron sixth ligand in a neighboring MP-8 molecule. In a similar way to the N-AcMP-8, the low spin state of N-AcMP-9 was produced by the deprotonation of the water molecule (pK(a)=9.6) situated at the sixth coordination position of the heme iron. Up to pH 8.5, the low spin states of MP-9 were aggregates in which the alpha-amino group of Lys13 replaced water at the sixth coordination position of a neighboring MP-9 molecule. Above pH 8.5, the epsilon-amino groups of Lys13 established intra-chain coordination and impaired the formation of aggregates. Such intra-chain interaction in MP9 was supported by molecular dynamics simulation. These MP-9 monomers might also exhibit OH(-) or histidinate at the fifth coordination position.

Modulation of cytochrome c spin states by lipid acyl chains: a continuous-wave electron paramagnetic resonance (CW-EPR) study of haem iron.

This work is a systematic study, showing a clear correlation between the nature of the lipid acyl chain and the spin states of cytochrome c interacting with different types of lipid membranes. According to the lipid acyl chain type, and the head group charge present in the bilayer, three spin states of cytochrome c were observed in different proportions: the native cytochrome c low spin state with rhombic symmetry (spin 1/2, g axially=3.07 and g radially=2.23), a low spin state with less rhombic symmetry (spin 1/2, g(1)=2.902, g(2)=2.225, and g(3)=1.510) and the high spin state (spin 5/2, g axially=6.0 and g radially=2.0). The proportion of the spin states of cytochrome c bound to bilayers was also dependent on the lipid/protein ratio, suggesting the existence of two or more protein sites interacting with the lipids. The lipid-induced alterations in the symmetry and spin states of cytochrome c exhibited partial reversibility when the ionic strength was increased, which reinforces the crucial role played by the electrostatic interaction with the lipid bilayer. Different cytochrome c spin states exhibited corresponding modifications in the haemprotein UV/visible spectra, particularly in the Q-band associated with loss of the 695 nm band and appearance of a band in the region of 600-650 nm. The observed reactivity of cytochrome c with oxidized forms of unsaturated lipids reinforces the possibility of the acyl chain insertion in the haemprotein structure.

Cierre de incisiones heridas quirúrgicas con sutura convencional vs. cianoacrilato, descripción clínica y estudio histológico en animales de experimentación reporte de casos / Suture technics in dentistry area

En el transcurso del tiempo las técnicas de sutura han ido evolucionando y ofreciendo diferentes alternativas de acuerdo a nuestra conveniencia en el campo odontológico,la cual tiene como finalidad confrontar o adherir dos bordes o extremos a una solución de continuidad en la mucosa oral. Sin embargo hasta el momento no se ha visto más allá de una aguja y de un hilo aumentando así el área de injuria en los tejidos orales,la cual proporciona buenos resultados ya que la utilización de materiales novedosos como el cianoacrilato es baja debido a que carece de bases sólidas que demuestren su efectividad en el proceso de cicatrización tanto clínico como histológico. Para salir del convencionalismo en cuanto a técnicas de sutura se refiere,quisimos explorar en este campo con el método de cianoacrilato(Histosella) puesto que es una alternativa que nos ofrece rapidez,economía,reducción de tiempo. Evaluando así los eventos de cicatrización clínicos e histológicos.para este estudio el equipo experiental fué de 4 perros criollos sin importar el sexo,pero teniendo en cuenta que la edad oscilará de 5 meses y medio a 7 meses;el peso de 2.5kg.a3.5Kg;con los esquemas de vacunación completos(Parvovirosis,triple,leptosporina,hepatitis,distemper(Moquillo))previamente desparasitado y con dieta controlada.Estos perros fueron sometidos a un ayuno de 12hrs antes de la intervención. Posteriormente se afeitó la parte ventro medial de pies,manos y cuello con el fín de obtener una zona apta para la aplicación de la venoclisis.