Muscle glycogen metabolism changes in rats fed early postnatal a fructose-rich diet after maternal protein malnutrition: effects of acute physical exercise at the maximal lactate steady-state intensity

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

Oxidation of Bovine Albumin by Hypochlorous and Hypobromous Acids: Structural and Functional Alterations

Aims: Hypochlorous (HOCl) and hypobromous (HOBr) acids are among the most powerful oxidants produced by the innate immune cells. Albumin is the predominant protein in most body fluids and is considered the most important antioxidant of blood plasma. Study Design: Oxidation of bovine albumin (BSA) and study of its structural and functional alterations. Place and Duration of Study: Faculty of Science and Faculty of Pharmaceutical Science, University of the State of Sao Paulo UNESP, between June and December 2012. Methodology: BSA was oxidized with excess of HOCl or HOBr and its structural and functional alterations were analyzed by spectroscopic techniques as UV-Vis absorption, intrinsic and synchronous fluorescence, fluorescence quenching, Rayleigh scattering and circular dichroism. Results: Both oxidants were able to deplete the intrinsic fluorescence of BSA, but HOBr was more effective than HOCl. The alterations in the synchronous fluorescence, UV-Vis absorption, and the appearance of a fluorescence band centered at 450 nm confirmed the difference between the oxidants. The oxidation did not induce aggregation of BSA as measured by Rayleigh scattering. The far-UV circular dichroism spectra showed a loss in the helical content and the near-UV-circular dichroism showed an alteration in the tertiary structure; HOBr was the more effective of the oxidants in this case. However, the oxidations did not induce significant alterations in the binding capacity of BSA, which was evaluated using hydrophobic (norfloxacin) and hydrophilic (ascorbic acid) drugs. Conclusion: These results suggest that, although highly susceptible to oxidation, the alterations did not inhibit BSA’s physiological function as a transport protein.

Changes in polyamine, total protein and total carbohydrate content and peroxidase activity during the lifetime of chrysanthemum faroe

The aim of the present work was to evaluate the changes in polyamine (PA) content, peroxidase (POX) activity and levels of total protein and total soluble carbohydrates throughout the lifetime of leaves and inflorescences of chrysantemum 'Faroe' treated with gibberellic acidd (GA3) (used in production practices) and kept at room temperature and cold storage. The treatments were composed of four doses of GA3 (0, 15, 30 and 45 mg L-1) applied at the beggining of flower bud formation (28 days after transplanting of seedlings). After harvesting, the stems (95% of the expanded ligule) were stored at 10ºC and 95% relative humidity for 48 hrs, or kept at room temperature. For biochemical analysis samples of leaves and inflorescences were collected at the 4th, 8th, 12th and 16th day after harvest. The application of GA3 in the field and cold storage increased the content of PAs. There was an increase in POX activity in leaves and inflorescences during postharvest and this increase was related to oxidation of the PAs studied. The amount of proteins and carbohydrates in chrysantemum 'Faroe' decreased during the storage at 25ºC and under cold conditions.

Cytochrome c-promoted cardiolipin oxidation generates singlet molecular oxygen

The interaction of cytochrome c (cyt c) with cardiolipin (CL) induces protein conformational changes that favor peroxidase activity. This process has been correlated with CL oxidation and the induction of cell death. Here we report evidence demonstrating the generation of singlet molecular oxygen [O-2((1)Delta(g))] by a cyt c-CL complex in a model membrane containing CL. The formation of singlet oxygen was directly evidenced by luminescence measurements at 1270 nm and by chemical trapping experiments. Singlet oxygen generation required cyt c-CL binding and occurred at pH values higher than 6, consistent with lipid-protein interactions involving fully deprotonated CL species and positively charged residues in the protein. Moreover, singlet oxygen formation was specifically observed for tetralinoleoyl CL species and was not observed with monounsaturated and saturated CL species. Our results show that there are at least two mechanisms leading to singlet oxygen formation: one with fast kinetics involving the generation of singlet oxygen directly from CL hydroperoxide decomposition and the other involving CL oxidation. The contribution of the first mechanism was clearly evidenced by the detection of labeled singlet oxygen [O-18(2)((1)Delta(g))] from liposomes supplemented with 18-oxygen-labeled CL hydroperoxides. However quantitative analysis showed that singlet oxygen yield from CL hydroperoxides was minor (<5%) and that most of the singlet oxygen is formed from the second mechanism. Based on these data and previous findings we propose a mechanism of singlet oxygen generation through reactions involving peroxyl radicals (Russell mechanism) and excited triplet carbonyl intermediates (energy transfer mechanism).

Cerebral White Matter Oxidation and Nitrosylation in Young Rodents With Kaolin-Induced Hydrocephalus

Hydrocephalus is associated with reduced blood flow in periventricular white matter. To investigate hypoxic and oxidative damage in the brains of rats with hydrocephalus, kaolin was injected into the cisterna magna of newborn 7- and 21-day-old Sprague-Dawley rats, and ventricle size was assessed by magnetic resonance imaging at 7, 21, and 42 days of age. In-situ evidence of hypoxia in periventricular capillaries and glial cells was shown by pimonidazole hydrochloride binding. Biochemical assay of thiobarbituric acid reaction and immunohistochemical detection of malondialdehyde and 4-hydroxy-2-nonenal indicated the presence of lipid peroxidation in white matter. Biochemical assay of nitrite indicated increased nitric oxide production. Nitrotyrosine immunohistochemistry showed nitrosylated proteins in white matter reactive microglia and astrocytes. Activities of the antioxidant enzymes catalase and glutathione peroxidase were not increased, and altered hypoxia-inducible factor 1 alpha was not detected by quantitative reverse transcription-polymerase chain reaction. Cerebral vascular endothelial growth factor expression determined by quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay was not changed, but vascular endothelial growth factor immunoreactivity was increased in reactive astrocytes of hydrocephalic white matter. To determine if nitric oxide synthase is involved in the pathogenesis, we induced hydrocephalus in 7-day-old wild-type and neuronal nitric oxide synthase-deficient mice. At 7 days, the wild-type and mutant mice exhibited equally severe ventriculomegaly and no behavioral differences, although increased glial fibrillary acidic protein was less in the mutant mice. We conclude that hypoxia, via peroxidation and nitrosylation, contributes to brain changes in young rodents with hydrocephalus and that compensatory mechanisms are negligible.

Age-dependent changes in rat lacrimal gland anti-oxidant and vesicular related protein expression profiles

Purpose: Anti-oxidation and exocytosis are important for maintaining exocrine tissue homeostasis. During aging, functional and structural alterations occur in the lacrimal gland (LG), including oxidative damage to proteins, lipids, and DNA. The aims of the present study were to determine in the aging LG: a) the effects of aging on LG structure and secretory activity and b) changes in the expression of oxidative stress markers. Methods: To address these goals, tear secretion composition and corneal impression cytology were compared between male Wistar rats of 2 (control) and 24 (aged) months. LG morphology and the expression levels of vitamin E and malonaldehyde (MDA) were evaluated to determine the anti-oxidant activity and lipid peroxidation, respectively. RT-PCR and western blot analysis were used for the analysis of Ras related in brain GTPase protein (Rab) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the secretory machinery (i.e.; Rab 3d, Rab 27, vesicle-associated membrane protein-2 (Vamp-2), and syntaxin). Results: Histological analysis of aged rats revealed a higher frequency of corneal epithelia metaplasia. In the acinar cells, organelles underwent degeneration, and lipofucsin-like material accumulated in the cytoplasm along with declines in the anti-oxidant marker vitamin E. Rab3d and Rab27b mRNA levels fell along with Rab3d protein expression, whereas syntaxin levels increased. Conclusions: These findings indicate that exocytotic and anti-oxidant mechanisms become impaired with age in the rat LG. In parallel with these structural alterations, functional declines may contribute to the pathophysiology caused by tear film modification in dry eye disease.

Protein Oxidative Stress and Dyslipidemia in Dialysis Patients

Our aim was to investigate and determine the associations between oxidative stress (OS), dyslipidemia and inflammation in patients treated with continuous ambulatory peritoneal dialysis (CAPD) and hemodialysis (HD) using observational cross-sectional study. Twenty patients in CAPD and 48 in HD for at least 8 weeks and aged =18 years were included in the study. Individuals with malignant or acute inflammatory disease were excluded. A control group of 17 healthy individuals was also recruited. The biochemical parameter evaluations were analyzed using colorimetric kits for albumin, serum glucose, total cholesterol (TC) and lipid fractions. To determine the inflammatory status, CRP, IL-6 and TNF-a were analyzed by automated chemiluminescence kits. Plasma advanced oxidation protein products (AOPP) were determined by spectrophotometry. Mean AOPP levels were significantly higher for the HD group compared to the control, and there was no difference in AOPP concentrations between the control and CAPD groups. Dialysis patients had levels of inflammatory parameters higher than controls, and showed a high prevalence of patients with dyslipidemia, especially in CAPD. In the HD group, AOPP was positively correlated with triglycerides (TG) and inversely associated with HDL. Also the HD group was observed to have negative associations between TNF-a and HDL, LDL and TC. In the CAPD group, CRP was inversely correlated with HDL. Hemodialysis patients had increased protein OS and associations of inflammation and dyslipidemia were also observed in these dialysis groups. A more detailed characterization of the relations between oxidative stress and other more traditional risk factors has therapeutic importance, since cardiovascular diseases are the leading cause of death among dialysis patients.

On the temperature stability of extracellular hemoglobin of Glossoscolex paulistus, at different oxidation states: SAXS and DLS studies

Glossoscolex paulistus hemoglobin (HbGp) was studied by dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). DLS melting curves were measured for met-HbGp at different concentrations. SAXS temperature studies were performed for oxy-, cyanomet- and met-HbGp forms, at several pH values. At pH 5.0 and 6.0, the scattering curves are identical from 20 to 60 degrees C, and R-g is 108 angstrom, independent of the oxidation form. At pH 7.0, protein denaturation and aggregation occurs above 55 degrees C and 60 degrees C, for oxy and met-HbGp, respectively. Cyanomet-HbGp, at pH 7.0, is stable up to 60 degrees C. At alkaline pH (8.0-9.0) and higher temperature, an irreversible dissociation process is observed, with a decrease of R-g, D-max and I(0). Analysis by p(r), obtained from GNOM, and OLIGOMER, was used to fit the SAXS experimental scattering curves by a combination of theoretical curves obtained for HbLt fragments from the crystal structure. Our results show clearly the increasing contribution of smaller molecular weight fragments, as a function of increasing pH and temperature, as well as, the order of thermal stabilities: cyanomet-> oxy- > met-HbGp. (C) 2012 Elsevier B.V. All rights reserved.

Release of NO from a nitrosyl ruthenium complex through oxidation of mitochondrial NADH and effects on mitochondria

Nitrosyl ruthenium complexes are promising NO donor agents with numerous advantages for the biologic applications of NO. We have characterized the NO release from the nitrosyl ruthenium complex [Ru(NO2)(bpy)(2)(4-pic)](+) (I) and the reactive oxygen/nitrogen species (ROS/RNS)-mediated NO actions on isolated rat liver mitochondria. The results indicated that oxidation of mitochondrial NADH promotes NO release from (I) in a manner mediated by NO2 formation (at neutral pH) as in mammalian cells, followed by an oxygen atom transfer mechanism (OAT). The NO released from (I) uncoupled mitochondria at low concentrations/incubation times and inhibited the respiratory chain at high concentrations/incubation times. In the presence of ROS generated by mitochondria NO gave rise to peroxynitrite, which, in turn, inhibited the respiratory chain and oxidized membrane protein-thiols to elicit a Ca2+-independent mitochondrial permeability transition; this process was only partially inhibited by cyclosporine-A, almost fully inhibited by the thiol reagent N-ethylmaleimide (NEM) and fully inhibited by the NO scavenger 2-(4-carboxyphenyl)-4,45,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). These actions correlated with the release of cytochrome c from isolated mitochondria as detected by Western blotting analysis. These events, typically involved in cell necrosis and/or apoptosis denote a potential specific action of (I) and analogs against tumor cells via mitochondria-mediated processes. (C) 2012 Elsevier Inc. All rights reserved.

Eliciting the mitochondrial unfolded protein response via NAD(+) repletion reverses fatty liver disease

With no approved pharmacological treatment, non-alcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease in western countries and its worldwide prevalence continues to increase along with the growing obesity epidemic. Here we show that a high-fat high-sucrose (HFHS) diet, eliciting chronic hepatosteatosis resembling human fatty liver, lowers hepatic NAD(+) levels driving reductions in hepatic mitochondrial content, function and ATP levels, in conjunction with robust increases in hepatic weight, lipid content and peroxidation in C57BL/6J mice. In an effort to assess the effect of NAD(+) repletion on the development of steatosis in mice, nicotinamide riboside (NR), a precursor for NAD(+) biosynthesis, was given to mice concomitant, as preventive strategy (NR-Prev), and as a therapeutic intervention (NR-Ther), to a HFHS diet. We demonstrate that NR prevents and reverts NAFLD by inducing a SIRT1- and SIRT3-dependent mitochondrial unfolded protein response (UPR(mt) ), triggering an adaptive mitohormetic pathway to increase hepatic β-oxidation and mitochondrial complex content and activity. The cell-autonomous beneficial component of NR treatment was revealed in liver-specific Sirt1 KO mice (Sirt1(hep-/-) ), while Apolipoprotein E-deficient (Apoe(-/-) ) mice challenged with a high-fat high-cholesterol diet (HFC), affirmed the use of NR in other independent models of NAFLD. CONCLUSION Our data warrant the future evaluation of NAD(+) boosting strategies to manage the development or progression of NAFLD. This article is protected by copyright. All rights reserved.

Identification of the prooxidant site of human ceruloplasmin: A model for oxidative damage by copper bound to protein surfaces

Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [14C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [14C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His426 because CpH426A almost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.

Conversion of cysteine to formylglycine: A protein modification in the endoplasmic reticulum

In sulfatases a Cα-formylglycine residue is found at a position where their cDNA sequences predict a cysteine residue. In multiple sulfatase deficiency, an inherited lysosomal storage disorder, catalytically inactive sulfatases are synthesized which retain the cysteine residue, indicating that the Cα-formylglycine residue is required for sulfatase activity. Using in vitro translation in the absence or presence of transport competent microsomes we found that newly synthesized sulfatase polypeptides carry a cysteine residue and that the oxidation of its thiol group to an aldehyde is catalyzed in the endoplasmic reticulum. A linear sequence of 16 residues surrounding the Cys-69 in arylsulfatase A is sufficient to direct the oxidation. This novel protein modification occurs after or at a late stage of cotranslational protein translocation into the endoplasmic reticulum when the polypeptide is not yet folded to its native structure.

A link between protein structure and enzyme catalyzed hydrogen tunneling

We present evidence that the size of an active site side chain may modulate the degree of hydrogen tunneling in an enzyme-catalyzed reaction. Primary and secondary kH/kT and kD/kT kinetic isotope effects have been measured for the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase at 25°C. As reported in earlier studies, the relationship between secondary kH/kT and kD/kT isotope effects provides a sensitive probe for deviations from classical behavior. In the present work, catalytic efficiency and the extent of hydrogen tunneling have been correlated for the alcohol dehydrogenase-catalyzed hydride transfer among a group of site-directed mutants at position 203. Val-203 interacts with the opposite face of the cofactor NAD+ from the alcohol substrate. The reduction in size of this residue is correlated with diminished tunneling and a two orders of magnitude decrease in catalytic efficiency. Comparison of the x-ray crystal structures of a ternary complex of a high-tunneling (Phe-93 → Trp) and a low-tunneling (Val-203 → Ala) mutant provides a structural basis for the observed effects, demonstrating an increase in the hydrogen transfer distance for the low-tunneling mutant. The Val-203 → Ala ternary complex crystal structure also shows a hyperclosed interdomain geometry relative to the wild-type and the Phe-93 → Trp mutant ternary complex structures. This demonstrates a flexibility in interdomain movement that could potentially narrow the distance between the donor and acceptor carbons in the native enzyme and may enhance the role of tunneling in the hydride transfer reaction.

Slow and fast dietary proteins differently modulate postprandial protein accretion

The speed of absorption of dietary amino acids by the gut varies according to the type of ingested dietary protein. This could affect postprandial protein synthesis, breakdown, and deposition. To test this hypothesis, two intrinsically 13C-leucine-labeled milk proteins, casein (CAS) and whey protein (WP), of different physicochemical properties were ingested as one single meal by healthy adults. Postprandial whole body leucine kinetics were assessed by using a dual tracer methodology. WP induced a dramatic but short increase of plasma amino acids. CAS induced a prolonged plateau of moderate hyperaminoacidemia, probably because of a slow gastric emptying. Whole body protein breakdown was inhibited by 34% after CAS ingestion but not after WP ingestion. Postprandial protein synthesis was stimulated by 68% with the WP meal and to a lesser extent (+31%) with the CAS meal. Postprandial whole body leucine oxidation over 7 h was lower with CAS (272 ± 91 μmol⋅kg−1) than with WP (373 ± 56 μmol⋅kg−1). Leucine intake was identical in both meals (380 μmol⋅kg−1). Therefore, net leucine balance over the 7 h after the meal was more positive with CAS than with WP (P < 0.05, WP vs. CAS). In conclusion, the speed of protein digestion and amino acid absorption from the gut has a major effect on whole body protein anabolism after one single meal. By analogy with carbohydrate metabolism, slow and fast proteins modulate the postprandial metabolic response, a concept to be applied to wasting situations.

Yeast flavin-containing monooxygenase is induced by the unfolded protein response

Flavin-containing monooxygenase from yeast (yFMO) carries out the O2- and NADPH-dependent oxidation of biological thiols, including oxidizing glutathione to glutathione disulfide. FMO provides a large fraction of the oxidizing necessary for proper folding of disulfide bond-containing proteins; deletion of the enzyme reduces proper folding of endogenous carboxypeptidase Y by about 40%. The enzyme is not essential to cell viability because other enzymes can generate a significant fraction of the oxidizing equivalents required by the cell. However, yFMO is vital to the yeast response to reductive stress. FMO1 deletion mutants grow poorly under reductive stress, and carboxypeptidase Y activity is less than 10% of that in a stressed wild type. The FMO1 gene appears to be under control of an unfolded protein response element and is inducible by factors, such as reductive stress, that elicit the unfolded protein response. Reductive stress can increase yFMO activity at least 6-fold. This increased activity allows the cell to process endogenous disulfide bond-containing proteins and also to allow correct folding of disulfide-bonded proteins expressed from multicopy plasmids. The unfolded protein response is mediated by the Hac1p transcription factor that mediates virtually all of the induction of yFMO triggered by exogenous reducing agents.