Changes In Protein Oxidation Research Articles

Free Fatty Acids and Oxidative Changes of a Raw Goat Milk Cheese through Maturation

Free fatty acids (FFA) and lipid and protein oxidation changes were studied throughout maturation process of a raw goat milk cheese with protected designation of origin. Cheeses were analyzed at 4 different times of maturation, at 1, 30, 60, and 90 d. All FFA significantly increased during maturation and the relative increase was higher for long-chain than medium- or short-chain FFA. At the end of maturation, oleic (C18:1 n9), butyric (C4:0), and palmitic (C16:0) acids were the most abundant. The higher levels of short-chain fatty acids (SCFA) regarding total FFA obtained at the end of Ibores cheese ripening compared with other raw goat milk cheeses, highlight the notable role of SCFA on the flavor of this cheese owing to their low-odor thresholds. Lipid oxidation values significantly increased during maturation process but low levels of malondialdehyde were reported; however, protein oxidation did not significantly change during ripening.

Simultaneous Detection of Changes in Protein Expression and Oxidative Modification as a Function of Age andAPOEGenotype

To better elucidate temporal changes in protein oxidation resulting from aging and the Alzheimer's disease-associated Apolipoprotein E (ApoE), we developed a 2D-DIGE-based method for simultaneously detecting differential expression and carbonyl oxidation of proteins. Specifically, we examined changes in the levels of oxidation and total protein expression in hippocampi from young-adult (25-30 weeks) and old (76-97 weeks) mice transgenic for the human Apolipoprotein E gene (APOE, APOE3, APOE4) isoforms, APOE3 or APOE4. Protein samples were labeled with either a fluorescent aminooxyacetamide (Alexa Fluor 488) to detect carbonyl modifications or with NHS-Cy3 to detect total protein expression. A protein sample used as an internal control was labeled with NHS-Cy5 and run on each gel. DIGE analysis revealed 38 differentially oxidized and 100 differentially expressed protein spots with significantly different levels (P < 0.05). For oxidized proteins, principal component analysis revealed two distinct clusters: one in which oxidation increased with age independent of APOE genotype, and the second in which oxidation was dependent on APOE genotype. For total protein expression, principal component analysis revealed a large overlap between changes with overall aging and between APOE genotypes. The use of a fluorescent tag to label oxidized proteins, in combination with a NHS-Cy3 to label total protein, makes it possible to determine changes in both protein oxidation and protein expression levels in a single experiment. These studies reveal that the expression levels of peroxiredoxin protein family members Prdx2, 3, and 6 are modified by age, APOE genotype, or both.

Protein oxidative changes in whole and skim milk after ultraviolet or fluorescent light exposure

We investigated how protein changes occur, at the primary or higher structural levels, when proteins are exposed to UV or fluorescent (FL) light while in the complex matrix, milk. Whole milk (WM) or skim milk (SM) samples were exposed to FL or UV light from 0 to 24h at 4°C. Protein oxidation was evaluated by the formation of protein carbonyls (PC), dityrosine bond (DiTyr), and changes in molecular weight (protein fragmentation and polymerization). Oxidative changes in AA residues were measured by PC. Dityrosine and N′-formylkynurenine (NFK), a carbonylation derivative of Trp, were measured by fluorometry. Protein carbonyls increased as a function of irradiation time for both WM and SM. The initial rate for PC formation by exposure to FL light (0.25 or 0.27nmol/h for WM and SM, respectively) was slower than that following exposure to UV light (1.95 or 1.20 nmol/h, respectively). The time course of NFK formation resembled that of PC. After 24h of UV exposure, SM had significantly higher levels of NFK than did WM. In contrast, WM samples irradiated with UV had higher levels of DiTyr than did SM samples, indicating different molecular pathways. The formation of intra- or intermolecular DiTyr bonds could be indicative of changes in the tertiary structure or oligomerization of proteins. The existence of NFK suggests the occurrence of protein fragmentation. Thus, proteolysis and oligomerization were analyzed by sodium dodecyl sulfate-PAGE. After 24h of exposing WM to UV or FL light, all the proteins were affected by both types of light, as evidenced by loss of material in most of the bands. Aggregates were produced only by UV irradiation. Hydrolysis by pepsin and enzyme-induced coagulation by rennet were performed to evaluate altered biological properties of the oxidized proteins. No effect on pepsin digestion or rennet coagulation was found in irradiated SM or WM. The oxidative status of proteins in milk and dairy products is of interest to the dairy industry and consumers. These findings provide knowledge that could be useful in determining the optimal lighting conditions in the dairy industry in general and in cheese making in particular.

Free fatty acids and oxidative changes of a Spanish soft cheese (PDO ‘Torta del Casar’) during ripening

SummaryFree fatty acids (FFA) and lipid and protein oxidation changes during ripening were studied in Torta del Casar cheese. This cheese with protected designation of origin (PDO) is made from raw ewe milk and uses vegetable rennet. Cheeses were analysed at four different stages of ripening at 1, 30, 60 and 90 days. Most FFA significantly increased throughout maturation, except valeric and margaric acids. Acetic acid content increased during ripening and was the most abundant FFA in Torta del Casar cheese at the end of ripening. Short chain fatty acids (SCFA) showed an important increase throughout maturation, especially butyric, isovaleric and isobutyric acids. Lipid oxidation values significantly increased during the first month and decreased in the last 2 months of maturation; however, protein oxidation did not significantly change during ripening. Changes in FFA, especially SCFA, could have great importance in Torta del Casar cheese final characteristics; however, oxidative reactions did not play an important role.

Age-associated changes in protein oxidation and proteasome activities in rat brain: Modulation by antioxidants

The free radical theory of ageing postulates that age-associated neurodegeneration is caused by an imbalance between pro-oxidants and antioxidants resulting in oxidative stress. The current study showed regional variation in brain susceptibility to age-associated oxidative stress as shown by increased lipofuscin deposition and protein carbonyl levels in male rats of age 15–16 months compared to control ones (3–5 months). The hippocampus is the area most vulnerable to change compared to the cortex and cerebellum. However, proteasomal enzyme activity was not affected by age in any of the brain regions studied. Treatment with melatonin or coenzyme Q10 for 4 weeks reduced the lipofuscin content of the hippocampus and carbonyl level. However, both melatonin and coenzyme Q10 treatments inhibited β-glutamyl peptide hydrolase activity. This suggests that these molecules can alter proteasome function independently of their antioxidant actions.

Copper related toxic effects on cellular protein metabolism in human astrocytes

Copper overload due to a defect in the ATPase 7B mediated copper excretion within hepatocytes produces the phenotype of Wilson disease. The overload of hepatocytes with copper results in necrotic liver cells and is accompanied by a high concentration of blood copper levels. That occurs to be the reason for increasing neurological copper concentration. Although copper is linked to oxidation, there are no data on the direct copper related effects in human brain cells. To test the copper induced changes in protein oxidation in human astrocyte like cells. We used U87 cells as model for human astrocytes. Cells were treated with increasing concentrations of copper(II)-chloride in Dulbeccos minimal essential medium. Subsequently, at different time points we investigated: cellular growth, cellular survival under copper treatment, the concentration of oxidized tryptophane in GADPH in vitro as well as the carbonyl concentration and the concentration of oxidized proteins in vivo in U87 glial cells. The viability of cells decreased with both increasing copper concentration and duration of treatment. The concentration of oxidized proteins was directly correlated to the increase of copper concentration and duration of exposure. These observations demonstrate the similarities between copper treatment and treatment with other commonly used oxidants, including hydrogen peroxide. Furthermore, the vulnerability of astrocytes towards copper exposure could be demonstrated. Therefore, these data give further insights into understanding of copper metabolism, which in turn is important to reveal the exact pathological mechanism in copper related diseases such as Wilson disease.

Correlation of oxidative stress and the loss of the nicotinic receptor alpha4 subunit in the temporal cortex of patients with Alzheimer's disease

The correlation between oxidative stress and the loss of nicotinic acetylcholine receptor (nAChR) α4 subunit has been investigated in the temporal cortex from patients with Alzheimer's disease (AD). The level of lipid peroxidation was significantly increased in AD brains, whereas there were no significant changes in protein oxidation either in whole tissues or the cellular membrane protein parts between AD brains and controls. The nAChR α4 subunit at protein level was significantly decreased in AD brains. Furthermore, there was a correlation between the increased levels of lipid peroxidation and the decreased numbers of the α4 subunit protein in AD brains. We suggest that lipid peroxidation might be a sensitive target in AD brain and related the mechanism of the loss of the nAChR α4 subunit in the disease.

Age-related changes in protein oxidation and proteolysis in mammalian cells.

Reactive oxygen species generated as by-products of oxidative metabolism, or from environmental sources, frequently damage cellular macromolecules. Proteins are recognized as major targets of oxidative modification, and the accumulation of oxidized proteins is a characteristic feature of aging cells. An increase in the amount of oxidized proteins has been reported in many experimental aging models, as measured by the level of intracellular protein carbonyls or dityrosine, or by the accumulation of protein-containing pigments such as lipofuscin and ceroid bodies. In younger individuals, moderately oxidized soluble cell proteins appear to be selectively recognized and rapidly degraded by the proteasome. An age-related accumulation of oxidized proteins could, therefore, be a result of declining activity of the proteasome. Previous research to investigate the notion of an age-related decline in the content and/or activity of the proteasome has generated contradictory results. The latest evidence, including our own recent findings, indicates that proteasome activity does, indeed, decline during aging as the enzyme complex is progressively inhibited by oxidized and cross-linked protein aggregates. We propose that cellular aging involves both an increase in (mitochondrial) oxidant production and a progressive decline in proteasome activity. Eventually so much proteasome is inactivated that oxidized proteins begin to accumulate rapidly and contribute to cellular dysfunction and senescence.

5-Aminosalicylic acid protection against oxidative damage to synaptosomal membranes by alkoxyl radicals in vitro.

The antioxidant properties of 5-aminosalicylic acid in vitro were evaluated in a synaptosomal membrane system prepared from gerbil cortical synaptosomes using EPR spin labeling and spectroscopic techniques. MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl) and 5-NS (5-nitroxide stearate) spin labels were used to assess changes in protein oxidation and membrane lipid fluidity, respectively. Synaptosomal membranes were subjected to oxidative stress by incubation with 1 mM azo-bis(isobutyronitrile) (AIBN) or 1 mM 2,2'-azobis(amidino propane) dihydrochloride (AAPH) at 37 degrees C for 30 minutes. The EPR analyses of the samples showed significant oxidation of synaptosomal proteins and a decrease in membrane fluidity. 5-Aminosalicylic acid also was evaluated by means of FRAP (the ferric reducing ability of plasma) test as a potential antioxidant. 5-Aminosalicylic acid also showed protection against the oxidation in gerbil cortical synaptosomes system caused by AIBN and AAPH. These results are consistent with the notion of antioxidant protection against free radical induced oxidative stress in synaptosomal membrane system by this agent.

Changes in macronutrient balance during over- and underfeeding assessed by 12-d continuous whole-body calorimetry

Alterations in energy balance must be accommodated by adjustments in the net storage of the major energy-yielding macronutrients: carbohydrate, protein, and fat. This study used continuous whole-body calorimetry to measure changes in energy expenditure and substrate oxidation during a 12-d imposed energy imbalance in six lean men on mixed diets (overfeeding: 16.5 MJ/d, +33%, n = 3; underfeeding: 3.5 MJ/d, -67%, n = 3). Changes in total energy expenditure (TEE) and its components were modest; TEE changed by +6.2% (overfeeding) and -10.5% (underfeeding). In consequence, body weight changed by +2.90 and -3.18 kg. Marked changes in metabolic fuel selection occurred over the course of the study. Carbohydrate intake (540 and 83 g/d for overfeeding and underfeeding, respectively) exerted direct autoregulatory feedback on carbohydrate oxidation (551 and 106 g/d at day 12 for overfeeding and underfeeding, respectively). Subjects were close to balance by day 5. Changes in protein oxidation were small and not sufficient to prevent the oxidation of body protein mass, or its accretion, in response to energy deficit or surplus. Fat oxidation (59 and 177 g/d for overfeeding and underfeeding, respectively) was not sensitive to dietary fat intake (150 and 20 g/d, for overfeeding and underfeeding, respectively), rather, its oxidation was inversely related to the oxidation of other substrates. Changes in fat balance accounted for 74.1% and 84.0% of the energy imbalance during overfeeding and underfeeding, respectively. This study shows a clear oxidative hierarchy for the macronutrients. Metabolic fuel selection is dominated by the need to maintain carbohydrate balance. This induces inappropriate counterregulatory alterations in fat oxidation during energy surplus.

Ammonia Quotient in Sockeye Salmon (Oncorhynchus nerka)

Ammonia quotients (A.Q., ratio of ammonia excreted to oxygen consumed) were computed from data on daily-fed and starved sockeye salmon (Oncorhynchus nerka). In 22-d starved salmon, where a diel pulse in oxygen consumption, but not in ammonia excretion, persisted, the A.Q. was 0.125 at the prepulse stage (0.11 in fed fish); the lowest A.Q. was 0.06 (0.05 in fed fish) at peak oxygen consumption. The highest A.Q. of 0.24 was obtained in fed fish at the peak of ammonia output. Based on a theoretical estimate of an aerobic maximum A.Q. of 0.33, it is estimated that protein oxidation changes from 18 to 38% in starved sockeye and 15 to 73% in fed fish. The latter value is possibly an overestimate because of the involvement of nonoxidative protein breakdown, but this anaerobic involvement in routine metabolism of sockeye may be negligible. Key words: ammonia excretion, ammonia quotient, Oncorhynchus nerka, daily metabolic pattern, protein catabolism, starvation effect, energy utilization, nitrogen-excretion ratio, Rhinomugil corsula