Assessing the structural and foaming property changes in egg yolk proteins due to malondialdehyde: Experimental and molecular docking studies.

This study evaluated the effects of varying levels of malondialdehyde (MDA) on the structural and foaming properties of the egg yolk proteins (EYPs), and the interaction between them was explored by molecular docking. The results showed that oxidative modification due to MDA increased the carbonyl content of EYPs by 4.49 times. Simultaneously, the total sulfhydryl content was reduced by 21.47%, and the solubility of EYPs was significantly decreased (p < 0.05). Continuous oxidation disorders the previously ordered structure of EYPs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that some proteins underwent crosslinking and aggregation with increased MDA oxidation, aligning with changes in particle size and zeta-potential. Moderate oxidation (<1 mmol/L) enhanced the foaming capacity and foam stability of EYPs. Additionally, molecular docking results uncovered favorable interactions between MDA and specific EYPs, primarily through hydrogen bonding. This research offers valuable insights into managing the functional and quality changes of yolk products during processing.

Secondary Lipid Oxidation Products as Modulators of Calpain-2 Functionality In Vitro.

The objective was to understand the impacts of secondary lipid oxidation products on calpain-2 activity and autolysis and, subsequently, to determine the quantity and localization of modification sites. 2-Hexenal and 4-hydroxynonenal incubation significantly decreased calpain-2 activity and slowed the progression of autolysis, while malondialdehyde had minimal impact on calpain-2 activity and autolysis. Specific modification sites were determined with LC-MS/MS, including distinct malondialdehyde modification sites on the calpain-2 catalytic and regulatory subunits. 2-Hexenal modification sites were observed on the calpain-2 catalytic subunit. Intact protein mass analysis with MALDI-MS revealed that a significant number of modifications on the calpain-2 catalytic and regulatory subunits are likely to exist. These observations confirm that specific lipid oxidation products modify calpain-2 and may affect the calpain-2 functionality. The results of these novel experiments have implications for healthy tissue metabolism, skeletal muscle growth, and post-mortem meat tenderness development.

Synthesizing Amino Acids Modified with Reactive Carbonyls in Silico to Assess Structural Effects Using Molecular Dynamics Simulations.

Protein carbonylation by reactive aldehydes derived from lipid peroxidation leads to cross-linking, oligomerization, and aggregation of proteins, causing intracellular damage, impaired cell functions, and, ultimately, cell death. It has been described in aging and several age-related chronic conditions. However, the basis of structural changes related to the loss of function in protein targets is still not well understood. Hence, a route to the in silico construction of new parameters for amino acids carbonylated with reactive carbonyl species derived from fatty acid oxidation is described. The Michael adducts for Cys, His, and Lys with 4-hydroxy-2-nonenal (HNE), 4-hydroxy-2-hexenal (HHE), and a furan ring form for 4-Oxo-2-nonenal (ONE), were built, while malondialdehyde (MDA) was directly attached to each residue. The protocol describes details for the construction, geometry optimization, assignment of charges, missing bonds, angles, dihedral angles parameters, and its validation for each modified residue structure. As a result, structural effects induced by the carbonylation with these lipid derivatives have been measured by molecular dynamics simulations on different protein systems such as the thioredoxin enzyme, bovine serum albumin and the membrane Zu-5-ankyrin domain employing root-mean-square deviation (RMSD), root mean square fluctuation (RMSF), structural secondary prediction (DSSP) and the solvent-accessible surface area analysis (SASA), among others.

Molecular dynamics of structural effects of reactive carbonyl species derivate of lipid peroxidation on bovine serum albumin.

BACKGROUND: Serum albumin is the most abundant protein in the Mammalia blood plasma at where plays a decisive role in the transport wide variety of hydrophobic ligands. BSA undergoes oxidative modifications like the carbonylation by the reactive carbonyl species (RCSs) 4-hydroxy-2-nonenal (HNE), 4 hydroxy-2-hexenal (HHE), malondialdehyde (MDA) and 4-oxo-2-nonenal (ONE), among others. The structural and functional changes induced by protein carbonylation have been associated with the advancement of neurodegenerative, cardiovascular, metabolic and cancer diseases. METHODS: To elucidate structural effects of protein carbonylation with RCSs on BSA, parameters for six new non-standard amino acids were designated and molecular dynamics simulations of its mono­carbonylated-BSA systems were conducted in the AMBER force field. Trajectories were evaluated by RMSD, RMSF, PCA, RoG and SASA analysis. RESULTS: An increase in the conformational instability for all proteins modified with local changes were observed, without significant changes on the BSA global three-dimensional folding. A more relaxed compaction level and major solvent accessible surface area for modified systems was found. Four regions of high molecular fluctuation were identified in all modified systems, being the subdomains IA and IIIB those with the most remarkable local conformational changes. Regarding essential modes of domain movements, it was evidenced that the most representatives were those related to IA subdomain, while IIIB subdomain presented discrete changes. CONCLUSIONS: RCSs induces local structural changes on mono­carbonylated BSA. Also, this study extends our knowledge on how carbonylation by RCSs induce structural effects on proteins.

Bioanalysis of Stress Biomarkers through Sensitive HILIC-MS/MS Method: A Stride toward Accurate Quantification of MDA, ACR, and CTA.

Quantifying reactive aldehyde biomarkers, such as malondialdehyde, acrolein, and crotonaldehyde, is the most preferred approach to determine oxidative stress. However, reported analytical methods lack specificity for accurately quantifying these aldehydes as certain methodologies may produce false positive results due to harsh experimental conditions. Thus, in this research work, a novel HILIC-MS/MS method with endogenous histidine derivatization is developed, which proves to have higher specificity and reproducibility in quantifying these aldehydes from the biological matrix. To overcome the reactivity of aldehyde, endogenous histidine is used for its derivatization. The generated adduct is orthogonally characterized by NMR and LC-HRMS. The method employed a hydrophilic HILIC column and multiple reaction monitoring (MRM) to accurately quantify these reactive aldehydes. The developed method is an unequivocal solution for quantifying stress in in vivo and in vitro studies.

Malondialdehyde-Induced Post-Translational Modification of Human Hemoglobin.

Lysine residues in proteins undergo multiple enzymatic and nonenzymatic post-translational modifications (PTMs). The terminal ε amine group of lysine residues in proteins is carbonylated chemically by carbonyl species such as glyoxal (GO; OCH-CHO, C2H2O2; MW 58) and methylglyoxal (MGO; OCH-C(=O)-CH3, C3H4O2; MW 72) that are derived from the metabolism of endogenous substances including glucose. The dicarbonyl species malondialdehyde (MDA, OCH-CH2-CHO, C3H4O2; MW 72) is generated by enzymatic and nonenzymatic peroxidation of polyunsaturated fatty acids (PUFAs). GO, MGO, and MDA occur in biological systems in their free forms and in their conjugated forms adducted to free amino acids and amino acid residues in proteins, notably to lysine. MDA is a C-H-acidic acid (pKa, 4.45). Biological MDA is widely used as a biomarker of lipid peroxidation. The most frequently analyzed biological samples for MDA are plasma and serum. Reportedly, MDA concentrations in plasma and serum samples of healthy and ill humans range by several orders of magnitude. The most severe preanalytical contributor is artificial formation of MDA in lipid-rich samples such as plasma and serum. In very few publications, plasma MDA concentrations were reported to lie in the lower mM-range.

Response to "Malondialdehyde-Induced Post-Translational Modification of Human Hemoglobin".

Although malondialdehyde and methylglyoxal have the same molecular formula, they have different chemistry in forming protein adducts. The major lysine adduct of malondialdehyde in hemoglobin is the N-propenal type, while that of methylglyoxal is N6-(1-carboxyethyl)lysine. This Letter provides evidence that the "methylglyoxal-like" hemoglobin adducts are not derived from malondialdehyde. This Letter also discusses the quantification of malondialdehyde-induced post-translational modifications in human hemoglobin by different mass spectrometry-based methods.

The MD17 datasets from the perspective of datasets for gas-phase "small" molecule potentials.

There has been great progress in developing methods for machine-learned potential energy surfaces. There have also been important assessments of these methods by comparing so-called learning curves on datasets of electronic energies and forces, notably the MD17 database. The dataset for each molecule in this database generally consists of tens of thousands of energies and forces obtained from DFT direct dynamics at 500 K. We contrast the datasets from this database for three "small" molecules, ethanol, malonaldehyde, and glycine, with datasets we have generated with specific targets for the potential energy surfaces (PESs) in mind: a rigorous calculation of the zero-point energy and wavefunction, the tunneling splitting in malonaldehyde, and, in the case of glycine, a description of all eight low-lying conformers. We found that the MD17 datasets are too limited for these targets. We also examine recent datasets for several PESs that describe small-molecule but complex chemical reactions. Finally, we introduce a new database, "QM-22," which contains datasets of molecules ranging from 4 to 15 atoms that extend to high energies and a large span of configurations.

Malondialdehyde as an Important Key Factor of Molecular Mechanisms of Vascular Wall Damage under Heart Diseases Development.

This mini review is devoted to a specific issue: the role of malondialdehyde (MDA)-a secondary product of free radical lipid peroxidation-in the molecular mechanisms of the formation of primary atherosclerotic vascular wall lesions. The principal difference between this review and the available literature is that it discusses in detail the important role in atherogenesis not of "oxidized" LDL (i.e., LDL particles containing lipohydroperoxides), but of LDL particles chemically modified by the natural low-molecular weight dicarbonyl MDA. To confirm this, we consider the data obtained by us earlier, indicating that "atherogenic" are not LDL oxidized as a result of free radical lipoperoxidation and containing lipohydroperoxy derivatives of phospholipids in the outer layer of particles, but LDL whose apoprotein B-100 has been modified due to the chemical reaction of terminal lysine residue amino groups of the apoB-100 with the aldehyde groups of the MDA (Maillard reaction). In addition, we present our original data proving that MDA injures endothelial glycocalyx that suppress the ability of the endothelium to control arterial tone according to changes in wall shear stress. In summary, this mini review for the first time exhaustively discloses the key role of MDA in atherogenesis.

A Simple HPLC/DAD Method Validation for the Quantification of Malondialdehyde in Rodent's Brain.

In the present study, a HPLC/DAD method was set up to allow for the determination and quantification of malondialdehyde (MDA) in the brain of rodents (rats). Chromatographic separation was achieved on Supelcosil LC-18 (3 µm) SUPELCO Column 3.3 cm × 4.6 mm and Supelco Column Saver 0.5 µm filter by using a mobile phase acetonitrile (A) and phosphate buffer (20 mM, pH = 6) (B). Isocratic elution was 14% for (A) and 86% for (B). The injection volume (loop mode) was 100 µL with an analysis time of 1.5 min. Flow rate was set at 1 mL/min. The eluted compound was detected at 532 nm by a DAD detector by keeping the column oven at room temperature. The results indicated that the method has good linearity in the range of 0.2-20 µg/g. Both intra- and inter-day precision, expressed as RSD, were ≤15% and the accuracies ranged between ±15%. The lower limit of quantification (LLOQ), stability, and robustness were evaluated and satisfied the validation criteria. The method was successfully applied in a study of chronic toxicology following different treatment regimens with haloperidol and metformin.

Resveratrol activates PI3K/AKT to reduce myocardial cell apoptosis and mitochondrial oxidative damage caused by myocardial ischemia/reperfusion injury.

Resveratrol is a kind of iPolyphenols widely existing in herbal medicine. Here we aim to investigate whether resveratrol can reduce the degree of myocardial ischemia/reperfusion (IR) injury and inhibit the development of oxidative stress, and elucidate the molecular mechanism of resveratrol in protecting myocardial cells. The primary rat cardiomyocytes were used to establish an ischemia/reperfusion model in vitro, and a series of routine biochemical experiments were conducted to explore the antioxidant and anti-apoptotic effects of resveratrol in myocardial ischemia-reperfusion injury. Compared with that of the simulated ischemia-refusion (SIR) group, cell viability in the SIR and resveratrol co-treatment groups increased significantly (P < 0.001), the release of lactate dehydrogenase (LDH) and creatine kinase MB (CKMB) decreased, the positive rate of reactive oxygen species (ROS) in cardiomyocytes decreased, and the concentration of catalase and glutathione peroxidase increased significantly (P < 0.001). Besides, resveratrol can activate PI3K/AKT signaling pathway. PI3K siRNA can inhibit the PI3K/AKT signaling mediated by resveratrol. The addition of resveratrol can significantly increase the activity of mitochondrial superoxide dismutase (SOD) and reduce the malondialdehyde (MDA), which indicates that the oxidative damage of mitochondria induced by resveratrol was significantly weakened. The mitochondrial functional changes induced by resveratrol can be reversed by PI3K siRNA. In conclusion, our study shows that resveratrol can reduce ROS in cardiomyocytes by PI3K/AKT signaling pathway activation, and effectively inhibit the apoptosis of cardiomyocytes, thus having a direct protective effect on cardiomyocytes under SR.

Gradient radiation breeding and culture domestication of menaquinone producing strains.

By comparing the survival rate and positive mutation rate of the primary mutagenic strain and progeny mutagenic strain under different radiation doses, the results showed that the tolerance of the mutagenic strain to radiation dose increased with the increase of the mutagenic generations. We adopted an improved gradient radiation breeding strategy to improve the breeding efficiency. The strains were treated with radiation in four stages. The first stage was low energy N+ ion implantation (ion energy 15 keV, dose 80 × 2.6 × 1013 cm-2). In the second stage, the energy and dose of N+ ion reached to 20 keV, 90 × 2.6 × 1013 cm-2. In the third stage, 60Co-γ radiation (dose of 1.56 kGy) was used. In the fourth stage, the radiation dose of 60Co-γ increased to 1.82 kGy. After each stage of radiation, the MK (Menaquinone) precursor 1, 4-dihydroxy-2-naphthalate (DHNA) was used as the stress factor to domesticate the mutant strains. By gradually increasing the concentration of DHNA in the culture medium, the substrate tolerance of Flavobacterium sp. was effectively improved. By measuring SOD (superoxide dismutase) activity and malondialdehyde, it showed that the cell damage caused by radiation mutagenesis to the offspring mutant was less than that of the primary mutant. Changes in membrane permeability and membrane potential of the mutant strains were reflected in changes in fluorescence intensity of luciferin diacetate and rhodamine 123, which could explain the enhanced substrate tolerance of strain F-2. After gradient radiation breeding and culture acclimation, the biomass of mutant Strain F-2 was 6.59 g/L, and the MK yield was 9.59 mg/L.

Chemical Characteristics and Oxidative Stability of Buffalo Mozzarella Cheese Produced with Fresh and Frozen Curd.

Milk and dairy products can have variable contents of antioxidant compounds that contribute to counteract the oxidation of lipids and proteins during processing and storage. The content of active antioxidant compounds is closely linked to their protection by oxidation. Freezing is one of the factors that can reduce antioxidant activity. Freezing of milk or curd is frequently used in case of the seasonality of milk production and/or seasonal increased demand for some products. In this paper, the effect of using frozen curd on the oxidative stability of buffalo Mozzarella cheese was evaluated. Samples of buffalo Mozzarella with different frozen curd content (0%, 5%, 20%, and 50%) were produced and analyzed at one and nine days. Mozzarella cheese with higher frozen curd content had a significant increase in redox potential parallel to the decrease in antioxidant activity, showing less protection from oxidation. Lipid and protein oxidation, expressed respectively by malondialdehyde and carbonyl content, increased significantly with increasing frozen curd. At nine days, carbonyls significantly increased while malondialdehyde content did not vary, showing that during storage, fat was more protected from oxidation than protein. The average carbonyl levels were comparable to those of some cooked cheeses, and the malondialdehyde levels were even lower. The results of this study stimulate the investigation of new strategies to decrease the oxidative damage in cheeses produced in the presence of factors decreasing oxidative stability.

Isolation and Functional Characterization of a Salt-Responsive Calmodulin-Like Gene MpCML40 from Semi-Mangrove Millettia pinnata.

Salt stress is a major increasing threat to global agriculture. Pongamia (Millettia pinnata), a semi-mangrove, is a good model to study the molecular mechanism of plant adaptation to the saline environment. Calcium signaling pathways play critical roles in the model plants such as Arabidopsis in responding to salt stress, but little is known about their function in Pongamia. Here, we have isolated and characterized a salt-responsive MpCML40, a calmodulin-like (CML) gene from Pongamia. MpCML40 protein has 140 amino acids and is homologous with Arabidopsis AtCML40. MpCML40 contains four EF-hand motifs and a bipartite NLS (Nuclear Localization Signal) and localizes both at the plasma membrane and in the nucleus. MpCML40 was highly induced after salt treatment, especially in Pongamia roots. Heterologous expression of MpCML40 in yeast cells improved their salt tolerance. The 35S::MpCML40 transgenic Arabidopsis highly enhanced seed germination rate and root length under salt and osmotic stresses. The transgenic plants had a higher level of proline and a lower level of MDA (malondialdehyde) under normal and stress conditions, which suggested that heterologous expression of MpCML40 contributed to proline accumulation to improve salt tolerance and protect plants from the ROS (reactive oxygen species) destructive effects. Furthermore, we did not observe any measurable discrepancies in the development and growth between the transgenic plants and wild-type plants under normal growth conditions. Our results suggest that MpCML40 is an important positive regulator in response to salt stress and of potential application in producing salt-tolerant crops.

Knockout of Auxin Response Factor SlARF4 Improves Tomato Resistance to Water Deficit.

Auxin response factors (ARFs) play important roles in various plant physiological processes; however, knowledge of the exact role of ARFs in plant responses to water deficit is limited. In this study, SlARF4, a member of the ARF family, was functionally characterized under water deficit. Real-time fluorescence quantitative polymerase chain reaction (PCR) and ß-glucuronidase (GUS) staining showed that water deficit and abscisic acid (ABA) treatment reduced the expression of SlARF4. SlARF4 was expressed in the vascular bundles and guard cells of tomato stomata. Loss of function of SlARF4 (arf4) by using Clustered Regularly Interspaced Short Palindromic Repeats/Cas 9 (CRISPR/Cas 9) technology enhanced plant resistance to water stress and rehydration ability. The arf4 mutant plants exhibited curly leaves and a thick stem. Malondialdehyde content was significantly lower in arf4 mutants than in wildtype plants under water stress; furthermore, arf4 mutants showed higher content of antioxidant substances, superoxide dismutase, actual photochemical efficiency of photosystem II (PSII), and catalase activities. Stomatal and vascular bundle morphology was changed in arf4 mutants. We identified 628 differentially expressed genes specifically expressed under water deficit in arf4 mutants; six of these genes, including ABA signaling pathway-related genes, were differentially expressed between the wildtype and arf4 mutants under water deficit and unlimited water supply. Auxin responsive element (AuxRE) elements were found in these genes' promoters indicating that SlARF4 participates in ABA signaling pathways by regulating the expression of SlABI5/ABF and SCL3, thereby influencing stomatal morphology and vascular bundle development and ultimately improving plant resistance to water deficit.

Expression of AhATL1, an ABA Transport Factor Gene from Peanut, Is Affected by Altered Memory Gene Expression Patterns and Increased Tolerance to Drought Stress in Arabidopsis.

Arachis hypogaea abscisic acid transporter like-1 (AhATL1) modulates abscisic acid (ABA) sensitivity by specifically influencing the importing of ABA into cells, and is a key player in plant stress responses. However, there is limited information on ABA transporters in crops. In this study, we found that the level of AhATL1 expression and AhATL1 distribution increased more rapidly in the second drought (D2) compared with in the first drought (D1). Compared with the first recovery (R1), the AhATL1 expression level and ABA content remained at a higher level during the second recovery (R2). The heterologous overexpression of AhATL1 in Arabidopsis changed the expression pattern of certain memory genes and changed the post response gene type into the memory gene type. Regarding the proline and water content of Col (Arabidopsis thaliana L. Heynh., Col-0), atabcg22, and AhATL1-OX during drought training, the second drought (D2) was more severe than the first drought (D1), which was more conducive to maintaining the cell osmotic balance and resisting drought. In summary, drought stress memory resulted in a rapid increase in the AhATL1 expression and AhATL1 distribution level, and then raised the endogenous ABA content and changed the post response gene type into the memory gene type, which enhanced the drought resistance and recovery ability.

Dual role (promotion and inhibition) of transglutaminase in mediating myofibrillar protein gelation under malondialdehyde-induced oxidative stress.

This study investigated the effects of transglutaminase (TGase) on the properties of myofibrillar protein (MP) and its heat-induced gels under malondialdehyde (MDA)-induced oxidation. The physicochemical characteristics, protein aggregation and rheological properties of MP were assessed. The gelling behaviours of MP were analysed with measurements of gel strength, cooking loss, microstructure and secondary structure. Under varying degrees of MDA oxidation, the addition of TGase always led to changes in the tertiary structure, loss of free amine and thiol groups, crosslinking of the myosin heavy chain, and decreasing solubility. However, the effect of TGase on MP gel quality differed. At 6 mmol/L MDA, the addition of TGase reduced the quality of MP gels by increasing cooking loss. However, at 12 mmol/L MDA, TGase reduced both the cooking loss and gel strength.

Combined application of ascorbic and oxalic acids delays postharvest browning of litchi fruits under controlled atmosphere conditions.

The effect of ascorbic acid [AA (40 mmol L-1)] and oxalic acid [OA (2 mmol L-1)] on browning of litchi fruit was investigated under 5% CO2 + 1% O2 controlled atmosphere (CA) and compared with air at 5 ± 1 °C for 28 days. The combined application of AA and OA suppressed browning index, soluble quinones, and activities of polyphenol oxidase and peroxidase under CA compared with control. The combination of CA along with AA + OA reduced weight loss and maintained higher anthocyanins, total phenolics, membrane integrity, ascorbate peroxidase, catalase, glutathione reductase and superoxide dismutase activities compared with control. In addition, AA + OA + CA combination showed markedly lower malondialdehyde, superoxide anion and hydrogen peroxide with substantially higher soluble solids content, ascorbic acid, titratable acidity and sensory quality compared with control. In conclusion, AA + OA combination could be considered appropriate to delay browning and to conserve litchi fruit visual appearance under CA storage conditions.

Food matrixes play a key role in the distribution of contaminants of lipid origin: A case study of malondialdehyde formation in vegetable oils during deep-frying.

Vegetable oils are increasingly replacing animal fats in diets, but malondialdehyde (MDA), a peroxidation product of these oils, has been regarded as toxic; this necessitated investigation of MDA formation during consumption. This study investigated MDA formation in four vegetable oils during frying French fries (FF) and fried chicken breast meat (FCBM) at 180 °C for 7 h. Results showed that MDA contents were lower in oils used for frying foods than in control oils, mainly because MDA was incorporated into the foods. MDA content was lower in FF, but higher in FCBM, due to the different food components. Model oil and food system analyses yielded similar results. MDA bound the hydrophobic helical structure in starch-based FF, but was exhibited greater reactivity with nucleophilic groups in protein-based FCBM, resulting in stronger interaction with FCBM than with FF. Our results indicated the existence of distinct mechanisms underlying MDA migration in different food matrixes.

Existence of natural mouse IgG mAbs recognising epitopes shared by malondialdehyde acetaldehyde adducts and Porphyromonas gingivalis.

Natural Abs are produced by B lymphocytes in the absence of external Ag stimulation. They recognise self, altered self and foreign Ags, comprising an important first-line defence against invading pathogens and serving as innate recognition receptors for tissue homeostasis. Natural IgG Abs have been found in newborns and uninfected individuals. Yet, their physiological role remains unclear. Previously, no natural IgG Abs to oxidation-specific epitopes have been reported. Here, we show the cloning and characterisation of mouse IgG mAbs against malondialdehyde acetaldehyde (MAA)-modified low-density lipoprotein. Sequence analysis reveals high homology with germline genes, suggesting that they are natural. Further investigation shows that the MAA-specific natural IgG Abs cross-react with the major periodontal pathogen Porphyromonas gingivalis and recognise its principle virulence factors gingipain Kgp and long fimbriae. The study provides evidence that natural IgGs may play an important role in innate immune defence and in regulation of tissue homeostasis by recognising and removing invading pathogens and/or modified self-Ags, thus being involved in the development of periodontitis and atherosclerosis.