Free Methionine Research Articles

The Level of Methionine Residues in Storage Proteins Is the Main Limiting Factor of Protein-Bound-Methionine Accumulation in Arabidopsis Seeds.

The low level of methionine, an essential sulfur-containing amino acid, limits the nutritional quality of seeds. Two main factors can control the level of protein-bound methionine: the level of free methionine that limits protein accumulation and the methionine residues inside the storage proteins. To reveal the main limiting factor, we generated transgenic Arabidopsis thaliana seed-specific plants expressing the methionine-rich sunflower seed storage (SSA) protein (A1/A2). The contents of protein-bound methionine in the water-soluble protein fraction that includes the SSA in A1/A2 were 5.3- and 10.5-fold, respectively, compared to control, an empty vector (EV). This suggests that free methionine can support this accumulation. To elucidate if the level of free methionine could be increased further in the protein-bound methionine, these lines were crossed with previously characterized plants having higher levels of free methionine in seeds (called SSE). The progenies of the crosses (A1S, A2S) exhibited the highest level of protein-bound methionine, but this level did not differ significantly from A2, suggesting that all the methionine residues of A2 were filled with methionine. It also suggests that the content of methionine residues in the storage proteins is the main limiting factor. The results also proposed that the storage proteins can change their content in response to high levels of free methionine or SSA. This was assumed since the water-soluble protein fraction was highest in A1S/A2S as well as in SSE compared to EV and A1/A2. By using these seeds, we also aimed at gaining more knowledge about the link between high free methionine and the levels of metabolites that usually accumulate during abiotic stresses. This putative connection was derived from a previous analysis of SSE. The results of metabolic profiling showed that the levels of 29 and 20 out of the 56 metabolites were significantly higher in SSE and A1, respectively, that had higher level of free methionine, compared A1S/A2S, which had lower free methionine levels. This suggests a strong link between high free methionine and the accumulation of stress-associated metabolites.

Characterisation of the periplasmic methionine sulfoxide reductase (MsrP) from Salmonella Typhimurium

The oxidation of free methionine (Met) and Met residues inside proteins leads to the formation of methionine sulfoxide (Met-O). The reduction of Met-O to Met is catalysed by a ubiquitous enzyme family: the methionine sulfoxide reductases (Msr). The importance of Msr systems in bacterial physiology and virulence has been reported in many species. Salmonella Typhimurium, a facultative intracellular pathogen, contains four cytoplasmic Msr. Recently, a periplasmic Msr enzyme (MsrP) has been identified in Escherichia coli. In the present study, the STM14_4072 gene from Salmonella was shown to encode the MsrP protein (StMsrP). We describe the experimental procedure and precautions for the production of this molybdo-enzyme. StMsrP was also demonstrated to reduce free Met-O and to catalyse the complete repair of an oxidized protein. More importantly, this study provides for the first time access to the exhaustive list of the Msr systems of a pathogen, including four cytoplasmic enzymes (MsrA, MsrB, MsrC, BisC) and one periplasmic enzyme (MsrP).

Parental Selenium Nutrition Affects the One-Carbon Metabolism and the Hepatic DNA Methylation Pattern of Rainbow Trout (Oncorhynchus mykiss) in the Progeny.

Selenium is an essential micronutrient and its metabolism is closely linked to the methionine cycle and transsulfuration pathway. The present study evaluated the effect of two different selenium supplements in the diet of rainbow trout (Onchorhynchus mykiss) broodstock on the one-carbon metabolism and the hepatic DNA methylation pattern in the progeny. Offspring of three parental groups of rainbow trout, fed either a control diet (NC, basal Se level: 0.3 mg/kg) or a diet supplemented with sodium selenite (SS, 0.8 mg Se/kg) or hydroxy-selenomethionine (SO, 0.7 mg Se/kg), were collected at swim-up fry stage. Our findings suggest that parental selenium nutrition impacted the methionine cycle with lower free methionine and S-adenosylmethionine (SAM) and higher methionine synthase (mtr) mRNA levels in both selenium-supplemented treatments. DNA methylation profiling by reduced representation bisulfite sequencing (RRBS) identified differentially methylated cytosines (DMCs) in offspring livers. These DMCs were related to 6535 differentially methylated genes in SS:NC, 6890 in SO:NC and 7428 in SO:SS, respectively. Genes with the highest methylation difference relate, among others, to the neuronal or signal transmitting and immune system which represent potential targets for future studies.

Serum choline in extremely preterm infants declines with increasing parenteral nutrition

PurposeCholine is an essential nutrient for fetal and infant growth and development. Parenteral nutrition used in neonatal care lack free choline but contain small amounts of lipid-bound choline in the form of phosphatidylcholine (PC). Here, we examined the longitudinal development of serum free choline and metabolically related compounds betaine and methionine in extremely preterm infants and how the concentrations were affected by the proportion of parenteral fluids the infants received during the first 28 postnatal days (PNDs).MethodsThis prospective study included 87 infants born at gestational age (GA) < 28 weeks. Infant serum samples were collected PND 1, 7, 14, and 28, and at postmenstrual age (PMA) 32, 36, and 40 weeks. The serum concentrations of free choline, betaine, and methionine were determined by 1H NMR spectroscopy.ResultsThe median (25th–75th percentile) serum concentrations of free choline, betaine, and methionine were 33.7 (26.2–41.2), 71.2 (53.2–100.8), and 25.6 (16.4–35.3) µM, respectively, at PND 1. The choline concentration decreased rapidly between PND one and PND seven [18.4 (14.1–26.4) µM], and then increased over the next 90 days, though never reaching PND one levels. There was a negative correlation between a high intake of parenteral fluids and serum-free choline.ConclusionCirculating free choline in extremely preterm infants is negatively affected by the proportion of parenteral fluids administered.Trial registrationClinicalTrials.gov Identifier NCT02760472, April 29, 2016, retrospectively registered.

Supplementation with free methionine or methionine dipeptide improves meat quality in broilers exposed to heat stress.

This study aimed to evaluate the effect of methionine dipeptide supplementation on the meat quality of broilers subjected to heat stress. A completely randomized 3 × 2 factorial design with four repetitions of each treatment was used. Three diets, unsupplemented (U), supplemented with methionine (M), and supplemented with methionine dipeptide (MM), were fed to 96 broilers subjected to thermal comfort (TC) or heat stress (HS, 32°C for 24h) conditions antemortem. Meat quality parameters, total antioxidant capacity (TAC), protein and lipid oxidation, and ryanodine receptor type 3 (RYR3) gene expression in breast muscle of 35-day-old broilers were evaluated. Methionine supplementation (M and MM) enhanced the nutritional quality of breast meat. Diet had a significant effect on breast meat pH, color (a*), and nitrogen and lipid contents. Interaction effects of diet and HS on TAC and protein oxidation were not observed. Diet and HS influenced lipid oxidation of breast meat after 7days of refrigerated storage. High RYR3 expression was observed in breast meat of broilers subjected to heat stress and fed the U diet. No differences were observed between M and MM diets in any of the parameters evaluated. The results showed that both sources of methionine (M and MM) can be supplemented in broiler diets with beneficial effects on breast yield and meat nutritional quality. In addition, HS has made chickens more susceptible to biomolecule oxidation, and MM can potentiate chicken TAC. Further study is needed to better understand the effects of MM on broilers.

Methionine Availability in the Arthropod Intestine Is Elucidated through Identification of Vibrio cholerae Methionine Acquisition Systems.

While only a subset of Vibrio cholerae strains are human diarrheal pathogens, all are aquatic organisms. In this environment, they often persist in close association with arthropods. In the intestinal lumen of the model arthropod Drosophila melanogaster, methionine and methionine sulfoxide decrease susceptibility to V. cholerae infection. In addition to its structural role in proteins, methionine participates in the methionine cycle, which carries out synthetic and regulatory methylation reactions. It is, therefore, essential for the growth of both animals and bacteria. Methionine is scarce in some environments, and the facile conversion of free methionine to methionine sulfoxide in oxidizing environments interferes with its utilization. To ensure an adequate supply of methionine, the genomes of most organisms encode multiple high-affinity uptake pathways for methionine as well as multiple methionine sulfoxide reductases, which reduce free and protein-associated methionine sulfoxide to methionine. To explore the role of methionine uptake and reduction in V. cholerae colonization of the arthropod intestine, we mutagenized the two high-affinity methionine transporters and five methionine sulfoxide reductases encoded in the V. cholerae genome. We show that MsrC is the sole methionine sulfoxide reductase active on free methionine sulfoxide. Furthermore, in the absence of methionine synthesis, high-affinity methionine uptake but not reduction is essential for V. cholerae colonization of the Drosophila intestine. These findings allow us to place a lower limit of 0.05 mM and an upper limit of 0.5 mM on the methionine concentration in the Drosophila intestine.IMPORTANCE Methionine is an essential amino acid involved in both biosynthetic and regulatory processes in the bacterial cell. To ensure an adequate supply of methionine, bacteria have evolved multiple systems to synthesize, import, and recover this amino acid. To explore the importance of methionine synthesis, transport, and recovery in any environment, all of these systems must be identified and mutagenized. Here, we have mutagenized every high-affinity methionine uptake system and methionine sulfoxide reductase encoded in the genome of the diarrheal pathogen V. cholerae We use this information to determine that high-affinity methionine uptake systems are sufficient to acquire methionine in the intestine of the model arthropod Drosophila melanogaster but are not involved in virulence and that the intestinal concentration of methionine must be between 0.05 mM and 0.5 mM.

Parenterally Delivered Methionyl-Methionine Dipeptide During Pregnancy Enhances Mammogenesis and Lactation Performance Over Free Methionine by Activating PI3K-AKT Signaling in Methionine-Deficient Mice

Background:Pregnancy-induced hypoaminoacidemia, ʟ-methionine (Met) included, disturbs embryogenesis and may also affect breast function. Supplementation with the dipeptideʟ-methionyl-Met (Met-Met) may improve lactation performance. Objective:We compared the effects of supplemental Met or Met-Met during pregnancy on mammogenesis and lactogenesis and investigated underlying mechanisms. Methods:In experiment 1, 9-wk-old ICR mice (n = 72, ~30 g) were divided into 3 groups. During the first 17 days of pregnancy (DP), the Control group was fed a diet with Met (8.2 g/kg) and saline was intraperitoneally injected, the Met group was fed a Met-devoid diet and 35% of the Met (92-mmoʟMet) as contained in the Control diet was intraperitoneally injected, and the Met-Met group was fed the same diet and 70-mmoʟMet plus 11-mmoʟMet-Met was intraperitoneally injected. All animals were fed the Control diet after DP17 and during lactation. Mammogenesis, lactogenesis, transcriptome at DP17, and milk performance during lactation were examined. In experiment 2, 9-wk-old ICR mice (n = 55, ~30 g) at DP0 were injected through the teat with adeno-associated virus for overexpression/inhibition of phosphoinositide-3-kinase regulatory subunit 1 (Pik3r1), divided into the Control, Met, and Met-Met groups and received the same treatment as experiment 1 to examine mammogenesis and lactogenesis at DP17. Results:In experiment 1, compared with the Met group, the Met-Met group showed higher (P < 0.05) mammary epithelium percentage (42%) and aS1-casein expression (84%) at DP17, milk yield (34%) and energy concentrations (8.7%) during lactation; transcriptomic analysis illustrated activated phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) signaling in the mammary glands of the Met-Met group (P-adj < 0.001). In experiment 2, overexpression ofPik3r1enhanced (P < 0.05) the protective effect of Met-Met over Met on mammogenesis and β-casein expression. Conclusion:Met-Met is more effective than Met in promoting mammogenesis and lactogenesis mainly by activation of PI3K-AKT signaling in Met-deficient mice.

Taurine requirement and metabolism response of tiger puffer Takifugu rubripes to graded taurine supplementation

An eight week feeding trial was conducted to investigate the effect of taurine supplementation on growth performance, sulfur amino acid concentrations, the activity and gene expression of enzymes related to taurine synthesis, and gene expression of taurine transporter (TAUT) in tiger puffer (Takifugu rubripes). Test diets were respectively supplemented with 0, 3, 6, 9, 12 and 20 g kg−1 taurine (the T0, T3, T6, T9, T12 and T20 diets) and a control diet was supplemented with 12 g kg−1 cysteine (the Cys diet). All experimental diets were fed to triplicate groups of 25 fish (initial weight 20.05 ± 0.06 g). Dose responses were observed in terms of specific growth rate and feed efficiency ratio. Dietary taurine requirement was estimated at 5.25 and 8.23 g kg−1 of dry matter of the diet based on regression analysis for specific growth rate and feed efficiency ratio, respectively. Free taurine concentrations of muscle and liver tissues increased with the increase of dietary taurine concentration. However, there was no significant difference in free taurine concentrations in muscle or liver between the Cys and T0 groups (p > .05). Free methionine concentration of liver tissue gradually increased with the increase of dietary taurine, which implied that exogenous taurine appeared to spare hepatic methionine. Cysteine sulfinic acid decarboxylase (CSD) activity and mRNA level in liver were down-regulated in the T9, T12 and T20 groups compared with the T0 group (p < .05), while no statistical difference was observed in cysteine dioxygenase (CDO) activity and expression between the T0 group and the taurine supplementation groups (the T3, T6, T9, T12 and T20 groups (p > .05). TAUT mRNA levels in muscle had a decrease trend in fish fed diets containing 1.7 to 8.2 g kg−1 taurine (the T0, T3 and T6 groups), while there was no significant difference when dietary taurine concentrations increased from 8.2 to 21.9 g kg−1 (the T6, T9, T12 and T20 groups). In conclusion, tiger puffer cannot synthesize taurine in the liver at the level of cysteine used in this study, whereas a sparing effect on the methionine demand may be observed in liver by dietary taurine supplementation. Our data indicates that taurine is a conditionally essential amino acid for tiger puffer, and the taurine requirement was estimated ranged from 5.25 to 8.23 g kg−1 of dry matter of the diet.

Formation of protein sub-visible particles during powder grinding of a monoclonal antibody

We have observed an interesting phenomenon in which grinding of freeze-dried monoclonal antibody X (mAb-X) formulation powder resulted to significant protein sub-visible particles (SbVPs) in the reconstituted liquid, which could only be observed by sensitive particle analytical methods such as MFI and DLS. Effects of grinding temperature and the free radical scavengers methionine and 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidin-yloxy free radical (CTPO) on the formation of SbVPs were also evaluated. Free radicals were observed by EPR and the amount of free radicals was correlated to the sample temperature prior to grinding. Formation of SbVPs could be partially inhibited by methionine and CTPO. The amount of SbVPs formed was dependent on the amount of free radicals/sample temperature prior to grinding. At higher temperatures, more free radicals and SbVPs formed. Other than the previously known protein degradation due to high temperature formed during mechanical grinding, we propose an unreported and supplementary mechanism, i.e., the formation of free radicals (i.e., due to break of CO or CS bonds) in the dried state during mechanical grinding, leading to protein particle formation in the reconstituted solution. Our observation suggested that mechanical grinding of protein powder should be avoided or used cautiously (i.e., grinding temperature, strength and time) and the effects on radical and particle formation be fully evaluated.

Influence of different cooking methods on the nutritional and potentially harmful components of peanuts

This study investigated the comparative effects of boiling, roasting, deep-frying methods on the content of nutritional and potentially harmful components in peanuts. After cooking, the contents of total reducing sugar, sucrose, unsaturated fatty acids and almost all individual amino acids were reduced. Free methionine disappeared after heating processing, whereas fructose, starch, cis-palmitoleic acid and saturated fatty acids were increased in processed samples. Micronutrients including flavonoids and phenolic reduced significantly after boiling process but increased after roasting process. Both of frying and roasting promoted the formation of potentially harmful components including HMF, acrylamide and furan. The overall compositional difference between samples were further displayed and identified by a combination application of HCA and PCA, which showed that the roasting and frying process had a significant impact on the nutritional composition of peanuts.

Atlantic salmon fed a nutrient package of surplus methionine, vitamin B12, folic acid and vitamin B6 improved growth and reduced the relative liver size, but when in excess growth reduced

Feeding plant‐based diet through smoltification of Atlantic salmon requires verification of the optimal level of 1C nutrients. Here, we fed Atlantic salmon plant‐based diets containing three different surplus amounts of the 1C nutrients; methionine, cobalamin (vitamin B12), pyridoxine (vitamin B6) and folic acid during 6 weeks in fresh water, through smoltification, followed by 3 months on‐growing period in salt water. The three diets were fed to fish dispersed in triplicate tanks throughout the experiment. Mean start body weight was 32 g. Dietary methionine levels in the diets were 6.7, 9.2 and 11.7 g/kg. Dietary B6 was 6.75, 8.45 and 11 mg/kg. Cobalamin was 0.16, 0.18 and 0.20 mg/kg. While dietary folic acid was 2.9, 4.8 and 6.3 mg/kg, diets are referred to as low, medium and high 1C diet. All other amino acids were similar between diets. The results showed no differences in growth or feed utilization in the fresh water period, but following the on‐growing salt water period, differences between diets occurred. The fish fed the medium 1C diet showed better growth, as compared to fish fed the low or high 1C diet (p = .009). The medium 1C fed fish showed a relative lower liver weight compared with fish fed low or high 1C diet (p = .025). Condition factor was better in fish fed the medium and high 1C diet as compared to those fed the low 1C diet (p = .0006). As expected, free methionine in liver, plasma and muscle increased by dietary methionine inclusion. Surplus vitamins only had minor effect on tissue concentrations. Based on these findings, we conclude that the micronutrient and methionine level presented in the medium 1C diet improved the growth, liver size and condition factor; however, more research is needed to evaluate the optimal requirement level for each of the 1C nutrients.

Dietary supplementation with free methionine or methionine dipeptide improves environment intestinal of broilers challenged with Eimeria spp.

This study examined the influence of a diet enriched with free methionine (dl-Met) or methionine dipeptide (dl-MMet) on the intestinal health of Eimeria-challenged (EC) and unchallenged (UC) broilers. A non-supplemented, methionine-deficient diet (NS) was used as control. Treatments were arranged in a 2 × 3 factorial completely randomized design with eight replications. Broilers in the EC group were infected with sporulated oocysts of Eimeria spp. (E. acervulina, E. maxima, E. praecox, and E. mitis) at 14 d of age. Performance analysis, light and electron microscopy of the jejunum, analysis of genes related to apoptosis and cell proliferation in the jejunum, and blood tests were performed at 6 days post-inoculation (dpi). EC broilers had poorer performance than UC broilers, regardless of diet (P < 0.001). Broilers fed the dl-Met diet had greater weight gain (P = 0.004) and lower feed conversion ratio (P = 0.019) than broilers fed other diets. Jejunal sections from EC broilers fed the NS diet showed short (P = 0.001) and wide villi (P < 0.001) with increased crypt depth (P < 0.001) and reduced villus / crypt ratio (P = 0.001), jejunal absorptive surface area (P < 0.001), number of neutral goblet cells (Eimeria challenge: P = 0.048; diet P = 0.016), and mucin 2 (MUC2) gene expression (P = 0.018). EC birds fed the dl-MMet diet had higher enterocyte height (P < 0.001). Birds fed the dl-MMet diet had low lamina propria width (P = 0.009). UC broilers fed the dl-Met diet had the highest number of acidic goblet cells (P = 0.005), whereas EC broilers assigned the dl-MMet diet showed the highest number of intraepithelial lymphocytes (P = 0.033). Reduced expression of caspase-3 (CASP3) (P = 0.005), B-cell lymphoma 2 (BCL2) (P < 0.001), mechanistic target of rapamycin (MTOR) (P < 0.001), and ribosomal protein S6 kinase B1 (RPS6KB1) (P < 0.001) genes was observed in EC animals. MTOR expression levels were highest in birds fed the dl-MMet diet (P = 0.004). Plasma activities of aspartate aminotransferase (AST) was influenced by both diet (P = 0.002) and Eimeria challenge (P = 0.005), with EC broilers assigned the NS diet showing the highest levels. EC broilers fed the NS diet had higher creatine kinase (CK) activity (P = 0.049). EC broilers had lower plasma uric acid (P = 0.004) and higher serum mucoproteins level (P < 0.001). These results indicate that methionine dipeptide supplementation is able to mitigate the harmful intestinal effects of Eimeria spp. in broilers.

Oxidative and Advanced Glycation End-Products Biomarkers and Their Association with the Risk of Cardiovascular Disease Outcomes in Type 1 Diabetes in DCCT/EDIC

Background: Type 1 diabetes confers a significant risk of cardiovascular disease (CVD), and new biomarkers of risk are needed for early detection. We have investigated the predictive value of oxidation products (OPs) and advanced glycation end-products (AGEs) for CVD events in the Diabetes Control and Complications Trial and the follow-up Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study. Methods: Using a case-control design, we measured plasma OPs including methionine sulfoxide [MetSO] and the AGE carboxyethyllysine [CEL] by LC/mass spectrometry in 459 participants (93 CVD cases and 366 controls) at three time points during DCCT, and at year 1-2 of EDIC. Cox proportional hazards models assessed the association between biomarkers as fixed or time-dependent covariates and the risk of subsequent CVD events. Prediction improvement was assessed using the C-statistic. Findings: Baseline CEL was associated with CVD (p=0·03, HR 1·15) when fully adjusted for age, HbA1c, BMI, HDL, LDL, sex, systolic and diastolic blood pressure, while MetSO at beginning of EDIC and as a time-dependent covariate, was strongly inversely associated with CVD outcomes (p < 0·001 and HR 0·58 and 0·61, respectively). MetSO also significantly increased the predictive c-statistic from 0·711 to 0·783 (p<0·001) when added to the other covariates. Interpretation: High levels of MetSO, a product of the free oxygen radical scavenger methionine, are associated with lower incidence of a primary CVD event during DCCT/EDIC· MetSO also significantly improved the predictive value over traditional risk factors, which may allow more timely institution of preventive therapies. Trial Registrations: NCT00360893, NCT00360815. Funding Statement: The DCCT/EDIC has been supported by cooperative agreement grants (1982-1993, 2012- 2017), and contracts (1982-2012) with the Division of Diabetes Endocrinology and Metabolic Diseases of the National Institute of Diabetes and Digestive and Kidney Disease (current grant numbers U01 DK094176 and U01 DK094157), and through support by the National Eye Institute, the National Institute of Neurologic Disorders and Stroke, the General Clinical Research Centers Program (1993-2007), and Clinical Translational Science Center Program (2006-present), Bethesda, Maryland, USA. Declaration of Interests: None for JML and IB as noted below. PJB and Scott Howell have the following Declarations of interest: PJB is Chief Scientific Officer and Scott Howell is the Laboratory Director of PreventAGE Healthcare LLC (PHC), and PJB prepared and submitted the SBIR that funded the study in PHC’s name. PHCs goal is to develop precise biomarkers that can predict diabetic complications. Although no commercialization of the content of this manuscript has taken place, it is possible that this could occur in the future. Further detail on this relationship is contained in the enclosed ICJME form and in the “declaration of interests” section. Ethics Approval Statement: All appropriate written consents, permissions, and releases have been obtained from the participants by the DCCT/EDIC central overview committee.

Dietary supplementation with free methionine or methionine dipeptide mitigates intestinal oxidative stress induced by Eimeria spp. challenge in broiler chickens

BackgroundThis study evaluated the effects of Eimeria spp. challenge and dietary supplementation with free methionine or methionine dipeptide on animal performance; expression of genes associated with the immune system, antioxidant system, and amino acid transport in the jejunum; and redox status of the jejunum of broiler chickens.MethodsA randomized, 2 × 3 factorial design was used, in which Eimeria spp. challenge was the first factor (Eimeria-challenged, EC, or unchallenged, UC, broilers) and methionine supplementation was the second factor (non-supplemented, NS; free dl-methionine, dl-Met; and methionine dipeptide, dl-methionyl-dl-methionine, dl-MMet). At 14 days of age, chickens were inoculated orally with sporulated oocysts of Eimeria acervulina, Eimeria praecox, Eimeria maxima, and Eimeria mitis. Birds were killed by cervical dislocation 144 h post-inoculation (PI), and the jejunum was collected for biochemical and molecular analyses.ResultsEC broilers had a 13% lower feed intake (FI), 37% lower body weight gain (BWG), and 39% higher feed conversion ratio (FCR) than UC broilers. Chickens fed the dl-Met diet had higher BWG (about 12% higher) and better FCR (about 12% lower) than chickens fed the NS diet. EC chickens had lower relative weight of the bursa of Fabricius (51.8%) and higher relative weights of the spleen and whole intestine (53.6% and 26.3%, respectively) than UC chickens. Eimeria spp. challenge led to an increase in the levels of oxidative substances, such as nitrite and thiobarbituric acid reactive substances (TBARS), in the jejunum of chickens 144 h PI. Among UC chickens, those fed the dl-Met diet had higher total antioxidant capacity (TAC) and lower catalase (CAT) and superoxide dismutase (SOD) activities. EC chickens that received the NS diet had higher carbonylated protein content (CP). This result was associated with their lower TAC and catalase activity. The lower TAC in EC chickens might have been due to reduced expression of catalase (CAT) and superoxide dismutase 1 (SOD1) genes. Chickens fed the dl-Met and dl-MMet diets had lower nitrite content. Eimeria spp. challenge suppressed neutral amino acid transporter 1 (B0AT1), peptide transporter 1 (PEPT1), toll-like receptor 5 (TLR5), interleukin 2 (IL2), and occludin (OCLN) gene expression and enhanced cationic amino acid transporter 1 (CAT-1) and interferon gamma (IFNG) gene expression. The highest PEPT1 expression level was observed in broilers fed the dl-MMet diet, and the lowest TLR5 expression level was found in broilers fed the NS diet.ConclusionOur results show for the first time that supplementation with methionine as free amino acid or dipeptide helps protect the intestinal cells of broilers under Eimeria spp. challenge from the oxidative damage induced by free radicals, mainly through modulation of the antioxidant system.

Polyamines are involved in GABA-regulated salinity-alkalinity stress tolerance in muskmelon

Salinity-alkalinity stress is a limiting environmental factor in muskmelon production. Treatment with γ-aminobutyric acid (GABA) protects crops against several abiotic stresses. Previous studies reported that GABA increased the content of polyamines (PAs) and improved the oxidative stress tolerance of muskmelon grown under salinity-alkalinity stress. However, it was unclear whether PAs were involved in GABA-regulated salinity-alkalinity stress tolerance. Here, we explored the roles of PAs in GABA-regulated Na+/K+ homeostasis and membrane lipid peroxidation. Our results showed that GABA pretreatment reduced the salinity-alkalinity stress-induced inhibition of muskmelon growth, significantly reduced the Na+/K+ ratio and enhanced the contents of free PAs, arginine, and methionine. GABA treatment upregulated the expression levels of PA-related genes under salinity-alkalinity stress. Application of exogenous spermidine reduced the salinity-alkalinity stress-induced inhibition of muskmelon growth, relative electrical conductivity, malondialdehyde content, and Na+/K+ ratio. By contrast, pretreatment of muskmelon plants with the Spd synthesis inhibitor dicyclohexylammonium sulfate (DCHA) eliminated GABA-remediated ion balance, membrane peroxidation injury, and plant growth under salinity-alkalinity stress conditions. In all, fed with GABA maintained the Na+/K+ balance via reducing Na+ content and increasing K+ uptake of muskmelon under salinity-alkalinity stress conditions, ultimately reduced membrane oxidation injury. And GABA provoked PAs levels involved in GABA-regulated Na+/K+ homeostasis and alleviation of membrane lipid peroxidation under salinity-alkalinity stress tolerance.

Effect of Fermentation on Enhancing the Nutraceutical Properties of Arthrospira platensis (Spirulina)

Arthrospira platensis (spirulina), a filamentous fresh-water planktonic cyanobacterium, possesses diverse biological activities and a unique nutritional profile, due to its high content of valuable nutrients. This study aimed to further improve the bioactive profile of spirulina, by fermenting it with the lactic acid bacterium Lactobacillus plantarum. In vitro comparison of the total phenolic content (TPC), C-phycocyanin, free methionine, DPPH radical scavenging capacity, ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) and protein fragmentation via SDS-PAGE in untreated versus 12 to 72 h fermented spirulina is reported here. After 36 h fermentation, TPC was enhanced by 112%, FRAP by 85% and ORAC by 36%. After 24 h, the DPPH radical scavenging capacity increased 60%, while the free methionine content increased by 94%, after 72 h. Past 36 h of fermentation, the total antioxidant capacity (TAC) diminished, possibly due to deterioration of the heat-sensitive antioxidants. However, protein fragmentation and free methionine content increased, linearly, with the fermentation time. Cyanobacterial peptides and other bioactive compounds trapped within the spirulina cell wall are released during fermentation and have a significant potential as a functional ingredient in nutraceuticals and pharmaceuticals, in addition to their nutritive value.

Voltammetric and mass spectrometry investigation of methionine oxidation

The electro-oxidation mechanism of free methionine and bound within different peptide sequences was investigated by voltammetry, at glassy carbon electrode, and mass spectrometry. It is proposed that the electro-oxidation of free methionine occurs in two steps, each involving the transfer of one electron and turns pH-independent from mild acid to mild alkaline electrolytes. The first oxidation reaction leads to the formation of a cation radical stabilized either through the amino group resulting in the dehydromethionine intermediate, or by interaction with a neutral methionine molecule leading to production of a dimer cationic radical. The dehydromethionine hydrolysis gave methionine sulfoxide as final oxidation product, whereas a future oxidation of methionine dimer cation radical, i.e. the second electro-oxidation step, results in a methionine dimer dication. Moreover, at high acid media, the protonated amino group influence the electro-oxidation process to take place via proton transfer mechanism. The presence of methionine sulfoxide and of the dimer cationic radical as oxidation products of methionine was confirmed by mass spectroscopy.

Impact of Tryptophan Oxidation in Complementarity-Determining Regions of Two Monoclonal Antibodies on Structure-Function Characterized by Hydrogen-Deuterium Exchange Mass Spectrometry and Surface Plasmon Resonance

PurposeTryptophan’s (Trp) unique hydrophobic and structural properties make it an important antigen binding motif when positioned in complementarity-determining regions (CDRs) of monoclonal antibodies (mAbs). Oxidation of Trp residues within the CDR can deleteriously impact antigen binding, particularly if the CDR conformation is altered. The goal of this study was to evaluate the conformational and functional impact of Trp oxidation for two mAb subtypes, which is essential in determining the structure-function relationship and establishing appropriate analytical control strategies during protein therapeutics development.MethodsSelective Trp oxidation was induced by 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) treatment in the presence of free methionine (Met). The native and chemically oxidized mAbs were characterized by hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational changes and surface plasmon resonance (SPR) for antigen-antibody binding.ResultsTreatment of mAbs with AAPH selectively oxidized solvent accessible Trp residues. Oxidation of Trp within or in proximity of CDRs increased conformational flexibility in variable domains and disrupted antigen binding.ConclusionsTrp oxidation in CDRs can adversely impact mAbs’ conformation and antigen binding. Trp oxidation should be carefully evaluated as part of critical quality attribute assessments. Oxidation susceptible Trp should be closely monitored during process development for mAbs to establish appropriate analytical control for manufacturing of drug substance and drug product.

Effect of Peroxide- Versus Alkoxyl-Induced Chemical Oxidation on the Structure, Stability, Aggregation, and Function of a Therapeutic Monoclonal Antibody

Current guidelines indicate that the effects of oxidation should be included as part of forced degradation studies on protein drugs. We probed the effect of 3 commonly used oxidants, hydrogen peroxide, tert-butyl hydroperoxide, and 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), on a therapeutic monoclonal IgG1 antibody (mAb8). Upon oxidation, mAb8 did not show noticeable changes in its secondary structure but showed minor changes in tertiary structure. Significant decrease in conformational stability was observed for all the 3 oxidized forms. Both hydrogen peroxide and tert-butyl hydroperoxide destabilized mainly the CH2 domain, whereas AAPH destabilized the variable domain in addition to CH2. Increased aggregation was found for AAPH-oxidized mAb8. In addition, a significant decrease in Fc receptor binding was observed for all 3 oxidized forms. Antibody dependent cell-mediated cytotoxicity, binding to target protein receptor, and cell proliferation activity were significantly reduced in the case of AAPH-oxidized mAb8. The presence of free methionine in the formulation buffer seems to alleviate the effect of oxidation. The results of this study show that the 3 oxidants differ in terms of their effects on the structure and function of mAb8 because of chemical modification of different sets of residues located in Fab versus Fc.

Investigation of potent odorants generated during the production of whey protein hydrolysates.

Whey protein hydrolysates (WPH) were prepared from whey protein concentrate (WPC) by partial or extensive hydrolysis. WPC and two WPH types were characterized by a descriptive sensory analysis. WPC exhibited a "milky", "sweet", and "mild milk fat" odor, whereas extensively hydrolyzed whey protein (E-WPH) demonstrated "cheese-like", "acrid odor", and "sour" olfactory characteristics. In E-WPH, 3-(methylthio)-propanal, phenylacetaldehyde, and 3-methylbutanoic acid were specifically detected with high flavor dilution factors upon aroma extract dilution analysis. These odor-active compounds likely contributed to the olfactory characteristics of E-WPH, and their concentration increased together with that of their respective putative precursors: free methionine, phenylalanine, and leucine.