Effects of Arginine Deimination and Citrulline Side-Chain Length on Peptide Secondary Structure Formation

Post-translational modifications expand the chemical functionality of peptides and proteins beyond that originating from the encoded amino acids, but studies on the structural effects of these modifications have been limited. Arginine undergoes deimination to give citrulline (Cit), converting the positively charged guanidinium moiety into a neutral urea group. Herein, we report the effect of Arg deimination on secondary structure formation. To understand the reason for the number of methylene units in Cit, the effect of Cit side-chain length on secondary structure formation was also studied. Ala-based peptides and β-hairpin peptides were used to study α-helix and β-sheet formation, respectively. Peptides containing Cit analogues were prepared by an orthogonal protecting group strategy coupled with solid-phase carbamylation. The CD data for the Ala-based peptides were analyzed by using modified Lifson–Roig theory, showing that the helix propensity of Arg decreased upon deimination and that either shortening or lengthening Cit also decreased the helix propensity. The β-hairpin peptides were analyzed by NMR methods, showing minimal change in strand formation energetics upon Arg deimination. Altering the Cit side-chain length did not affect strand formation energetics either. These results should be useful for the preparation of urea-bearing systems and the design of peptides incorporating urea-bearing residues with varying side-chain length.     

l‐Citrulline Supplementation‐Increased Skeletal Muscle PGC‐1α Expression Is Associated with Exercise Performance and Increased Skeletal Muscle Weight

Scope

l ‐citrulline has recently been reported as a more effective supplement for promoting intracellular nitric oxide (NO) production compared to l ‐arginine. Here, the effect of l ‐citrulline on skeletal muscle and its influence on exercise performance were investigated. The underlying mechanism of its effect, specifically on the expression of skeletal muscle peroxisome proliferator‐activated receptor‐gamma coactivator‐1α (PGC‐1α), was also elucidated.

Methods and results

Six‐week‐old ICR mice were orally supplemented with l ‐citrulline (250 mg kg^?1) daily, and their performance in weight‐loaded swimming exercise every other day for 15 days, was evaluated. In addition, mice muscles were weighed and evaluated for the expression of PGC‐1α and PGC‐1α‐regulated genes. Mice orally supplemented with l ‐citrulline had significantly higher gastrocnemius and biceps femoris muscle mass. Although not statistically significant, l ‐citrulline prolonged the swimming time to exhaustion. PGC‐1α upregulation was associated with vascular endothelial growth factor α (VEGFα) and insulin‐like growth factor 1 (IGF‐1) upregulation. VEGFα and IGF‐1 are important for angiogenesis and muscle growth, respectively, and are regulated by PGC‐1α. Treatment with NG‐nitro‐l ‐arginine methyl ester hydrochloride (l ‐NAME), a nitric oxide synthesis inhibitor, suppressed the l ‐citrulline‐induced PGC‐1α upregulation in vitro.

Conclusion

Supplementation with l ‐citrulline upregulates skeletal muscle PGC‐1α levels resulting in higher skeletal muscle weight that improves time to exhaustion during exercise.

     

Bioactive compounds from flesh and by-product of fresh-cut watermelon cultivars

BACKGROUND: The fresh‐cut industry produces thousands of tons of waste in non‐edible portions that present an environmental and management problem. These by‐products could be reused, in particular, to obtain bioactive compounds. In this study, five different fresh‐cut watermelon cultivars were assessed for their flesh and by‐product bioactive contents. RESULTS: The amount of by‐product varied between 31.27 and 40.61% of initial fresh weight (f.w.) depending on the cultivar. Watermelon cultivars were poor sources of total antioxidant, and the content was similar between rind and flesh samples (46.96 vs 43.46 mg ascorbic acid equivalent antioxidant capacity kg^?1 f.w.). However, the rind had a moderate total phenolic content higher than that of the flesh (458 vs 389 mg chlorogenic acid equivalent kg^?1 f.w.) and a much higher content of the amino acid citrulline (3.34 vs 2.33 g kg^?1 f.w.), which has potential bioactive properties. CONCLUSION: Watermelon rind offers quantitative interest as a natural source of citrulline, particularly Fashion, a dark‐skinned, seedless cultivar. More research is required on the efficient extraction of citrulline from watermelon rind and its suitability as an additive to drinks, juices or others products to produce new functional food products with valid health claims. Copyright © 2010 Society of Chemical Industry     

瓜氨酸生产相关基因簇在谷氨酸棒杆菌中的组成型表达

构建1种组成型载体并将载体应用在表达瓜氨酸相关基因簇argCJBDF上。通过去除pXMJ19诱导型启动子上游阻遏蛋白lacI基因的方法,构建组成型质粒pXMJ19-lacI,并将谷氨酸棒杆菌中合成瓜氨酸途径的基因簇argCJBDF克隆到改造过的组成型载体中,实现瓜氨酸合成相关基因簇argCJBDF在谷氨酸棒杆菌的组成型表达。结果表明:重组菌在30℃摇瓶发酵72 h后,N-乙酰谷氨酸激酶的酶活达到(0.323±0.015)U/mg,瓜氨酸的产量达到4.33 g/L。成功构建的组成型表达载体,实现了外源基因簇argCJBDF在谷氨酸棒杆菌中的组成型表达。     

Contribution of citrulline to the formation of ethyl carbamate during Chinese rice wine production

Ethyl carbamate is a well-known carcinogen and widely occurs in Chinese rice wine. To provide more clues to minimise ethyl carbamate accumulation, the levels of possible precursors of ethyl carbamate in Chinese rice wine were investigated by HPLC. Studies of the possible precursors of ethyl carbamate in Chinese raw rice wine with various additives and treatments indicated that significant amounts of urea can account for ethyl carbamate formation. It was also recognised that citrulline is another important precursor that significantly affects ethyl carbamate production during the boiling procedure used in the Chinese rice wine manufacturing process. Besides urea and citrulline, arginine was also found to be an indirect ethyl carbamate precursor due to its ability to form urea and citrulline by microorganism metabolism.     

发酵酒精饮品中氨基甲酸乙酯前体物的代谢调控机制研究进展

氨基甲酸乙酯（ethyl carbamate,EC）又称脲烷,是一种广泛存在于发酵酒精饮品中的氨基甲酰类物质。由于EC目前被证实能对实验动物造成多位点致癌,并且对人类具有潜在致癌性,因此其对发酵食品安全的影响是目前发酵工业中面临的重要问题。EC主要由氨基甲酰类物质和乙醇自发作用形成,其中重要的前体物包括尿素和瓜氨酸,并且二者均来源于精氨酸的分解代谢,因此对这三者代谢调控机制的研究能够为EC抑制途径的探索提供新的思路,本文主要就尿素、瓜氨酸及精氨酸分别在酿酒酵母及乳酸菌中的代谢调控机制进行系统的概述,并对未来EC的相关研究进行展望。     

Effects of storage time and temperature on the concentration of ethyl carbamate and its precursors in wine

A 3-year study was carried out on the effects of time and temperature on the concentration of ethyl carbamate in wine. The study monitored the changing concentration of ethyl carbamate and of urea and citrulline, which are two major precursors of ethyl carbamate in wine. In addition to the formation of ethyl carbamate, both urea and citrulline decayed in other reactions. Kinetic analysis was carried out to model the formation of ethyl carbamate and its dependence on the concentrations of ethanol, urea and citrulline. This led to the development of an equation that can be used to predict the concentration of ethyl carbamate in wine at the point of consumption, resulting from any given storage time and temperature profile. The results were in good agreement with data obtained from similar studies.     

Effect of ageing process on carcinogen ethyl carbamate (EC), its main precursors and aroma compound variation in Hakka Huangjiu produced in southern China

Wine ageing is of great importance to the quality and organoleptic properties of Huangjiu. The aim of the present work was to investigate the effect of ageing temperature on the content changes in ethyl carbamate (EC) and its precursors including citrulline, arginine, ornithine and urea in Hakka Huangjiu. Aroma compound changes were also detected using headspace solid-phase microextraction coupled with gas chromatography spectrometry. Results indicated that with the increase in ageing temperature, citrulline and EC concentrations increased, whereas arginine and ornithine concentrations decreased. Urea was decreased slightly from 4 to 20 °C while increased at 37 °C. Pearson test demonstrated that ageing temperature had significant correlation with EC and arginine. The concentrations of aroma compounds, especially esters, alcohols, acids and heterocyclic compounds were different in different ageing temperatures. Ageing temperature of Hakka Huangjiu was suggested setting in a lower temperature as for strictly controlling contents of urea and citrulline, as well as EC.