Polyamine dependence of Chinese hamster ovary cells in serum-free culture is due to deficient arginase activity

We recently isolated a Chinese hamster ovary cell line which grows well without serum but requires the exogenous polyamines putrescine, spermidine or spermine for continuous replication. Here we show that these cells are defective in the arginase-catalyzed synthesis of ornithine, the precursor of polyamines, and that ornithine can replace polyamines in the medium for supporting growth of the cells. The activities of two other key enzymes of polyamine biosynthesis, ornithine decarboxylase and adenosylmethionine decarboxylase, are clearly detectable and show increase during polyamine starvation. In ornithine- and polyamine-free medium cellular putrescine and spermidine are rapidly depleted while the concentration of spermine decreases only moderately. We show further that the cells are able to grow in serum-containing medium without added ornithine or polyamines. This is explained by our finding that serum contains arginase which synthesizes ornithine from arginine in the medium. All the sera from different animal species tested contained arginase activity although in greatly varying amounts. Serum-free medium is therefore essential for expression of arginase deficiency in cells in tissue culture. The eventual importance of polyamines for serum-free cultures in general is discussed.     

Nitrogenous excretion and arginase specific activity of kuruma shrimp Marsupenaeus japonicus exposed to elevated ambient nitrite

Nitrogenous excretion and arginase specific activity were measured while Marsupenaeus japnoicus Bate (7.4±1.2 g) were exposed to 0 (control), 0.36 and 1.39 mM nitrite at 30‰ (g kg⁻¹) salinity for 24 h. Excretions of total-N, organic-N, urea-N, and ammonia-N increased significantly with an increase of ambient nitrite. Arginase specific activities of hepatopancreas and hemolymph increased directly with ambient nitrite. The fact that M. japonicus following exposure to 1.39 mM nitrite increased its arginase specific activity indicated an argininolysis in reducing joint toxicities of metabolic ammonia and incorporated nitrite.     

Development of chromatographic and free radical scavenging activity fingerprints by thin‐layer chromatography for selected Salvia species

Introduction – Plant‐derived free radical scavengers have become the subject of intensive scientific interest. Recently, the concept of coupling chromatographic fingerprints with biological fingerprinting analysis has gained much attention for the quality control of plant extracts. However, identification of free radical scavenging activity of each single compound in a complex mixture is a difficult task. Thin‐layer chromatography with post‐chromatographic derivatisation with the methanol solution of DPPH can be a valuable tool in such analyses. Objective – Development of chromatographic and free radical scavenging fingerprints of nineteen Salvia species grown and cultivated in Poland. Methodology – Chromatography was performed on the silica gel layers with use of two eluents, one for the resolution of the less polar compounds, and the other one for the resolution of the medium and highly polar ones. The plates were sprayed with the vanillin–sulfuric acid reagent to produce chemical fingerprints, and with DPPH solution to generate free radical scavenging fingerprints. Results – With four Salvia species, it was revealed that their strong free radical scavenging properties are not only due to the presence of polar flavonoids and phenolic acids, but also due to the presence of several free radical scavengers in the less polar fraction. Because of the similarities in both the chromatographic and the free radical scavenging fingerprints, S. triloba can be introduced as a possible equivalent of the pharmacopoeial species, S. officinalis. Conclusion – Fingerprints developed in the experiments proved useful for the analysis of complex extracts of the different Salvia species. Copyright © 2010 John Wiley & Sons, Ltd.     

Free radical scavenging activity of novel thiazolidine‐2,4‐dione derivatives

Free radical activity towards superoxide anion radical (), hydroxyl radical (HO^?) and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) of a series of novel thiazolidine‐2,4‐dione derivatives (TSs) was examined using chemiluminescence, electron paramagnetic resonance (EPR) and EPR spin trapping techniques. 5,5‐Dimethyl‐1‐pyrroline‐N‐oxide (DMPO) was applied as the spin trap. Superoxide radical was produced in the potassium superoxide/18‐crown‐6 ether dissolved in dimethyl sulfoxide. Hydroxyl radical was generated in the Fenton reaction (Fe(II) + H₂O₂. It was found that TSs showed a slight scavenging effect (15–38% reduction at 2.5 mmol/L concentration) of the DPPH radical and a high scavenging effect of (41–88%). The tested compounds showed inhibition of HO^? ‐dependent DMPO‐OH spin adduct formation (the amplitude of EPR signal decrease ranged from 20 to 76% at 2.5 mmol/L concentration. Our findings present new group compounds of relatively high reactivity towards free radicals. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis and activity of a coumarin‐based fluorescent probe for hydroxyl radical detection

As a type of reactive oxygen species (ROS), hydroxyl radical (·OH) is closely associated with many kinds of diseases. The present study aimed to develo p a novel OH fluorescent probe based on coumarin, a new compound that has not been previously reported. This probe exhibited good linear range and selectivity for ·OHl, and is able to avoid interference from some metal ions and other kinds of ROS (H₂O₂, O₂^.‐, ¹O₂, and HClO). Meanwhile, this probe has been used to evaluate the ·OH‐scavenging efficiency of different compounds, such as isopropyl alcohol, cytosine, uracil, Tempo, Glutathione (GSH), and dimethyl sulfoxide (DMSO). Therefore, the present study shows that this probe not only can effectively measure the level of ·OH, but also can assess the ·OH‐scavenging efficiency of different compounds. Furthermore this current study suggested that following further optimization, this probe may be potentially applied in the diagnosis of oxidative stress in human body.     

Novel hydroxyl radical scavenging antioxidant activity assay for water-soluble antioxidants using a modified CUPRAC method

Reactive oxygen species (ROS) such as superoxide anion, hydroxyl ((*)OH), peroxyl, and alkoxyl radicals may attack biological macromolecules giving rise to oxidative stress-originated diseases. Since (*)OH is very short-lived, secondary products resulting from (*)OH attack to various probes are measured. Although the measurement of aromatic hydroxylation with HPLC/electrochemical detection is more specific than the low-yield TBARS test, it requires sophisticated instrumentation. As a more convenient and less costly alternative, we used p-aminobenzoate, 2,4- and 3,5-dimethoxybenzoate probes for detecting hydroxyl radicals generated from an equivalent mixture of Fe(II)+EDTA with hydrogen peroxide. The produced hydroxyl radicals attacked both the probe and the water-soluble antioxidants in 37 degrees C-incubated solutions for 2h. The CUPRAC (i.e., our original method for total antioxidant capacity assay) absorbance of the ethylacetate extract due to the reduction of Cu(II)-neocuproine reagent by the hydroxylated probe decreased in the presence of (*)OH scavengers, the difference being proportional to the scavenging ability of the tested compound. A rate constant for the reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. The second-order rate constants of the scavengers were determined with competition kinetics by means of a linear plot of A(0)/A as a function of C(scavenger)/C(probe), where A(0) and A are the CUPRAC absorbances of the system in the absence and presence of scavenger, respectively, and C is the molar concentration of relevant species. The 2,4- and 3,5-dimethoxybenzoates were the best probes in terms of linearity and sensitivity. Iodide, metabisulfite, hexacyanoferrate(II), thiourea, formate, and dimethyl sulfoxide were shown by the modified CUPRAC assay to be more effective scavengers than mannitol, glucose, lysine, and simple alcohols, as in the TBARS assay. The developed method is less lengthy, more specific, and of a higher yield than the classical TBARS assay. The hydroxyl radical scavenging rate constants of ascorbic acid, formate, and hexacyanoferrate(II) that caused interference in other assays could be easily found with the proposed procedure.     

Activation of Leishmania (Leishmania) amazonensis arginase at low temperature by binuclear Mn center formation of the immobilized enzyme on a Ni resin

In Leishmania, arginase is responsible for the production of ornithine, a precursor of polyamines required for proliferation of the parasite. In this work, the activation kinetics of immobilized arginase enzyme from L. (L.) amazonensis were studied by varying the concentration of Mn²⁺ applied to the nickel column at 23 °C. The intensity of the binding of the enzyme to the Ni²⁺ resin was directly proportional to the concentration of Mn²⁺. Conformational changes of the enzyme may occur when the enzyme interacts with immobilized Ni²⁺, allowing the following to occur: (1) entrance of Mn²⁺ and formation of the metal bridge; (2) stabilization and activation of the enzyme at 23 °C; and (3) an increase in the affinity of the enzyme to Ni²⁺ after the Mn²⁺ activation step. The conformational alterations can be summarized as follows: the interaction with the Ni²⁺ simulates thermal heating in the artificial activation by opening a channel for Mn²⁺ to enter.     

Evaluation of hydroxyl radical-scavenging property of purpurogallin using high pressure liquid chromatography

Purpurogallin (PPG) has been used as an additive to edible and non-edible oils or fats to retard oxidation. Its antioxidant mechanism is not known. We investigated the ability of PPG to scavenge exogenously generated hydroxyl radicals (·OH) using a sensitive high pressure liquid chromatographic (HPLC) method. ·OH was generated by photolysis of H₂O₂ (1.25–10 moles) with UV light and was trapped with salicylic acid (500 nmoles). Salicylic acid is hydroxylated to produce ·OH adduct products 2,3-and 2,5-dihydroxybenzoic acid (DHBA). H₂O₂ produced concentration-dependent ·OH as estimated by generation of 2,3- and 2,5-DHBA. PPG (100, 200, 300, 400, 500 and 600 nmoles) produced concentration-dependent decreases in ·OH adduct products (approximately 70% inhibition with 600 nmoles of PPG). It did not affect the peak of standard 2,3- and 2,5-DHBA indicating that the decrease in the adduct product generated by H₂O₂ is due to scavenging of ·OH. These results indicate that photolysis of H₂O₂ by UV light produces ·OH and that PPG scavenges ·OH.     

Probing antioxidant activity of 2′-hydroxychalcones: Crystal and molecular structures, in vitro antiproliferative studies and in vivo effects on glucose regulation

In order to better understand the antioxidant behavior of a series of polyphenolic 2′-hydroxychalcones, we describe the results of several chemical and biological studies, in vitro and in vivo. Single crystal X-ray methods elucidated their molecular structures and important intermolecular interactions such as H-bonding and molecular stacking in the crystal structures that contribute to our knowledge in explaining antioxidant activity. The results of experiments using the 1,1-diphenyl-2-dipicrylhydrazyl (DPPH) UV–vis spectroscopic method indicate that a hydroxyl group in position 5′ induces the highest antioxidant activity. Consequently, 2,2′,5′-trihydroxychalcone was selected for further study in vitro towards ROS scavenging in L-6 myoblasts and THP-1 human monocytes, where it shows an excellent antioxidant activity in a concentration range lower than that reported by most studies of related molecules. In addition, this chalcone shows a very selective activity: it inhibits the proliferation of leukemic cells, but it does not affect the normal L-6 myoblasts and human fibroblasts. In studying 2,2′,5′-trihydroxychalcone's effect on weight gain and serum glucose and insulin levels in Zucker fatty (fa⁻/fa⁻) rats we found that supplementing the diet with a 10 mg/kg dose of this chalcone (3 times weekly) blunted the increase in glucose that co-occurs with weight gain over the 6-week treatment period. It is concluded that 2,2′,5′-trihydroxychalcone has the potential to serve as a protective agent for some debilitating diseases.     

Preparation and characterization of molecular weight fractions of glycosaminoglycan from sea cucumber Thelenata ananas using free radical depolymerization

A glycosaminoglycan from sea cucumber Thelenata anana (THG) was isolated as a polymer of molecular weight of around 70 kDa. Its low molecular weight derivatives were first prepared by free radical depolymerization with hydrogen peroxide in the presence of copper(II) ion. The parameters of the process were investigated by a high-performance gel permeation chromatography. Analyses of chemical composition and molecular weight distribution indicated that the fragmentation of the main-chain of THG occurred randomly, obeyed pseudo first-order kinetics, and produced species with rather narrow and unimodal distribution of molar mass. The characterization of different molecular weight fractions was investigated by using viscometry and atomic force microscopy (AFM). Analysis of molecular weight and intrinsic viscosity in terms of the known theories for unperturbed wormlike cylinder yielded 1201 ± 110 nm⁻¹, 15.3 ± 1.5 nm, and 1.5 ± 0.3 nm for molar mass per unit contour length M_L, persistence length q, and diameter d, respectively. The M_L and d values were approximately consistent with those observed by AFM. The present data suggest that THG may dissolve in 0.1 M aqueous NaCl as single-stranded helical chains.     

Cobalt(II) ion as a promoter of hydroxyl radical and possible ‘crypto-hydroxyl’ radical formation under physiological conditions. Differential effects of hydroxyl radical scavengers

Co(II) ions react with hydrogen peroxide under physiological conditions to form a ‘reactive species’ that can hydroxylate aromatic compounds (phenol and salicylate) and degrade deoxyribose to thiobarbituric-acid-reactive material. Catalase decreases the formation of this species but superoxide dismutase or low concentrations of ascorbic acid have little effect. EDTA, present in excess over the Co(II), can accelerate deoxyribose degradation and aromatic hydroxylation. In the presence of EDTA, deoxyribose degradation by the reactive species is inhibited competitively by scavengers of the hydroxyl radical (OH), their effectiveness being related to their second-order rate constants for reaction with OH. In the absence of EDTA the scavengers inhibit only at much higher concentrations and their order of effectiveness is changed. It is suggested that, in the presence of EDTA, hydroxyl radical is formed ‘in free solution’ and attacks deoxyribose or an aromatic molecule. In the absence of EDTA, OH radical is formed in a ‘site-specific’ manner and is difficult to intercept by OH scavengers. The relationship of these results to the proposed ‘crypto OH’ radical is discussed.     

Synthesis,molecular docking and kinetic studies of novel quinolinyl based acyl thioureas as mushroom tyrosinase inhibitors and free radical scavengers

The enzyme tyrosinase plays a vital role in melanin biosynthesis and enzymatic browning of vegetables and fruits. A series of novel quinolinyl thiourea analogues (11a-j) were synthesized by reaction of 3-aminoquinoline and corresponding isothiocyanates, in moderate to excellent yields with different substitutions and their inhibitory effect on mushroom tyrosinase and free radical scavenging activity were evaluated. The compound N-(quinolin-3-ylcarbamothioyl)hexanamide (11c) exhibited the maximum tyrosinase inhibitory effect (IC₅₀ = 0.0070 ± 0.0098 µM) compared to other derivatives and the reference Kojic acid (IC₅₀ = 16.8320 ± 0.0621 µM). The docking studies were carried out and the compound (11c) showed most negative estimated free energy of −7.2 kcal/mol in mushroom tyrosinase active site. The kinetic analysis revealed that the compound (11c) inhibits the enzyme tyrosinase non-competitively to form the complex of enzyme and inhibitor. The results revealed that 11c could be identified as putative lead compound for the design of efficient tyrosinase inhibitors.     

Free radical scavenging activity of Lactobacillus fermentum in vitro and its antioxidative effect on growing–finishing pigs

Aims:  To evaluate the free radical-scavenging capacity of Lactobacillus fermentum and its effects on antioxidant enzyme levels in finishing pigs.
Methods and Results:  The free radical-scavenging activity of Lact. fermentum was analysed in vitro . The tested Lactobacillus showed a high scavenging ability against DPPH (1,1-diphenyl-2-picrylhydrazyl), superoxide and hydroxyl radicals which was dose dependent. Subsequently, 108 crossbred pigs weighing 20·67 BW, were allotted to dietary treatments including a basal diet or the basal diet supplemented with either aureomycin or 10·2 × 10⁷  Lact. fermentum CFU g⁻¹ diet. Supplementation of Lact. fermentum increased total antioxidant capacity ( P  < 0·01) in serum from 50 kg pigs, while serum superoxide dismutase ( P  = 0·01) and glutathione peroxidase ( P  < 0·01) increased, and malondialdehyde levels decreased ( P  < 0·01) in 90 kg pigs. Hepatic catalase ( P  = 0·04), muscle superoxide dismutase ( P  < 0·01) and copper–zinc-superoxide dismutase were enhanced ( P  = 0·01), whereas malondialdehyde levels were reduced ( P  = 0·05) by Lact. fermentum .
Conclusions:  The free radical-scavenging capacity of Lact. fermentum was dose dependent and its supplementation improved the antioxidant status of pigs.
Significance and Impact of the Study:  Lactobacillus fermentum could be used to alleviate oxidative stress and increase pig performance and improve pork quality.     

Measurement of OH radical CT for inactivating Cryptosporidium parvum using photo/ferrioxalate and photo/TiO2 systems

Aim: This study investigates the inactivation of Cryptosporidium parvum using the OH radical and reports the OH radical CT (OH radical concentration × contact time) values for C. parvum inactivation. Methods and Results: Although a wealth of information has demonstrated the efficacy of the microbial inactivation activity of the OH radical, no studies have performed a quantitative estimation of the OH radical for C. parvum inactivation. The CT value of the OH radical required for 2 log C. parvum inactivation was measured with two OH radical‐generating systems, photo/ferrioxalate and photo/TiO₂. The OH radical was approx. 10⁴–10⁷‐fold more effective for microbial inactivation than other popular chemical disinfectants such as ozone, chlorine dioxide and free chlorine. Conclusions: The OH radical appears to be suitable for microbial inactivation with a calculated CT value required for 2 log C. parvum inactivation of 9·3 × 10^?5 mg min l^?1. Significance and Impact of the Study: This study is the first report of an investigation on the role of the OH radical in the photo/ferrioxalate and photo/TiO₂ systems and on the OH radical CT required for C. parvum inactivation.     

雪松不同部位中原花青素的含量测定及其清除DPPH自由基的活性

雪松(Cedrus deodara)是松科(Pianaceae)雪松属(Cedrus)树种的泛称,为广泛分布于喜马拉雅山脉的常绿树种,包括雪松(C.deodara)、黎巴嫩雪松(C.libani)、短叶雪松(C.brevifolia)和北非雪松(C.atlantica)等。雪松的针叶药用历史悠久,主要化学成分为黄酮类、苯丙素类、有机酸类、三萜类、甾体类、多糖及针叶胶等,具有抗肿瘤、抗氧化、改善记忆、抗菌及抗病毒等多种功效。为了测定雪松不同部位中原花青素的含量,并考察其清除DPPH自由基活性的能力,该研究以Fe3+离子为催化剂,用正丁醇-盐酸法分别测定雪松松针、花序、小枝、木质部、树皮中原花青素的含量,并对不同部位提取液清除DPPH自由基的能力进行测定。结果表明:松针、花序、木质部、树皮、小枝中原花青素的含量分别为12.93%、12.93%、6.41%、12.00%、3.37%,在质量浓度(以生药计)为0.8 mg·m L-1时,其对DPPH自由基的清除率均达到或接近90%。该研究结果提示雪松各个部位的原花青素含量丰富,其提取液的体外抗氧化活性很高,这为雪松的进一步开发利用提供了很好的参考价值。     

Optimization of enzyme-assisted improvement of polyphenols and free radical scavenging activity in red rice bran: A statistical and neural network-based approach

The current study is focused on optimizing the parameters involved in enzymatic processing of red rice bran for maximizing total polyphenol (TP) and free radical scavenging activity (FRSA). The sequential optimization strategies using central composite design (CCD) and artificial neural network (ANN) modeling linked with genetic algorithm (GA) was performed to study the effect of incubation time (60–90?min), xylanase concentration (5–10?mg/g), cellulase concentration (5–10?mg/g) on the response, i.e., total polyphenol and FRSA. The result showed that incubation time has a negative effect on the response, while the square effect of xylanase and cellulase showed positive effect on the response. A maximum TP of 2,761?mg ferulic acid Eq/100?g bran and FRSA of 778.4?mg Catechin Eq/100?g bran was achieved with incubation time (min)?=?60.491; xylanase (mg/g)?=?5.4633; cellulase (mg/g)?=?11.5825. Furthermore, ANN-GA-based optimization showed better predicting capabilities as compared to CCD.     

The rate of free radical production as a determinant of the rate of aging: evidence from the comparative approach

The relationship of oxidative stress with maximum life span (MLSP) in different vertebrate species is reviewed. In all animal groups the endogenous levels of enzymatic and non-enzymatic antioxidants in tissues negatively correlate with MLSP and the most longevous animals studied in each group, pigeon or man, show the minimum levels of antioxidants. A possible evolutionary reason for this is that longevous animals produce oxygen radicals at a low rate. This has been analysed at the place where more than 90% of oxygen is consumed in the cell, the mitochondria. All available work agrees that, across species, the longer the life span, the lower the rate of mitochondrial oxygen radical production. This is true even in animal groups that do not conform to the rate of living theory of aging, such as birds. Birds have low rates of mitochondrial oxygen radical production, frequently due to a low free radical leak in their respiratory chain. Possibly the low rate of mitochondrial oxygen radical production of longevous species can decrease oxidative damage at targets important for aging (like mitochondrial DNA) that are situated near the places of free radical generation. A low rate of free radical production can contribute to a low aging rate both in animals that conform to the rate of living (metabolic) theory of aging and in animals with exceptional longevities, like birds and primates. Available research indicates there are at least two main characteristics of longevous species: a high rate of DNA repair together with a low rate of free radical production near DNA. Simultaneous consideration of these two characteristics can explain part of the quantitative differences in longevity between animal species. Accepted: 12 December 1997