Adapter-Directed Display: A Modular Design for Shuttling Display on Phage Surfaces

A novel adapter-directed phage display system was developed with modular features. In this system, the target protein is expressed as a fusion protein consisting of adapter GR1 from the phagemid vector, while the recombinant phage coat protein is expressed as a fusion protein consisting of adapter GR2 in the helper phage vector. Surface display of the target protein is accomplished through specific heterodimerization of GR1 and GR2 adapters, followed by incorporation of the heterodimers into phage particles. A series of engineered helper phages were constructed to facilitate both display valency and formats, based on various phage coat proteins. As the target protein is independent of a specific phage coat protein, this modular system allows the target protein to be displayed on any given phage coat protein and allows various display formats from the same vector without the need for reengineering. Here, we demonstrate the shuttling display of a single-chain Fv antibody on phage surfaces between multivalent and monovalent formats, as well as the shuttling display of an antigen-binding fragment molecule on phage coat proteins pIII, pVII, and pVIII using the same phagemid vectors combined with different helper phage vectors. This adapter-directed display concept has been applied to eukaryotic yeast surface display and to a novel cross-species display that can shuttle between prokaryotic phage and eukaryotic yeast systems.     

Growth of human mesenchymal stem cells (MSCs) on films of enzymatically modified chitosan

Mesenchymal stem cells (MSCs) are known to be an attractive cell source for tissue engineering and regenerative medicine. One of the main limiting steps for clinical use or biotechnological purposes is the expansion step. The research of compatible biomaterials for MSCs expansion is recently regarded as an attractive topic. The aim of this study was to create new functional biomaterial for MSCs expansion by evaluating the impact of chitosan derivative films modified by enzymatic approach. First, chitosan particles were enzymatically modified with ferulic acid (FA) or ethyl ferulate (EF) under an eco‐friendly procedure. Then, films of chitosan and its modified derivatives were prepared and evaluated by physicochemical and biological properties. Results showed that the enzymatic grafting of FA or EF onto chitosan significantly increased hydrophobic and antioxidant properties of chitosan films. The MSCs cell viability on chitosan derivative films also increased depending on the film thickness and the quantity of grafted phenols. Furthermore, the cytotoxicity test showed the absence of toxic effect of chitosan derivative films towards MSCs cells. Cell morphology showed a well attached and spread phenotype of MSCs cells on chitosan derivative films. On the other hand, due to the higher phenol content of FA‐chitosan films, their hydrophobic, antioxidant properties and cell adhesion were improved in comparison with those of EF‐chitosan films. Finally, this enzymatic process can be considered as a promising process to favor MSCs cell growth as well as to create useful biomaterials for biomedical applications especially for tissue engineering. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:491–500, 2016     

卷丝苣苔和勐醒芒毛苣苔抗氧化活性研究

利用3种方法DPPH、ABTS和FRAP分析两种苦苣苔科植物卷丝苣苔和勐醒芒毛苣苔提取物总抗氧化作用.在4种提取物中,卷丝苣苔甲醇提取物对DPPH自由基的清除能力(IC50=4.92μg/mL)比阳性对照BHT作用强(IC50=18.79 μg/mL);卷丝苣苔甲醇提取物对ABTS自由基的清除能力(IC50=11.10 μg/mL)比BHT(IC50=6.04 μg/mL)略低;卷丝苣苔甲醇提取物还原Fe3+的能力(FRAP=2403.77±38.05 μmol TE/g)比BHT(FRAP=1748.49±3.46 μmol TE/g)高.在4种提取物中,卷丝苣苔甲醇提取物抗氧化能力最高.     

Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer

Cancer prevention and treatment using traditional Chinese medicines have attracted increasing interest. This study characterizes antioxidant activity and phenolic compounds of traditional Chinese medicinal plants associated with anticancer, comprising 112 species from 50 plant families. The improved ABTS(*+) method was used to systematically assess the total antioxidant capacity (Trolox equivalent antioxidant capacity, TEAC) of the medicinal extracts. The TEAC values and total phenolic content for methanolic extracts of herbs ranged from 46.7 to 17,323 micromol Trolox equivalent/100 g dry weight (DW), and from 0.22 to 50.3 g of gallic acid equivalent/100 g DW, respectively. A positive, significant linear relationship between antioxidant activity and total phenolic content (all R(2) values>/=0.95) showed that phenolic compounds were the dominant antioxidant components in the tested medicinal herbs. Major types of phenolic compounds from most of the tested herbs were preliminarily identified and analyzed, and mainly included phenolic acids, flavonoids, tannins, coumarins, lignans, quinones, stilbenes, and curcuminoids. These medicinal herbs exhibited far stronger antioxidant activity and contained significantly higher levels of phenolics than common vegetables and fruits. Traditional Chinese medicinal plants associated with anticancer might be potential sources of potent natural antioxidants and beneficial chemopreventive agents.     

Non-enzymatic antioxidant capacity assays: Limitations of use in biomedicine

Abstract

The ‘Total antioxidant capacity’ (TAC) is a parameter frequently used for characterization of food products and of the antioxidant status of the body. This mini-review shows shortcomings of TAC assays and points of concern that should be considered when performing and interpreting results of such assays. The term TAC is not optimal since the assay measures only part of antioxidant capacity, usually excluding enzymatic activities. Antioxidant and oxidant-regenerating enzymes in blood cells and the blood vessel wall have a profound impact on the antioxidant properties of blood plasma, which is not reflected in the in vitro assays of isolated plasma. The term ‘Non-enzymatic antioxidant capacity’ (NEAC) is suggested as more relevant than TAC. NEAC is estimated by various methods, which yield different values and results obtained using different methods do not always show satisfactory correlation. One reason for the discrepancy of results is the use of different oxidants in NEAC assays. The use of hydroxyl radical as the oxidant is not recommended in view of the high and non-specific reactivity of this species.     

Anticholinesterse and antioxidant investigations of crude extracts,subsequent fractions,saponins and flavonoids of atriplex laciniata L.: potential effectiveness in Alzheimer’s and other neurological disorders

Background

Atriplex laciniata L. was investigated for phenolic, flavonoid contents, antioxidant, anticholinesterase activities, in an attempt to explore its effectiveness in Alzheimer’s and other neurological disorders. Plant crude methanolic extract (Al.MeF), subsequent fractions; n-hexane (Al.HxF), chloroform (Al.CfF), ethyl acetate (Al.EaF), aqueous (Al.WtF), Saponins (Al.SPF) and Flavonoids (Al.FLVF) were investigated for DPPH, ABTS and H₂O₂ free radical scavenging activities. Further these extracts were subjected to acetylcholinesterase (AChE) & butyrylcholinesterase (BChE) inhibitory activities using Ellman’s assay. Phenolic and Flavonoid contents were determined and expressed in mg Gallic acid GAE/g and Rutin RTE/g of samples respectively.

Results

In DPPH free radicals scavenging assay, Al.FLVF, Al.SPF and Al.MeF showed highest activity causing 89.41 ± 0.55, 83.37 ± 0.34 and 83.37 ± 0.34% inhibition of free radicals respectively at 1 mg/mL concentration. IC₅₀ for these fractions were 33, 83 and 82 μg/mL respectively. Similarly, plant extracts showed high ABTS scavenging potential, i.e. Al.FLVF (90.34 ± 0.55), Al.CfF (83.42 ± 0.57), Al.MeF (81.49 ± 0.60) with IC₅₀ of 30, 190 and 70 μg/ml respectively. further, H₂O₂ percent scavenging was highly appraised in Al.FLVF (91.29 ± 0.53, IC₅₀ 75), Al.SPF (85.35 ± 0.61, IC₅₀ 70) and Al.EaF (83.48 ± 0.67, IC₅₀ 270 μg/mL). All fractions exhibited concentration dependent AChE inhibitory activity as; Al.FLVF, 88.31 ± 0.57 (IC₅₀ 70 μg/mL), Al.SPF, 84.36 ± 0.64 (IC₅₀ 90 μg/mL), Al.MeF, 78.65 ± 0.70 (IC₅₀ 280 μg/mL), Al.EaF, 77.45 ± 0.46 (IC₅₀ 270 μg/mL) and Al.WtF 72.44 ± 0.58 (IC₅₀ 263 μg/mL) at 1 mg/mL. Likewise the percent BChE inhibitory activity was most obvious in Al.FLVF 85.46 ± 0.62 (IC₅₀ 100 μg/mL), Al.CfF 83.49 ± 0.46 (IC₅₀ 160 μg/mL), Al.MeF 82.68 ± 0.60 (IC₅₀ 220 μg/mL) and Al.SPF 80.37 ± 0.54 (IC₅₀ 120 μg/mL).

Conclusions

These results stipulate that A. laciniata is enriched with phenolic and flavonoid contents that possess significant antioxidant and anticholinestrase effects. This provide pharmacological basis for the presence of compounds that may be effective in Alzheimer’s and other neurological disorders.     

Endogenous and Dietary Indoles: A Class of Antioxidants and Radical Scavengers in the ABTS Assay

Indoles are very common in the body and diet and participate in many biochemical processes. A total of twenty-nine indoles and analogs were examined for their properties as antioxidants and radical scavengers against 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ABTS^?+ radical cation. With only a few exceptions, indoles reacted nonspecifically and quenched this radical at physiological pH affording ABTS. Indoleamines like tryptamine, serotonin and methoxytryptamine, neurohormones (melatonin), phytohormones (indoleacetic acid and indolepropionic acid), indoleamino acids like l-tryptophan and derivatives (N-acetyltryptophan, l-abrine, tryptophan ethyl ester), indolealcohols (tryptophol and indole-3-carbinol), short peptides containing tryptophan, and tetrahydro-β-carboline (pyridoindole) alkaloids like the pineal gland compound pinoline, acted as radical scavengers and antioxidants in an ABTS assay-measuring total antioxidant activity. Their trolox equivalent antioxidant capacity (TEAC) values ranged from 0.66 to 3.9?mM, usually higher than that for Trolox and ascorbic acid (1?mM). The highest antioxidant values were determined for melatonin, 5-hydroxytryptophan, trp-trp and 5-methoxytryptamine. Active indole compounds were consumed during the reaction with ABTS^?+ and some tetrahydropyrido indoles (e.g. harmaline and 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid ethyl ester) afforded the corresponding fully aromatic β-carbolines (pyridoindoles), that did not scavenge ABTS^?+. Radical scavenger activity of indoles against ABTS^?+ was higher at physiological pH than at low pH. These results point out to structural compounds with an indole moiety as a class of radical scavengers and antioxidants. This activity could be of biological significance given the physiological concentrations and body distribution of some indoles.     

The antioxidant activities of seasonings used in Asian cooking. Powerful antioxidant activity of dark soy sauce revealed using the ABTS assay

Scavenging of the ABTS (2,2′-azinobis[3-ethylbenzothiazoline-6-sulphonate])-derived nitrogen-centred radical cation (ABTS^?+) was used to compare the total antioxidant activities of several seasonings used in Asian cooking. The results were expressed as Trolox equivalent antioxidant capacity (TEAC). The TEAC activities of dark soy sauces were found to be exceptionally high. In evaluating the TEAC of commercial products, attention must be paid to the addition of preservatives by manufacturers to the seasonings tested. Sodium benzoate (a preservative added to several seasonings) did not react significantly with ABTS^?+, but the sulphite content of certain white wines may have led to an over-estimation of their TEAC.     

Antioxidant profile of dihydroxy- and trihydroxyphenolic acids-A structure–activity relationship study

Eight structurally similar dihydroxy and trihydroxyphenolic acids (protocatechuic acid, 3,4-dihydroxyphenylacetic acid, hydrocaffeic acid, caffeic acid, gallic acid, 3,4,5-trihydroxyphenylacetic acid, 3-(3,4,5-trihydroxyphenyl)propanoic acid and 3-(3,4,5-trihydroxyphenyl)propenoic acid) were examined for their total antioxidant capacity (TAC). Furthermore, their ability to scavenge peroxyl radicals, generated by AAPH in liposomes, was determined. The antioxidant/pro-oxidant activity of the compounds was screened using the 2′-deoxyguanosine assay. All compounds behave as radical scavengers, with 3,4,5-trihydroxyphenylacetic acid being the most potent. Nevertheless, in the lipid peroxidation assay an inverse ranking order was observed, 3,4-dihydroxyphenylacetic acid being the most effective compound. All the dihydroxylated compounds showed a pro-oxidant behaviour leading to an increase of 50% in 8-OH-dG induction. From the structure–antioxidant activity relationship studies performed it may be concluded that the number of phenolic groups and the type of the alkyl spacer between the carboxylic acid and the aromatic ring strongly influence the antioxidant activity.     

Antioxidants and ROS scavenging ability in ten Darjeeling tea clones may serve as markers for selection of potentially adapted clones against abiotic stress

Ten Darjeeling tea clones (BT15/263, RR17/144, B777, T253, B157, Sundaram, HV39, AV2, K1/1 and TTV1) were collected from the experimental garden of Darjeeling Tea Research and Development Centre, Kurseong. Total phenol, flavonoids and two antioxidating enzymes (peroxidase and superoxide dismutase) were estimated. The total phenol ranged between 241 and 28 GAE mg g⁻¹ of leaf dry weight. The highest amount obtained in four clones, B15/263 (241.47), RR17/144 (221.2), B777 (154.54) and B157 (140.23 mg g⁻¹). Flavonoids were estimated as Catechin equivalent (CE) and ranged between 56.88 and 20.81 CE mg g⁻¹ leaf dry weight. Higher amounts occurred in BT15/263 (56.88 mg g⁻¹), B777 (56.69) and RR17/144 (48.63). Antioxidant activities were measured following DPPH and ABTS free radicle scavenging procedures and the results were well according to total polyphenol content among the clones (in total phenols, ranges of correlation in DPPH assay were r² = 0.990–0.989, p ≤ 0.05; in flavonoids r² = 0.954, p ≤ 0.01–0.987, p ≤ 0.05). Similarly, ABTS percent scavenging results were quiet significant. The IC₅₀ values were determined for both DPPH and ABTS assay. PAGE expressions of isoforms in two antioxidative enzymes and quantification of them also varied much among the investigated clones. The incidence of total phenols, flavonoids, PRX and SOD and ROS scavenging assay in in-situ condition, might be used as biochemical markers towards the superior adaptability against abiotic stress. In the present work, four clones (B15/263, B777, RR17/144 and B157) would be designated as comparatively better suited to the predicted abiotic stress.