A globotriaosylceramide (Gb3Cer) mimic peptide isolated from phage display library expressed strong neutralization to Shiga toxins

A Gb₃-trisaccharide mimic peptide was selected with biopanning from a phage display library against anti-Gb₃ antibody to neutralize Shiga toxins (Stxs). Biopanning was carried out on a microplate immobilized with a Fab fragment of anti-Gb₃ antibody and a subtraction procedure screening was applied to enhance specificity. The selected phage clones showed strong affinity to anti-Gb₃ antibody and to Stxs. Among these clones, a 9-mer sequence WHWTWLSEY was determined as the strongest Gb₃ mimic peptide and chemically synthesized. The peptide bound strongly to Stx-1 and Stx-2, though the binding was inhibited with Gb₃Cer. Surface plasmon resonance (SPR) and fluorescent spectroscopy determined that the affinity of the peptide to both Stxs was strong. Neutralization activity was confirmed by in vitro assay with HeLa cells. The Gb₃ mimic peptide potentially has great promise for use against Stxs.     

Structure-Function Studies of a Melanocarpus albomyces Laccase Suggest a Pathway for Oxidation of Phenolic Compounds

Melanocarpus albomyces laccase crystals were soaked with 2,6-dimethoxyphenol, a common laccase substrate. Three complex structures from different soaking times were solved. Crystal structures revealed the binding of the original substrate and adducts formed by enzymatic oxidation of the substrate. The dimeric oxidation products were identified by mass spectrometry. In the crystals, a 2,6-dimethoxy-p-benzoquinone and a C-O dimer were observed, whereas a C-C dimer was the main product identified by mass spectrometry. Crystal structures demonstrated that the substrate and/or its oxidation products were bound in the pocket formed by residues Ala191, Pro192, Glu235, Leu363, Phe371, Trp373, Phe427, Leu429, Trp507 and His508. Substrate and adducts were hydrogen-bonded to His508, one of the ligands of type 1 copper. Therefore, this surface-exposed histidine most likely has a role in electron transfer by laccases. Based on our mutagenesis studies, the carboxylic acid residue Glu235 at the bottom of the binding site pocket is also crucial in the oxidation of phenolics. Glu235 may be responsible for the abstraction of a proton from the OH group of the substrate and His508 may extract an electron. In addition, crystal structures revealed a secondary binding site formed through weak dimerization in M. albomyces laccase molecules. This binding site most likely exists only in crystals, when the Phe427 residues are packed against each other.     

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.     

Lipopolysaccaride-binding peptides obtained by phage display method

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. It has strong toxicity and might cause sepsis or septic shock. Thus early detection of LPS and neutralization of LPS toxicity are required. We obtained several new LPS-binding peptides using a phage display method. We synthesized 3 of these peptides and analyzed their binding affinity and capacity to LPS. One of these peptides, named Li5-001, showed high binding affinity to LPS and lipid A; the K_d values were 10 and 1 nM, respectively. Li5-001 showed a high binding capacity to LPS, and was estimated to bind 130 ng LPS/mg, which is higher than that of polymyxin B (80 ng LPS/mg); however, its LPS-neutralizing activity was low. Li5-001 coupled with beads will be useful for eliminating endotoxin contamination from pharmaceuticals. Its low LPS-neutralizing activity allows to be used in the Limulus amebocyte lysate test without eluting LPS from the Li5-001 coupled beads.     

Antioxidative effect of melatonin on DNA and erythrocytes against free-radical-induced oxidation

The aim of this work is to investigate the antioxidative effect of melatonin (N-acetyl-5-methoxytryptamine) on the oxidation of DNA and human erythrocytes induced by 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). First, the 50% inhibition concentration (IC50) of melatonin is measured by reacting with two radical species, i.e., 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS*+) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH). The IC50 of melatonin are 75microM and 300microM when melatonin reacts with ABTS*+ and DPPH, respectively. Especially, the reactions of melatonin with ABTS*+ and DPPH are the direct evidence for melatonin to trap radicals. Then, melatonin is applied to protect DNA and human erythrocytes against oxidative damage and hemolysis induced by 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). The presence of melatonin prolongs the occurrence of the oxidative damage of DNA and hemolysis of erythrocytes, generating an inhibition period (t(inh)). The proportional relationship between t(inh) and the concentration of melatonin ([MLT]) is treated by the chemical kinetic equation, t(inh)=(n/R(i))[MLT], in which n means the number of peroxyl radical trapped by an antioxidant, and R(i) stands for the initiation rate of the radical reaction. It is found that every molecule of melatonin can trap almost two radicals in protecting DNA and erythrocytes. Furthermore, quantum calculation proves that the indole-type radical derived from melatonin is much stable than amide-type radical. Finally, melatonin is able to accelerate hemolysis of erythrocytes induced by hemin, indicating that melatonin leads to the collapse of the erythrocyte membrane in the presence of hemin. This may provide detailed information for the usage of melatonin and helpful reference for the design of indole-related drugs.     

Biobleaching of wheat straw-rich soda pulp with alkalophilic laccase from gamma-proteobacterium JB: optimization of process parameters using response surface methodology

An alkalophilic laccase from gamma-proteobacterium JB was applied to wheat straw-rich soda pulp to check its bleaching potential by using response surface methodology based on central composite design. The design was employed by selecting laccase units, ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) concentration and pH as model factors. The results of second order factorial design experiments showed that all three independent variables had significant effect on brightness and kappa number of laccase-treated pulp. Optimum conditions for biobleaching of pulp with laccase preparation (specific activity, 65 nkat mg(-1) protein) were 20 nkat g(-1) of pulp, 2mM ABTS and pH 8.0 which enhanced brightness by 5.89% and reduced kappa number by 21.1% within 4h of incubation at 55 degrees C, without further alkaline extraction of pulp. Tear index (8%) and burst index (18%) also improved for laccase-treated pulp as compared to control raw pulp. Treatment of chemically (CEH1H2) bleached pulp with laccase showed significant effect on release of chromophores, hydrophobic and reducing compounds. Laccase-prebleaching of raw pulp reduced the use of hypochlorite by 10% to achieve brightness of resultant hand sheets similar to the fully chemically bleached pulp.     

A simple method for monitoring chromatography column eluates for laccase activity during enzyme purification

A simple and economical method is described that allows rapid detection of laccase activity in chromatography column fractions during enzyme purification. Aliquots of column eluants are applied to filter paper coated with 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) containing a numbered grid, and incubated at ambient temperature for 20 min. Indications of enzyme activity are simply observed by a colour change. This method avoids having to manually assay each fraction of a chromatographic run for enzyme activity.     

Simultaneous detection of the antioxidant and pro-oxidant activity of dietary polyphenolics in a peroxidase system

The ability to reduce the peroxidase (myeloglobin/H₂O₂)-generated ABTS_•+ [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) radical cation] has been used to rank the antioxidant activity of various agents including dietary flavonoids and chalcones. Surprisingly, we found that in the presence of catalytic concentrations of the phenol B-ring containing flavonoids, apigenin, naringenin and the chalcone phloretin, the formation of the ABTS_•+ was initially increased. The enhanced formation of the ABTS_•+ was attributed to the peroxidase/H₂O₂ mediated generation of polyphenolic phenoxyl radicals that were able to co-oxidize ABTS. The relative ABTS_•+ generating ability of these dietary polyphenolics correlated with their ability to co-oxidize NADH to the NAD* radical with the resultant generation of superoxide. This pro-oxidant activity was not observed for either luteolin or eriodyctiol, which are B-ring catecholic analogues of apigenin and naringenin, respectively, suggesting that these antioxidants are incapable of the transition metal-independent generation of reactive oxygen species. This pro-oxidant activity of the polyphenolics therefore needs to be taken into account when quantifying antioxidant activity.     

Cytoprotective and antioxidant properties of organic selenides for the myelin-forming cells,oligodendrocytes

Here a new series of twenty-one organoselenides, of potential protective activity, were synthesized and tested for their intrinsic cytotoxicity, anti-apoptotic and antioxidant capacities in oligodendrocytes. Most of the organoselenides were able to decrease the ROS levels, revealing antioxidant properties. Compounds 5b and 7b showed a high glutathione peroxidase (GPx)-like activities, which were 1.5 folds more active than ebselen. Remarkably, compound 5a diminished the formation of the oligodendrocytes SubG1 peak in a concentration-dependent manner, indicating its anti-apoptotic properties. Furthermore, based on the SwissADME web interface, we performed an in-silico structure-activity relationship to explore the drug-likeness of these organoselenides, predicting the pharmacokinetic parameters for compounds of interest that could cross the blood-brain barrier. Collectively, we present new organoselenide compounds with cytoprotective and antioxidant properties that can be considered as promising drug candidates for myelin diseases.     

Radical scavenging and anti-inflammatory activities of (hetero)arylethenesulfonyl fluorides: Synthesis and structure-activity relationship (SAR) and QSAR studies

A series of (hetero)arylethenesulfonyl fluorides (1–58) were synthesized and screened for their in vitro antioxidant (DPPH, ABTS and DMPD methods) and anti-inflammatory activities. The results revealed that compounds 4, 15, 16, 24, 25, 26, 38, 39, 40, and 54 exhibited excellent antioxidant activity using all the three performed antioxidant methods, which were superior to the standard antioxidants ascorbic acid and gallic acid. Compounds 6–9, 11, 18, 19, 21, 22, 30, 39, 40, 44, 45, 48–50, 54, 55 and 57 displayed promising anti-inflammatory activity, which were better than the reference drug indomethacin. Preliminary structure–activity relationship (SAR) revealed that compounds containing electron donating (OH and OCH₃) groups on the phenyl ring possessed excellent antioxidant properties while compounds containing electron-withdrawing (Cl, NO₂, F and Br) groups on the phenyl ring were found to be most potent anti-inflammatory agents. The presence of SO₂F group played a crucial role in increases both antioxidant and anti-inflammatory activities.