LysK,the enzyme lysing Staphylococcus aureus cells: Specific kinetic features and approaches towards stabilization

LysK, the enzyme lysing cells of Staphylococcus aureus, can be considered as perspective antimicrobial agent. Knowledge of LysK properties and behavior would allow optimizing conditions of its storage as well as formulating strategy towards its stabilization. Reaction of LysK with substrate (suspension of autoclaved Staphylococcus aureus cells) has been found to be adequately described by the two-stage Michaelis–Menten kinetic scheme. Ionization of the enzyme and enzyme–substrate complex is important for revealing catalytic activity, which is controlled by two ionogenic groups with pK 6.0 and 9.6. Ionization energy of the group with pK 6.0 is of 30 kJ/mol, thus, pointing out on His residue; pK 9.6 might be attributed to metal ion or metal-bound water molecule. At temperatures lower than 40 °C, LysK stability depends on its concentration, pH and presence of low molecular weight additives. Results of electrophoresis under native and denaturing conditions as well as sedimentation analysis strongly suggest that aggregation is behind LysK inactivation. Decrease in the enzyme concentration, as well as addition of low molecular mass polyols (glycerol, sorbitol, sucrose, trehalose) and Ca²⁺ cations resulted in an enhanced (more than 100 times) stability of LysK. Dramatic stability decline observed in a narrow temperature range (40–42 °C) was accompanied by changes in LysK secondary structure as confirmed by CD spectroscopy studies. According to computer modeling data, Cys and His residues and metal cation might play a crucial role for LysK catalytic activity. Our data on the enzyme activity in the presence of ethylenediaminetetraacetic acid and different metal cations confirmed the importance of metal cation in LysK catalysis.     

Facile purification of mono-PEGylated interleukin-1 receptor antagonist and its characterization with multi-angle laser light scattering

Recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was chemically conjugated with succinimidyl carbonate monomethoxyl polyethylene glycols of 5 kDa (SC-PEG_5k) and 10 kDa (SC-PEG_10k) molecular weight. A facile purification of the conjugates was achieved by one-step cationic exchange chromatography. The purity of mono-PEGylated protein was greater than 95%. The purified conjugate was characterized by multi-angle laser light scattering (MALLS) for determining the apparent gyration radius (r_g) and hydrodynamic radius (r_h). MALLS results showed that the conjugation of PEG markedly enhanced r_g and r_h of parent protein (r_g: from 15.7 to 48.2 nm for the PEG_5k and 81.9 nm for the PEG_10k; r_h: from 4.2 to 58.4 nm for the PEG_5k and 102.3 nm for the PEG_10k). The PEGylated rhIL-1ra retained 44.6% of binding activities to the cell receptor for PEG_5k and 40.2% for PEG_10k, compared to the original protein.     

Electrostatically Induced Recruitment of Membrane Peptides into Clusters Requires Ligand Binding at Both Interfaces

Protein recruitment to specific membrane locations may be governed or facilitated by electrostatic attraction, which originates from a multivalent ligand. Here we explored the energetics of a model system in which this simple electrostatic recruitment mechanism failed. That is, basic poly-L-lysine binding to one leaflet of a planar lipid bilayer did not recruit the triply-charged peptide (O-Pyromellitylgramicidin). Clustering was only observed in cases where PLL was bound to both channel ends. Clustering was indicated (i) by the decreased diffusional PLL mobility D_PLL and (ii) by an increased lifetime τ_PLL of the clustered channels. In contrast, if PLL was bound to only one leaflet, neither D_PLL nor τ_P changed. Simple calculations suggest that electrostatic repulsion of the unbound ends prevented neighboring OPg dimers from approaching each other. We believe that a similar mechanism may also operate in cell signaling and that it may e.g. contribute to the controversial results obtained for the ligand driven dimerization of G protein-coupled receptors.     

A novel thermostable α-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b: Purification and characterization

High levels of an extracellular α-galactosidase are produced by the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b when grown in submerse culture and induced by sucrose. The enzyme was purified 114-fold from the culture supernatant by (NH₄)₂SO₄ fractionation, and by chromatographical steps including Sepharose CL-6B gel filtration, DEAE-Sepharose FF anion-exchange, Q-Sepharose FF anion-exchange and Superose 12 gel filtration. The purified enzyme exhibits apparent homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and iso-electric focusing (IEF). The native molecular weight of the monomeric α-galactosidase is 93 kDa with an isoelectric point of 3.9. The enzyme displays a pH and temperature optimum of 5–5.5 and 65 °C, respectively. The purified enzyme retains more than 90% of its activity at 45 °C in a pH range from 5.5 to 9.0. The enzyme proves to be a glycoprotein and its carbohydrate content is 5.3%. Kinetic parameters were determined for the substrates p-nitrophenyl-α-galactopyranoside, raffinose and stachyose and very similar K_m values of 1.13 mM, 1.61 mM and 1.17 mM were found. Mn⁺⁺ ions activates enzyme activity, whereas inhibitory effects can be observed with Ca⁺⁺, Zn⁺⁺ and Hg⁺⁺. Five min incubation at 65° with 10 mM Ag⁺ results in complete inactivation of the purified α-galactosidase. Amino acid sequence alignment of N-terminal sequence data allows the α-galactosidase from Thermomyces lanuginosus to be classified in glycosyl hydrolase family 36.     

Purification and characterization of lectin from fruiting body of Ganoderma lucidum: Lectin from Ganoderma lucidum

A novel 114 kDa hexameric lectin was purified from the fruiting bodies of the mushroom Ganoderma lucidum. Biochemical characterization revealed it to be a glycoprotein having 9.3% neutral sugar and it showed hemagglutinating activity on pronase treated human erythrocytes. The lectin was stable in the pH range of 5–9 and temperature up to 50 °C. The hemagglutinating activity was inhibited by glycoproteins that possessed N-as well as O-linked glycans. Chemical modification of the G. lucidum lectin revealed contribution of tryptophan and lysine to binding activity. The thermodynamics of binding of bi- and triantennary N-glycans to G. lucidum lectin was studied by spectrofluorimetry. The lectin showed very high affinity for asialo N-linked triantenary glycan and a preference for asialo glycans over sialylated glycans. The binding was accompanied with a large negative change in enthalpy as well as entropy, indicating primarily involvement of polar hydrogen, van der Waals and hydrophobic interactions in the binding.     

Thermostable properties of the periplasmic selenate reductase from Thauera selenatis

Selenate reductase (SER) from Thauera selenatis is a member of a distinct class of the TAT-translocated type II molybdoenzymes and is closely related to a group of thermostable nitrate reductases (pNAR) found in hyperthermophilic archaea. In the present study the thermostable and thermo-active properties of SER, isolated with either molybdenum (Mo) or tungsten (W) at the active site, are reported. Results show that the purified Mo–SER complex is stable and active upon heat-shock incubation for 10 min at temperatures up to 60 °C. At temperatures greater than 65 °C all three subunits (SerABC) are readily denatured. The optimum temperature for maximum activity recorded was also determined to be 65 °C. T. selenatis can grow readily on a tungstate rich medium up to concentrations of 1 mM. SER isolated from periplasmic fractions from cells grown on 1 mM tungstate displayed selenate reductase activities with a 20-fold reduction in V_max (0.01 μmol [S]/min/mg) and a 23-fold increase in substrate binding affinity (K_m 0.7 μM). The thermo-stability and pH dependence of W–SER was shown to be similar to that observed for Mo–SER. By contrast, the optimum reaction temperature for W–SER exceeded the maximum temperature tested (>80 °C). The combined data from the kinetic analysis and thermal activity profiles provide evidence that W can substitute for Mo at the active site of SER and retain detectable selenate reductase activity. It is argued that despite the similarity in their catalytic and electron conducting subunits, the presence of a membrane anchor in the archaeal pNAR system appears pivotal to the enhanced hyperthermostability. The fact that Mo–SER is thermostable up to 65 °C however, could be advantageous when designing selenate contamination remediation strategies.     

Effects of temperature, salinity, and irradiance on the growth of the dinoflagellate Akashiwo sanguinea

The effects of temperature, salinity, and irradiance on the growth of the dinoflagellate Akashiwo sanguinea were examined in the laboratory. The irradiance at the light compensation point (I₀) was 14.40 μmol m^− 2 s^− 1 and the irradiance at growth saturation (I_s) was 114 μmol m^− 2 s^− 1. We exposed A. sanguinea to 48 combinations of temperature (5-30 °C) and salinity (5-40) under saturating irradiance; it exhibited its maximum growth rate of 1.13 divisions/day at a combination of 25 °C and salinity of 20. A. sanguinea was able to grow at temperatures from 10 to 30 °C and salinities from 10 to 40. This study revealed that A. sanguinea was a eurythermal and euryhaline organism; in Japan it should have formed blooms in early summer, when salinity was relatively low. In addition, it was noteworthy that A. sanguinea had markedly cold-durability, retaining the motile form of vegetative cells for more than 50 days at 5 °C and at salinities of 25-30.     

Isolation and characterization of a fish F-type lectin from gilt head bream (Sparus aurata) serum

A novel fucose-binding lectin, designated SauFBP32, was purified by affinity chromatography on fucose–agarose, from the serum of the gilt head bream Sparus aurata. Electrophoretic mobility of the subunit revealed apparent molecular weights of 35 and 30 kDa under reducing and non-reducing conditions, respectively. Size exclusion analysis suggests that the native lectin is a monomer under the selected experimental conditions. Agglutinating activity towards rabbit erythrocytes was not significantly modified by addition of calcium or EDTA; activity was optimal at 37 °C, retained partial activity by treatment at 70 °C, and was fully inactivated at 90 °C. On western blot analysis, SauFBP showed intense cross-reactivity with antibodies specific for a sea bass (Dicentrarchus labrax) fucose-binding lectin. In addition, the similarity of the N-terminal sequence and a partial coding domain to teleost F-type lectins suggests that SauFBP32 is a member of this emerging family of lectins.     

A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification,characterization, and expression

The carboxylesterase, a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 85 °C and 8.0, respectively. The enzyme showed remarkable thermostability: 41% of its activity remained after 5 days of incubation at 80 °C. In addition, the purified enzyme exhibited stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity towards various PNP esters and short acyl chain triacylglycerols such as tributyrin (C_4:0). Among the PNP esters tested, the best substrate was PNP-caprylate (C₈) with K_m and k_cat values of 71 μM and 14,700 s⁻¹, respectively. The carboxylesterase gene consisted of 915 bp corresponding to 305 amino acid residues. We demonstrated that active recombinant S. solfataricus carboxylesterase could be expressed in Escherichia coli. The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site.     

Structural basis for thermostability revealed through the identification and characterization of a highly thermostable phosphotriesterase-like lactonase from Geobacillus stearothermophilus

A new enzyme homologous to phosphotriesterase was identified from the bacterium Geobacillus stearothermophilus (GsP). This enzyme belongs to the amidohydrolase family and possesses the ability to hydrolyze both lactone and organophosphate (OP) compounds, making it a phosphotriesterase-like lactonase (PLL). GsP possesses higher OP-degrading activity than recently characterized PLLs, and it is extremely thermostable. GsP is active up to 100 °C with an energy of activation of 8.0 kcal/mol towards ethyl paraoxon, and it can withstand an incubation temperature of 60 °C for two days. In an attempt to understand the thermostability of PLLs, the X-ray structure of GsP was determined and compared to those of existing PLLs. Based upon a comparative analysis, a new thermal advantage score and plot was developed and reveals that a number of different factors contribute to the thermostability of PLLs.     

Multigenerational analysis of temperature and salinity variability affects on metabolic rate,generation time,and acute thermal and salinity tolerance in Daphnia pulex

Climate change, sea level rise, and human freshwater demands are predicted to result in elevated temperature and salinity variability in upper estuarine ecosystems. Increasing levels of environmental stresses are known to induce the cellular stress response (CSR). Energy for the CSR may be provided by an elevated overall metabolic rate. However, if metabolic rate is constant or lower under elevated stress, energy for the CSR is taken from other physiological processes, such as growth or reproduction. This study investigated the examined energetic responses to the combination of temperature and salinity variability during a multigenerational exposure of partheogenetically reproducing Daphnia pulex. We raised D. pulex in an orthogonal combination of daily fluctuations in temperature (15, 15–25, 15–30 °C) and salinity (0, 0–2, 0–5). Initially metabolic rates were lower under all variable temperature and variable salinity treatments. By the 6th generation there was little metabolic variation among low and intermediate temperature and salinity treatments, but metabolic suppression persisted at the most extreme salinity. When grown in the control condition for the 6th generation, metabolic suppression was only observed in D. pulex from the most extreme condition (15–30 °C, 0–5 salinity). Generation time was influenced by acclimation temperature but not salinity and was quickest in specimens reared at 15–25 °C, likely due to Q₁₀ effects at temperatures closer to the optima for D. pulex, and slowest in specimens reared at 15–30 °C, which may have reflected elevated CSR. Acute tolerance to temperature (LT₅₀) and salinity (LC₅₀) were both highest in D. pulex acclimated to 15–30 °C and salinity 0. LT₅₀ and LC₅₀ increased with increasing salinity in specimens raised at 15 °C and 15–25 °C, but decreased with increasing salinity in specimens raised at 15–30 °C. Thus, increasing temperature confers cross-tolerance to salinity stress, but the directionality of synergistic effects of temperature and salinity depend on the degree of environmental variability. Overall, the results of our study suggest that temperature is a stronger determinant of metabolism, growth, and tolerance thresholds, and assessment of the ecological impacts of environmental change requires explicit information regarding the degree of environmental variability.     

Polymeric micelles of PEG-PE as carriers of all-trans retinoic acid for stability improvement

The topical application of all-trans retinoic acid (ATRA) is an effective treatment for several skin disorders, including photo-aging. Unfortunately, ATRA is susceptible to light, heat, and oxidizing agents. Thus, this study aimed to investigate the ability of polymeric micelles prepared from polyethylene glycol conjugated phosphatidylethanolamine (PEG-PE) to stabilize ATRA under various storage conditions. ATRA entrapped in polymeric micelles with various PEG and PE structures was prepared. The critical micelle concentrations were 97–243 μM, depending on the structures of the PEG and PE molecules. All of the micelles had particle diameters of 6–20 nm and neutral charges. The highest entrapment efficiency (82.7%) of the tested micelles was exhibited by ATRA in PEG with a molecular weight of 750 Da conjugated to dipalmitoyl phosphatidylethanolamine (PEG₇₅₀-DPPE) micelles. The PEG₇₅₀-DPPE micelle could significantly retard ATRA oxidation compared to ATRA in 75% methanol/HBS solution. Up to 87% of ATRA remained in the PEG₇₅₀-DPPE micelle solution after storage in ambient air for 28 days. This result suggests that PEG₇₅₀-DPPE micelle can improve ATRA stability. Therefore, ATRA in PEG₇₅₀-DPPE micelle is an interesting alternative structure for the development of cosmeceutical formulations.     

Phage Lysin LysK Can Be Truncated to Its CHAP Domain and Retain Lytic Activity against Live Antibiotic-Resistant Staphylococci

A truncated derivative of the phage endolysin LysK containing only the CHAP (cysteine- and histidine-dependent amidohydrolase/peptidase) domain exhibited lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus. This is the first known report of a truncated phage lysin which retains high lytic activity against live staphylococcal cells.     

Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis

The serine alkaline protease, SAPB, from Bacillus pumilus CBS is characterized by its high thermoactivity, pH stability and high catalytic efficiency (k_cat/K_m) as well as its excellent stability and compatibility with an alkaline environment under harsh washing conditions. Based on sequence alignments and homology-modeling studies, the present study identified five amino acids Leu31, Thr33, Asn99, Phe159 and Gly182 being putatively important for the enzymatic behaviour of SAPB. To corroborate the role of these residues, 12 mutants were constructed by site-directed mutagenesis and then purified and characterized. The findings demonstrate that the single mutants F159T, F159S and G182S and combined double substitutions were implicated in the decrease of the optimum pH and temperature to 8.0–9.0 and 50 °C, respectively, and that mutant F159T/S clearly affected substrate affinity and catalytic efficiency. With regards to the single L31I, T33S and N99Y and combined double and triple mutations, the N99Y mutation strongly improved the half-life times at 50 °C and 60 °C to 660 and 295 min from of 220 and 80 min for the wild-type enzyme, respectively. More interestingly, this mutation also shifted the optimum temperature from 65 °C to 75 °C and caused a prominent 31-fold increase in k_cat/K_m with N-succinyl-l-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF). The L31I and T33S mutants were observed to improve mainly the optimum pH from 11.0 to 11.5 and from 11.0 to 12.0, respectively. Kinetic studies of double and triple mutants showed that the cumulative effect of polar uncharged substitutions had a synergistic effect on the P1 position preference using synthetic peptide substrates, which confirms the implication of these amino acids in substrate recognition and catalytic efficiency.     

Enhancement of lipase stability and productivity through chemical modification and its application to latex-based polymer emulsions

The primary focus of this research was to employ amino-group specific chemical modifications for improving the productivity and stability of two commercially produced lipases, Lipase-A from Candida antarctica (CAL_UM) and Greasex from Humicola lanuginosa (HLL_UM), for application in a latex-based paint formulation. The modified lipases showed higher percentage increase (benzoic anhydride-modified, HLL_BA, 150%; PEG-modified, HLL_PEG,162% at 75 °C) as well as higher absolute productivities 41, 50, 52 and 53 μmole substrate mg⁻¹ lipase for unmodified, CAL_PEG, HLL_PEG and HLL_BA, respectively at 37 °C. The half-lives of thermal inactivation for all modified variants were improved from 40 to 166% at 50, 60 and 70 °C relative to unmodified lipases. The higher thermal stability and catalytic efficiency (k_cat/K_m) with concomitant lower activity (k_cat) indicates that enhanced productivity is likely to be due to the modified enzymes being better able to resist thermal denaturation over the time course of the productivity experiments. Importantly, both lipases, CAL_BA (60%) and HLL_BA (55%) retained the highest activity in paint compared with CAL_UM (36%) and HLL_UM (39%) after 20 weeks incubation at 25 °C. The long term stability of the modified lipases illustrates their potential value for commercial paint and other industrial applications.     

N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT)

The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~ 55 kDa), higher than predicted (~ 50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 °C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~ 35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~ 47 kDa protein; substitution of both sites gave a smaller (~ 35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH₂ or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains.     

Identification of Paenibacillus sp. 2S-6 and application of its xylanase on biobleaching

A Paenibacillus sp. strain 2S-6 was isolated from the black liquor of the first brownstock washing stage of kraft pulping process and identified by its 16S rDNA sequence. This bacterial strain utilized a variety of saccharides and polysaccharides as carbon source, but neither lignin nor lipids. Crude xylanase from Paenibacillus sp. 2S-6 was produced in a 5 L laboratory fermenter at 37 °C, pH 7. After 24 h, up to 10.5 IU xylanase per mg of protein in the crude extract of fermentation broth was obtained. After two-stage ultrafiltration, the optimal activity of partially purified xylanase reached 60.51 IU/mg at 50 °C, pH 6. A major band indicating molecular weight of 33 kDa was shown on SDS-PAGE for the partially purified xylanase. After 4 h at 60 °C, 48.99% and 31.25% residual xylanase activities were demonstrated at pH 7 and 9, respectively. Efficacy of its xylanase on the bleaching agent saving was demonstrated by using 5 IU xylanase per gram oven-dried pulp prior to bleaching, referred as biobleaching. Identical levels of brightness and higher levels of viscosity were obtained for the xylanase pretreated eucalypt kraft pulps followed by a 20% reduction of the bleaching agent dosage in the first step of a commercial C₇₀/D₃₀-E_o-D bleaching sequence.     

Biochemical characterisation of triose phosphate isomerase from the liver fluke Fasciola hepatica

Triose phosphate isomerase (TPI) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, a reaction in the glycolytic pathway. TPI from the common liver fluke, Fasciola hepatica, has been cloned, sequenced and recombinantly expressed in Escherichia coli. The protein has a monomeric molecular mass of approximately 28 kDa. Crosslinking and gel filtration experiments demonstrated that the enzyme exists predominantly as a dimer in solution. F. hepatica TPI is predicted to have a β-barrel structure and key active site residues (Lys-14, His-95 and Glu-165) are conserved. The enzyme shows remarkable stability to both proteolytic degradation and thermal denaturation. The melting temperature, estimated by thermal scanning fluorimetry, was 67 °C and this temperature was increased in the presence of either dihydroxyacetone phosphate or glyceraldehyde 3-phosphate. Kinetic studies showed that F. hepatica TPI demonstrates Michaelis–Menten kinetics in both directions, with K_m values for dihydroxyacetone phosphate and glyceraldehyde 3-phosphate of 2.3 mM and 0.66 mM respectively. Turnover numbers were estimated at 25,000 s⁻¹ for the conversion of dihydroxyacetone phosphate and 1900 s⁻¹ for the conversion of glyceraldehyde 3-phosphate. Phosphoenolpyruvate acts as a weak inhibitor of the enzyme. F. hepatica TPI has many features in common with mammalian TPI enzymes (e.g. β-barrel structure, homodimeric nature, high stability and rapid kinetic turnover). Nevertheless, recent successful identification of specific inhibitors of TPI from other parasites, suggests that small differences in structure and biochemical properties could be exploited in the development of novel, species-specific inhibitors.     

Effects of docosahexaenoic acid on in vitro amyloid beta peptide 25–35 fibrillation

Amyloid β peptide_25–35 (Aβ_25–35) encompasses one of the neurotoxic domains of full length Aβ_1–40/42, the major proteinaceous component of amyloid deposits in Alzheimer's disease (AD). We investigated the effect of docosahexaenoic acid (DHA, 22:6, n-3), an essential brain polyunsaturated fatty acid, on the in vitro fibrillation of Aβ_25–35 and found that it significantly reduced the degree of fibrillation, as shown by a decrease in the intensity of both the thioflavin T and green fluorescence in confocal microscopy. Transmission electron microscopy revealed that DHA-incubated samples were virtually devoid of structured fibrils but had an amorphous-like consistency, whereas the controls contained structured fibers of various widths and lengths. The in vitro fibrillation of Aβ_25–35 appeared to be pH-dependent, with the strongest effect seen at pH 5.0. DHA inhibited fibrillation at all pHs, with the strongest effect at pH 7.4. It also significantly decreased the levels of Aβ_25–35 oligomers. Nonreductive gradient gel electrophoresis revealed that the molecular size of the oligomers of Aβ_25–35 was 10 kDa (equivalent to decamers of Aβ_25–35) and that DHA dose-dependently reduced these decamers. These results suggest that DHA decreases the in vitro fibrillation of Aβ_25–35 by inhibiting the oligomeric amyloid species and, therefore, Aβ_25–35-related neurotoxicity or behavioral impairment could be restrained by DHA.