Enzymatic transformation of 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) by immobilized α-cyclodextrin glucanotransferase from recombinant Escherichia coli

This work aims to produce 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin with immobilized α-cyclodextrin glucanotransferase (α-CGTase) from recombinant Escherichia coli. Molecular sieve (SBA-15) was used as an adsorbent, and sodium alginate was used as a carrier, and glutaraldehyde (GA) was used as a cross-linker. The effects of several key variables on α-CGTase immobilization were examined, and optimal immobilization conditions were determined as the following: glutaraldehyde (GA, cross-linker) 0.01% (v/v), SBA-15 (adsorbent) 2 g/L, CaCl₂ 3 g/L, sodium alginate 20 g/L, adsorption time 3 h, and immobilization time 1 h. In comparison with free α-CGTase, immobilized α-CGTase had a similar optimal pH (5.5) and a higher optimal temperature (45 °C). The continuous production of AA-2G from ascorbic acid and β-cyclodextrin in the presence of immobilized α-CGTase was carried out, and the highest AA-2G production reached 21 g/L, which was 2-fold of that with free α-CGTase. The immobilization procedure developed here was efficient for α-CGTase immobilization, which was proved to be a prospective approach for the enzymatic production of AA-2G.     

Statistical optimization and multiple objective programming of lysozyme refolding catalyzed by recombinant DsbA in vitro

DsbA (disulfide bond formation protein A) is essential for disulfide bond formation directly affecting the nascent peptides folding to the correct conformation in vivo. In this paper, recombinant DsbA protein was employed to catalyze denatured lysozyme refolding and inhibit the aggregation of folding intermediates in vitro. Statistical methods, i.e., Plackett–Burman design and small central composite design, were adopted to screen out important factors affecting the refolding process and correlating these parameters with the refolding efficiency including both protein recovery and specific activity of refolded lysozyme. Four important parameters: initial lysozyme concentration, urea concentration, KCl concentration and GSSG (glutathione disulfide) concentration were picked out and operating conditions were optimized by introducing the effectiveness coefficient method and transforming the multiple objective programming into an ordinary constrained optimization issue. Finally, 99.7% protein recovery and 25,600 U/mg specific activity of lysozyme were achieved when 281.35 μg/mL denatured lysozyme refolding was catalyzed by an equivalent molar of DsbA at the optimal settings. The results indicated that recombinant DsbA protein could effectively catalyze the oxidized formation and reduced isomerization of intramolecular disulfide bonds in the refolding of lysozyme in vitro.     

Sharper angle,higher risk? The effect of cutting angle on knee mechanics in invasion sport athletes

IntroductionCutting is an important skill in team-sports, but unfortunately is also related to non-contact ACL injuries. The purpose was to examine knee kinetics and kinematics at different cutting angles.Material and methods13 males and 16 females performed cuts at different angles (45°, 90°, 135° and 180°) at maximum speed. 3D kinematics and kinetics were collected. To determine differences across cutting angles (45°, 90°, 135° and 180°) and sex (female, male), a 4 × 2 repeated measures ANOVA was conducted followed by post hoc comparisons (Bonferroni) with alpha level set at α ≤ 0.05 a priori.ResultsAt all cutting angles, males showed greater knee flexion angles than females (p < 0.01). Also, where males performed all cutting angles with no differences in the amount of knee flexion −42.53° ± 8.95°, females decreased their knee flexion angle from −40.6° ± 7.2° when cutting at 45° to −36.81° ± 9.10° when cutting at 90°, 135° and 180° (p < 0.01). Knee flexion moment decreased for both sexes when cutting towards sharper angles (p < 0.05). At 90°, 135° and 180°, males showed greater knee valgus moments than females. For both sexes, knee valgus moment increased towards the sharper cutting angles and then stabilized compared to the 45° cutting angle (p < 0.01). Both females and males showed smaller vGRF when cutting to sharper angles (p < 0.01).ConclusionIt can be concluded that different cutting angles demand different knee kinematics and kinetics. Sharper cutting angles place the knee more at risk. However, females and males handle this differently, which has implications for injury prevention.     

Influence of the microenvironment of thiol groups in low molecular mass thiols and serum albumin on the reaction with methylglyoxal

Methylglyoxal (MG), a reactive α-oxoaldehyde that is produced in higher quantities in diabetes, uremia, oxidative stress, aging and inflammation, reacts with the thiol groups (in addition to the amino and guanidino groups) of proteins. This causes protein modification, formation of advanced glycated end products (AGEs) and cross-linking. Low molecular mass thiols can be used as competitive targets for MG, preventing the reactions mentioned above. Therefore, this paper investigated how the microenvironment of the thiol group in low molecular mass thiols (cysteine, N-acetylcysteine (NAcCys), carboxymethylcysteine (CMC) and glutathione (GSH)) and human serum albumin (HSA) affected the thiol reaction with MG. The SH group reaction course was monitored by ¹H-NMR spectroscopy and spectrophotometric quantification. Changes in the HSA molecules were monitored by SDS-PAGE. The microenvironment of the SH group had a major effect on its reactivity and on the product yield. The reactivity of SH groups decreased in the order Cys > GSH > NAcCys. CMC did not react. The percentages of the reacted SH groups in the equilibrium state were almost equal, regardless of the ratio of thiol compound/MG (1:1, 1:2, 1:5): 38.1 ± 0.9%; 38.2 ± 0.7% and 39.0 ± 0.8% for Cys; 26.5 ± 0.6%; 26.6 ± 2.6% and 27.4 ± 2.5% for GSH; 10.8 ± 0.9%; and 11.2 ± 0.7% and 12.2 ± 0.9% for NAcCys, respectively. Our results explain why substances containing α-amino-β-mercapto-ethane as a pharmacophore are successful scavengers of MG. In equilibrium, HSA SH reacted in high percentages both with an insufficient amount and with an excess of MG (55% and 65%, respectively). An analysis of the hydrophobicity of the microenvironment of the SH group on the HSA surface showed that it could contribute to high levels of SH modification, leading to an increase in the scavenging activity of the albumin thiol.     

Equilibria and kinetics of protein transfer to and from affinity-based reverse micelles of Span 85 modified with Cibacron Blue F-3GA

Sorbitan trioleate was modified with Cibacron Blue F-3GA (CB) to create an affinity surfactant and to form affinity-based reverse micelles in n-hexane. The partitioning equilibria and the extraction kinetics of lysozyme and bovine serum albumin (BSA) were then examined. The solubilization capacity of the reverse micellar system for lysozyme increased linearly with increasing the CB concentration from 0.1 to 0.5 mmol L⁻¹. In contrast, the capacity for BSA at 0.5 mmol L⁻¹ of coupled CB was only about one-fifth that for lysozyme. It indicates a strong steric hindrance effect of the micelles for the high molecular mass protein. The overall volumetric mass transfer coefficient of lysozyme in the forward extraction increased from 0.43 × 10⁻³ to 1.25 × 10⁻³ s⁻¹ with increasing CB concentration from 0.1 to 0.5 mmol L⁻¹. Due to the high molecular mass of BSA, its volumetric mass transfer coefficient in the forward extraction was only one-sixth that of lysozyme. The ratio of the coefficient in the back extraction to that in the forward extraction was less than 0.03, much lower than those in other micellar systems. It indicates that the interfacial resistance in this system was severer than in others.     

A centrifugal ultrafiltration strategy for isolating the low-molecular weight (≤ 25 K) component of human plasma proteome

The low-molecular weight fraction (LMF) of the human plasma proteome is an invaluable source of biological information, especially in the context of identifying plasma-based biomarkers of disease. In this study, a separation and enrichment strategy based on centrifugal ultrafiltration was developed for the LMF (i.e., ≤ 25 K) of plasma routinely prepared from normal, healthy volunteers. Four commercially-available filter membranes of similar nominal molecular weight cut-off (NMWC), but differing membrane chemistries and filter orientations (Microcon®, Millipore; Centrisart®, Sartorius; Amicon Ultra®, Millipore; Vivaspin®, Sartorius), were evaluated. Of these filtration devices, only the Sartorius Vivaspin® tangential membrane, NMWC 20 K was effective in the non-retention of M_r > 50 K, and recovery and enrichment of low-M_r components from human plasma. This filter membrane device was further optimized with respect to plasma buffer composition, centrifugal force, duration and temperature. Optimal ultrafiltration conditions were obtained using 100 µL of normal plasma in 10% acetonitrile, and a centrifugation force of 4000 × g for 35 min at 20 °C. In this LMF, 44 proteins (from 266 unique peptides) were identified using a combination of 1D-SDS-PAGE / nano-LC-MS/MS and a stringent level of identification (FDR < 1%). We report the identification of several proteins (e.g., protein KIAA0649 (Q9Y4D3), rheumatoid factor D5, serine protease inhibitor A3, and transmembrane adapter protein PAG) previously not reported in extant high-confidence Human Proteome Organization (HUPO) Plasma Proteome Project datasets. When compared with the low-M_r human plasma/serum proteome datasets of Zhou et al. (Electrophoresis, 2004. 25, 1289–98), Gundry et al. (Proteomics Clin. Appl., 2007. 1, 73–88) and Villanueva et al. (Anal Chem, 2004. 76, 1560–70), 64% of our identifications (28 proteins) were novel; these include cofilin-1, PPIase A, and the SH3 domain-binding glutamic acid-rich-like protein 3. In addition to intact proteins, many peptide fragments from high-abundance proteins (e.g., fibrinogen, clusterin, Factor XIIIa, transferrin, kinogen-1, and inter-alpha-trypsin inhibitor), presumably derived by ex vivo proteolysis, were observed.     

Synthesis of thermo-sensitive polyacrylamide derivatives for affinity precipitation and its application in purification of lysozyme

The thermo-sensitive N-alkyl substituted polyacrylamide polymer was synthesized by radical polymerization and its lower critical solution temperature (LCST) was controlled to be 28 °C. The thermo-sensitive recovery of polymer was over 95% in the presence of 0.05 M NaClO₄. Cibacron Blue F3GA was covalently immobilized onto the polymer via the nucleophilic reaction between the active chlorine atom of its triazine ring and the hydroxyl group of the polymer. The ligands density was 30 μmol g⁻¹ polymer. The adsorption capacity of lysozyme on the polymer was 3.4 mg g⁻¹polymer in affinity precipitation process. And over 90% of adsorbed lysozyme was eluted by 0.5 M KSCN at pH 8.0. When the affinity polymer was applied in the purification of lysozyme from egg white, the purification factor was 28 and lysozyme yield was 80% or so.     

Light-induced disulfide dimerization of recoverin under ex vivo and in vivo conditions

Despite vast knowledge of the molecular mechanisms underlying photochemical damage of photoreceptors, linked to progression of age-related macular degeneration, information on specific protein targets of the light-induced oxidative stress is scarce. Here, we demonstrate that prolonged intense illumination (halogen bulb, 1500 lx, 1–5 h) of mammalian eyes under ex vivo (cow) or in vivo (rabbit) conditions induces disulfide dimerization of recoverin, a Ca²⁺-dependent inhibitor of rhodopsin kinase. Western blotting and mass spectrometry analysis of retinal extracts reveals illumination time-dependent accumulation of disulfide homodimers of recoverin and its higher order disulfide cross-linked species, including a minor fraction of mixed disulfides with intracellular proteins (tubulins, etc.). Meanwhile, monomeric bovine recoverin remains mostly reduced. These effects are accompanied by accumulation of disulfide homodimers of visual arrestin. Histological studies demonstrate that the light-induced oxidation of recoverin and arrestin occurs in intact retina (illumination for 2 h), while illumination for 5 h is associated with damage of the photoreceptor layer. A comparison of ex vivo levels of disulfide homodimers of bovine recoverin with redox dependence of its in vitro thiol–disulfide equilibrium (glutathione redox pair) gives the lowest estimate of redox potential in rod outer segments under illumination from −160 to −155  mV. Chemical crosslinking and dynamic light scattering data demonstrate an increased propensity of disulfide dimer of bovine recoverin to multimerization/aggregation. Overall, the oxidative stress caused by the prolonged intense illumination of retina might affect rhodopsin desensitization via concerted disulfide dimerization of recoverin and arrestin. The developed herein models of eye illumination are useful for studies of the light-induced thiol oxidation of visual proteins.     

Recover of peripheral nerve function after prolong hypothermic cardiac arrest in a porcine model with extra corporeal life support

ObjectivesSurviving long lasting cardiac arrest following accidental hypothermia has been reported after treatment with extra corporeal life support (ECLS), but there is a risk of neurologic injury. Most surviving hypothermia patients have a prolonged stay in the intensive care unit, where most patients experience polyneuropathy. Theoretically, accidental hypothermic cardiac arrest may in itself contribute to polyneuropathy. This study was designed to examine the impact of three hours of cardiac arrest at a core temperature of 20 °C followed by reanimation of peripheral nerve function.MethodsSeven pigs were cannulated for ECLS and cooled to a core temperature of 20 °C followed by three hours of circulatory arrest where the extremities were packed with ice. After three hours, ECLS was started and rewarming was performed. During the process, neural testing of the ulnar nerve (a somatic nerve) and of the vagus nerve (an autonomic nerve) were performed and blood was drawn for analysis of p-potassium, serum-neuron-specific enolase, and S100b protein.ResultsThe ulnar nerve was cooled from 34.9±1.6 °C to 12.8±3.8 °C and the vagus nerve from 36.2±1.2 °C to 15.4±1.4 °C. Physiologic function of both somatic and autonomic nerves were strongly affected by cooling, but recovered to almost normal levels during rewarming, even after three hours of hypothermic cardiac arrest. P-potassium rose from 3.9 (3.6–4.6) mmol/l to 8.1 (7.2–9.1) mmol/l after three hours of cardiac arrest, but normalized after recirculation. There was no rise in serum-neuron-specific enolase, but a slight rise in S100b protein during three hours of hypothermic cardiac arrest was observed. All pigs obtained return of spontaneous circulation (ROSC).ConclusionsReanimation after three hours of hypothermic cardiac arrest using ECLS was possible with no or, if present, minor damage to the two nerves tested.     

Continuous degradation of maltose by enzyme entrapment technology using calcium alginate beads as a matrix

Maltase from Bacillus licheniformis KIBGE-IB4 was immobilized within calcium alginate beads using entrapment technique. Immobilized maltase showed maximum immobilization yield with 4% sodium alginate and 0.2 M calcium chloride within 90.0 min of curing time. Entrapment increases the enzyme–substrate reaction time and temperature from 5.0 to 10.0 min and 45 °C to 50 °C, respectively as compared to its free counterpart. However, pH optima remained same for maltose hydrolysis. Diffusional limitation of substrate (maltose) caused a declined in V_max of immobilized enzyme from 8411.0 to 4919.0 U ml^?1 min^?1 whereas, K_m apparently increased from 1.71 to 3.17 mM ml^?1. Immobilization also increased the stability of free maltase against a broad temperature range and enzyme retained 45% and 32% activity at 55 °C and 60 °C, respectively after 90.0 min. Immobilized enzyme also exhibited recycling efficiency more than six cycles and retained 17% of its initial activity even after 6th cycles. Immobilized enzyme showed relatively better storage stability at 4 °C and 30 °C after 60.0 days as compared to free enzyme.     

Supermacroporous hydrophobic affinity cryogels for protein chromatography

N-Methacryloyl-l-tryptophan (MATrp) containing poly(2-hydroxyethyl methacrylate) based supermacroporous cryogel [PHEMATrp] was prepared for lysozyme purification form chicken egg white. MATrp was synthesized by reacting methacryloyl chloride with l-tryptophan methyl ester and provided hydrophobic functionality to the cryogel. PHEMATrp cryogel with 60–100 μm pore size was obtained by free radical polymerization of HEMA and MATrp having a specific surface area of 50 m²/g. PHEMATrp cryogel was characterized by swelling studies, FTIR and SEM. The equilibrium swelling ratios of the cryogels were 7.18 g H₂O/g for PHEMA and 6.99 g H₂O/g for PHEMATrp. Lysozyme adsorption experiments were investigated under different conditions in continuous system (i.e., medium pH, flow-rate, protein concentration, temperature, salt type). Lysozyme adsorption capacity of PHEMA and PHEMATrp cryogels from aqueous solutions was estimated as 2.9 and 46.8 mg/g (0.49 and 7.85 mg/mL), respectively. Lysozyme molecules were desorbed with 0.5 M ethylene glycol solution with 91% recovery. It was observed that PHEMATrp cryogel can be used without significant decrease in lysozyme adsorption capacity after five adsorption–desorption cycles. PHEMATrp cryogel was used for the purification of lysozyme from chicken egg white. Purity of lysozyme was estimated by SDS-PAGE. Possible denaturation of purified lysozyme was checked with fluorimetric measurements. Specific activity of the purified lysozyme was found as 43,140 U/mg using Micrococcus lysodeikticus as substrate.     

Redox properties of a thioredoxin-like Arabidopsis protein,AtTDX

AtTDX is an enzyme present in Arabidopsis thaliana which is composed of two domains, a thioredoxin (Trx)-motif containing domain and a tetratricopeptide (TPR)-repeat domain. This enzyme has been shown to function as both a thioredoxin and a chaperone. The midpoint potential (E_m) of AtTDX was determined by redox titrations using the thiol-specific modifiers, monobromobimane (mBBr) and mal-PEG. A NADPH/Trx reductase (NTR) system was used both to validate these E_m determination methods and to demonstrate that AtTDX is an electron-accepting substrate for NTR. Titrations of full-length AtTDX revealed the presence of a single two-electron couple with an E_m value of approximately ?260 mV at pH 7.0. The two cysteines present in a typical, conserved Trx active site (WCGPC), which are likely to play a role in the electron transfer processes catalyzed by AtTDX, have been replaced by serines by site-directed mutagenesis. These replacements (i.e., C304S, C307S, and C304S/C307S) resulted in a complete loss of the redox process detected using either the mBBr or mal-PEG method to monitor disulfide/dithiol redox couples. This result supports the conclusion that the couple with an E_m value of ?260 mV is a disulfide/dithiol couple involving Cys304 and Cys307. Redox titrations for the separately-expressed Trx-motif containing C-domain also revealed the presence of a single two-electron couple with an E_m value of approximately ?260 mV at 20 °C. The fact that these two E_m values are identical, provides additional support for assignment of the redox couple to a disulfide/dithiol involving C304 and C307. It was found that, while the disulfide/dithiol redox chemistry of AtTDX was not affected by increasing the temperature to 40 °C, no redox transitions were observed at 50 °C and higher temperatures. In contrast, Escherichia coli thioredoxin was shown to remain redox-active at temperatures as high as 60 °C. The temperature-dependence of the AtTDX redox titration is similar to that observed for the redox activity of the protein in enzymatic assays.     

Tennis players show a lower coactivation of the elbow antagonist muscles during isokinetic exercises

PurposePrevious studies have suggested that muscle coactivation could be reduced by a recurrent activity (training, daily activities). If this was correct, skilled athletes should show a specific muscle activation pattern with a low level of coactivation of muscles which are typically involved in their discipline. In particular, the aim of this study was to verify the hypothesis that the amount of antagonist activation of biceps brachii (BB) and triceps brachii (TB) is different between tennis players and non-players individuals during maximal isokinetic contractions.MethodsTen young healthy men and eight male tennis players participated in the study. The surface electromyographic signals (sEMG) were recorded from the BB and TB muscles during three maximal voluntary isometric contractions (MVC) of elbow flexors and extensors and a set of three maximal elbow flexions and extensions at 15°, 30°, 60°, 120°, 180° and 240°/s. Normalized root mean square (RMS) of sEMG was calculated as an index of sEMG amplitude.ResultsAntagonist activation (%RMSmax) of TB was significantly lower in tennis players (from 14.0 ± 7.9% at MVC to 16.3 ± 8.9% at 240°/s) with respect to non-players (from 27.7 ± 19.7% at MVC to 38.7 ± 17.6% at 240°/s) at all angular velocities. Contrary to non-players, tennis players did not show any difference in antagonist activation between BB and TB muscles.ConclusionsTennis players, with a constant practice in controlling forces around the elbow joint, learn how to reduce coactivation of muscles involved in the control of this joint. This has been shown by the lower antagonist muscular activity of triceps brachii muscle during isokinetic elbow flexion found in tennis players with respect to non-players.     

Characterization of variant diphtheria toxin–interleukin-3 fusion protein,DTIL3K116W,for phase I clinical trials

We have produced clinical grade of DTIL3K116W, a variant diphtheria toxin–interleukin-3 fusion protein, for treatment of acute myeloid leukemia. The product was filter sterilized, aseptically vialed, and stored at ?80 °C. It was characterized by Coomassie-stained SDS-PAGE, endotoxin assay, cytotoxicity assay, sterility, mass spectroscopy, receptor binding affinity, ADP-ribosylation, inhibition of normal human CFU-GM, disulfide bond analysis, immunoblots, stability, size exclusion chromatography–HPLC, sequencing, and immunohistochemistry. Vialed product was sterile in 0.25 M NaCl/5 mM Tris, pH 7.9, and had a protein concentration of 1.08 mg/ml. Purity by SDS-PAGE was >99%. Aggregates by HPLC were <1%. Endotoxin levels were 0.296 EU/mg. Peptide mapping and mass spectroscopy confirmed its composition and molecular weight. The vialed drug kept reactivity with anti-IL3 and DT antibodies. Potency study revealed a 48-h EC₅₀ of 0.5 pM on TF1/H-ras cell. Its binding properties were confirmed by competitive experiments showing IC₅₀ of 1.4 nM. ADP-ribosylation activity was equivalent to DTGM-CSF. Drug did not react with tested frozen human tissue sections by immunohistochemistry. There was no evidence of loss of solubility, proteolysis aggregation, or loss of potency over 6 months at ?80 °C. Further, the drug was stable at 4 and 25 °C in the plastic syringe and administration tubing for 48 h.     

Immobilization of alkaline serine endopeptidase from Bacillus licheniformis on SBA-15 and MCF by surface covalent binding

An industrial enzyme, alkaline serine endopeptidase, was immobilized on surface modified SBA-15 and MCF materials by amide bond formation using carbodiimide as a coupling agent. The specific activities of free enzyme and enzyme immobilized on SBA-15 and MCF were studied using casein (soluble milk protein) as a substrate. The highest activity of free enzyme was obtained at pH 9.5 while this value shifted to pH 10 for SBA-15 and MCF immobilized enzyme. The highest activity of immobilized enzymes was obtained at higher temperature (60 °C) than that of the free enzyme (55 °C). Kinetic parameters, Michaelis–Menten constant (K_m) and maximum reaction velocity (V_max), were calculated as K_m = 13.375, 11.956, and 8.698 × 10^?4 mg/ml and V_max = 0.156, 0.163 and 0.17 × 10^?3 U/mg for the free enzyme and enzyme immobilized on SBA-15 and MCF, respectively. The reusability of immobilized enzyme showed 80% of the activity retained even after 15 cycles. Large pore sized MCF immobilized enzyme was found to be more promising than the SBA-15 immobilized enzyme due to the availability of larger pores of MCF, which offer facile diffusion of substrate and product molecules.     

Ethyl isovalerate synthesis using Candida rugosa lipase immobilized on silica nanoparticles prepared in nonionic reverse micelles

Uniform and monodispersed silica nanoparticles were synthesized with a mean diameter of 100 ± 20 nm as analyzed by Transmission Electron Microscopy (TEM). Glutaraldehyde was used as a coupling agent for efficient binding of the lipase onto the silica nanoparticles. For the hydrolysis of pNPP at pH 7.2, the activation energy within 25–40 °C for free and immobilized lipase was 7.8 and 1.25 KJ/mol, respectively. The V_max and K_m of immobilized lipase at 25 °C for pNPP hydrolysis were found to be 212 μmol/min/mg and 0.3 mM, whereas those for free lipase were 26.17 μmol/min and 1.427 mM, respectively. The lower activation energy of immobilized lipase in comparison to free lipase suggests a change in conformation of the enzyme leading to a requirement for lower energy on the surface of the nanoparticles. A better yield (7 fold higher) of ethyl isovalerate was observed using lipase immobilized onto silica nanoparticles in comparison to free lipase.     

Improved esterification activity of Candida rugosa lipase in organic solvent by immobilization as Cross-linked enzyme aggregates (CLEAs)

Cross-linked enzyme aggregates (CLEA^®s) were prepared from Candida rugosa lipase (CrL) using glutaraldehyde as the cross-linker. The optimum conditions of the immobilization process were determined (precipitant: ethanol, crosslinker concentration: 25 mM, enzyme concentration: 50 mg/ml, crosslinking time: 45 min.). CLEAs were shown to have several advantages compared to the free enzyme. They were more stable at 50 °C and 60 °C and had good reusability; retaining 40% of their initial activity after 15 recycles in aqueous media and remaining constant at that level thereafter, suggesting some initial leaching in water. The CLEAs catalyzed esterification reactions in cyclohexane, affording higher conversions than with the free enzyme, especially when longer fatty acids and alcohols were used as substrates.     

Peroxidación lipídica en la membrana de los linfocitos y oxidación proteica en el suero de personas ancianas, ¿marcadores potenciales de fragilidad y dependencia? Resultados preliminares

ObjectiveTo study the relationships between lipid peroxidation of the lymphocyte membrane, protein oxidation and different markers of frailty and dependence.MethodsThe sample consisted of 15 elderly patients in an intermediate and long-term care center, who had not suffered any acute process recently. The geriatric assessment included, functional capacity (Barthel and Lawton indexes), comorbidity (Charlson index), and cognitive function (Mini Mental State Examination of Folstein). The frailty was estimated by the Hospital Admission Risk Profile (high risk of frailty 4-5 points, intermediate/low 0-3 points) and Frailty Scale of Rockwood (mild frailty < 6, intermediate frailty/severe ≥ 6). Lipid peroxidation was studied by determination of conjugated dienes and trienes. Analysis of protein oxidation was performed by determining malondialdehyde bound to plasma proteins, corrected by total protein quantification.ResultsElderly patients at high risk of frailty according to Hospital Admission Risk Profile presented mean values of conjugated dienes of 7.94 ± 1.32%, trienes of 1.75 ± 0.51%, and malondialdehyde bound to plasma proteins of 141.9 ± 27.3 nmol/g. In the group of intermediate/low risk, these values were 4.96 ± 2.77% (P = .035), 1.37 ± 0.78% (P = .337) and 96.4 ± 31.5 nmol/g (P = .022), respectively. In those with intermediate/severe frailty according to the Frailty Scale of Rockwood, these values were 7.06 ± 2.18%; 1.73 ± 0.50% and 119.6 ± 37.9 nmol/g, respectively, and in those with mild frailty 2.56 ± 1.48% (P = 014); 0.61 ± 0.58% (P = 020) and 173.2 ± 51.9 nmol/g (P = .144), respectively. There was good correlation between the Hospital Admission Risk Profile score and malondialdehyde bound to plasma proteins (r = 0.70; P = 01) and between the Frailty Scale of Rockwood score and conjugated dienes (r = 0.65; P = 01).ConclusionsElderly patients with a higher degree of frailty appear to have greater levels of lipid peroxidation, which could be considered a marker of frailty.     

Combined effects of temperature and salinity on functional responses of haemocytes and survival in air of the clam Ruditapes philippinarum

The combined effects of temperature and salinity on both immune responses and survival in air of the clam, Ruditapes philippinarum, were evaluated for the first time. The animals were kept for 7 days at three differing temperature (5 °C, 15 °C, 30 °C) and salinity values (18 psu, 28 psu, 38 psu), and effects of the resulting 9 experimental conditions on total haemocyte count (THC), Neutral Red uptake (NRU), haemolymph protein concentration, and lysozyme activity in both haemocyte lysate (HL) and cell-free haemolymph (CFH) were evaluated. The survival-in-air test was also performed. Two-way ANOVA analysis revealed that temperature influenced significantly THC and NRU, whereas salinity and temperature/salinity interaction affected NRU only. Temperature and salinity did not influence significantly HL and CFH lysozyme activity, as well as haemolymph total protein content. Survival-in-air test is widely used to evaluate general stress conditions in clams. In the present study, temperature and salinity were shown to influence the resistance to air exposure of R. philippinarum. The highest LT₅₀ (air exposure time resulting in 50% mortality) value was recorded in clams kept at 18 psu and 15 °C, whereas the lowest value was observed in clams kept at 28 psu and 30 °C. Overall, results obtained demonstrated that temperature and salinity can affect some functional responses of haemocytes from R. philippinarum, and suggested a better physiological condition for animals kept at 15 °C temperature and 18 psu salinity.