Kinetic and thermodynamic characterization of the cold activity acquired upon single amino-acid substitution near the active site of a thermostable α-glucosidase

The α-glucosidase of Bacillus sp. SAM1606, a thermophilic bacterium, is a thermostable enzyme that has maximal activity at an apparent optimal temperature between 65 and 70 °C and only very low activity at low temperatures (0–25 °C). In this study, we identified Thr272, which is located adjacent to Glu271 (a catalytic residue) and Gly273 (a determinant of specificity), as a determinant of the optimal temperature, as substitution of Thr272 with other residues significantly altered the temperature–activity profile of the enzyme. Substitution of Thr272 with other amino acids, in particular bulky hydrophobic residues such as valine, methionine and phenylalanine, resulted in a significant downward shift (by 30 °C) of the apparent optimal temperature with an increase in catalytic activity at low temperatures. The observed downward shift of the apparent optimal temperature was not due to instability of the mutants at 40–65 °C, as the mutants were stable at temperatures up to 65 °C. Among the mutants examined, T272V displayed the highest k_cat values at 10–25 °C, which was at least 11-fold greater than the k_cat value observed for the wild-type enzyme. The thermodynamic characteristics of reactions catalyzed by T272V, T272M, T272F, and wild type at 25 °C were examined in greater detail. The T272V, T272M and T272F mutants displayed large K_s (or K_m) values and reduced and values at 25 °C, consistent with the general features of cold adaptation. The observed cold activities of T272V, T272M and T272F most likely arose from local flexibility of the active site at low temperatures due to loss of a Thr272-mediated hydrogen bond. However, this hydrogen-bond loss likely permits reversible conformational changes of the active site to less active forms at elevated temperatures (e.g., 60 °C). This may explain why catalytic activities for T272V, T272M and T272F at high temperatures (e.g., 60 °C) were lower than those at low temperature (e.g., 25 °C), even though the mutant enzymes appeared stable at 60 °C.     

Clicker increases resistance to extinction but does not decrease training time of a simple operant task in domestic dogs (Canis familiaris)

Despite its popularity among pet owners and professional trainers, we are not aware of any studies that have investigated the efficacy of clicker training in canines. To this end, we taught 35 basenjis to nose-touch an orange traffic cone. Upon meeting pre-determined criteria, dogs progressed through: (1) training trials, wherein correct responses were followed immediately with a click plus food (clicker group) or food alone (control group); (2) strengthening trials, wherein dogs received the same reinforcement protocol as in training trials, except nose-touching behaviour was variably reinforced; and (3) extinction trials, wherein food was withheld from both groups, but dogs in the clicker group received a click alone for nose-touches. We found that the clicker and control groups did not differ with regard to the number of trials or the time required to meet training or strengthening criteria (P > 0.05 for all). However, the clicker group required significantly more trials (log₁₀ transformed means ± S.E. = 1.6 ± 0.03 trials versus 1.4 ± 0.03 trials, P < 0.001) and more time (log₁₀ transformed means ± S.E. = 2.85 ± 0.03 s versus 2.73 ± 0.03 s, P = 0.008) to reach extinction criterion. Additionally, younger dogs required fewer training (, P = 0.001) and strengthening (, P = 0.029) trials and less training (, P = 0.005) and strengthening (, P = 0.013) time to meet criteria than did older dogs. However, no age effect was found on extinction for either the number or duration of trials (P > 0.05 for both), implying that persistence in previously reinforced behaviour did not influence the age sensitivity found in task acquisition. Overall, these results suggest that, whereas the clicker may prolong behaviour without primary reinforcement, it does not reduce the training time of a simple operant task in dogs when primary reinforcement is briefly delayed. We speculate that the clicker may be most useful in maintaining established behaviours when primary reinforcement is unavailable or when its delivery is impractical. Additionally, we found that basenji dogs may become progressively impaired with age in the acquisition of stimulus-reward contingencies.     

Measurement of the apparent diffusivity of ethylene glycol in mouse ovaries through rapid MRI and theoretical investigation of cryoprotectant perfusion procedures

Successful organ cryopreservation will significantly benefit human health and biomedical research. One of the major challenges to this accomplishment is the need for optimization of cryoprotectant agent (CPA) perfusion procedures that involve highly complicated mass transfer processes in organs. The diffusivity of CPA is of critical importance for designing perfusion procedures to minimize the associated toxicity and osmotic damage. However, to date there have been no attempts to measure the CPA diffusivity in organs. In this study, we established a simple CPA diffusion model for relatively small organs, e.g., mouse ovaries, defined the apparent diffusivity () of CPA for these organs, and established a practical approach to measure the value of through magnetic resonant imaging (MRI). Using rapid MRI techniques and water saturation analyses, the distribution of ethylene glycol (EG) concentration in the centric cross-section of mouse ovaries was measured at a series of time points during perfusion, and these data were fit to the integral form of the mass transfer equation in the established model. These fits resulted in a value of for EG in mouse ovaries of 6.1 ± 1.4 × 10⁻⁷ cm²/s (mean ± SD). Based on these results, we proposed a modified perfusion procedure that may improve the survival of small organs or thin tissues during equilibrium cooling processes and assessed its efficiency through theoretical analyses.     

Photonic crystal nanostructures for optical biosensing applications

We present the design, fabrication and optical investigation of photonic crystal (PhC) nanocavity drop filters for use as optical biosensors. The resonant cavity mode wavelength and Q-factor are studied as a function of the ambient refractive index and as a function of adsorbed proteins (bovine serum albumin) on the sensor surface. Experiments were performed by evanescent excitation of the cavity mode via a PhC waveguide. This in turn is coupled to a ridge waveguide that allows the introduction of a fluid flow cell on a chip. A response of ∂λ/∂c=(4.54±0.66)×10⁵ nm/M is measured leading to a measured detection limit as good as  fg or  pg/mm²in the sensitive area.     

Sawdust pellets from coniferous species as adsorbents for NO2 removal

Carbonaceous adsorbents based on sawdust pellets from coniferous tree species were obtained by carbonisation at different temperatures and different periods of time. The effect of NO₂ adsorption in dry and wet condition on the sorption ability of the chars obtained was tested. The results have shown that NO₂ sorption properties of chars depend on the conditions of pyrolysis and the conditions of adsorption. The best NO₂ sorption capacity of 18.3 and 43.1 mg/g in dry and wet conditions, respectively, was noted for the char sample pyrolysed at 800 °C for 60 min. The FTIR spectra of the exhausted samples reveal a great increase in the intensity of the band at 1380 cm⁻¹ assigned to the vibrations of –NO₂, –ONO₂ or , while in the DTG curves a new peak appears in the range 200–400 °C assigned to the release of nitrogen compounds of low stability in high temperatures.     

Kinetic studies on the Rhizomucor miehei lipase catalyzed esterification reaction of oleic acid with 1-butanol in a biphasic system

The kinetics of the esterification of oleic acid with 1-butanol catalyzed by free Rhizomucor miehei lipase in a biphasic system was studied in a batch reactor. The reaction appeared to proceed via a Ping Pong bi–bi mechanism with 1-butanol inhibition. The kinetic constants of the model were determined from experiments at 30 °C with initial concentrations of oleic acid and 1-butanol in the organic phase and 0.05–0.2 g L⁻¹ enzyme in the aqueous phase. The model was used to simulate the batch concentration profiles of the product as well as the initial reaction rates. Agreement of the model with both the batch concentration profiles (average error of 7.2%) and the initial reaction rate per experiment (average error of 16.0%) was good.     

Kinetics and mechanism of the oxidation of oxalic acid by tetrachloroaurate(III) ion

The oxidation of oxalic acid by tetrachloroaurate(III) ion in 0.005 ? [HClO₄] ? 0.5 mol dm⁻³ is first order in and a fractional order in [oxalic acid], the reactive entities being AuCl₃(OH)⁻ and ions. The pseudo first-order rate, k_obs, with respect to [Au(III)], is retarded by increasing [H⁺] and [Cl⁻]. The retardation by H⁺ ion is caused by the dissociation equilibrium . A mechanism in which a substitution complex, is formed from AuCl₃(OH)⁻ and ions prior to its rate limiting disproportionation into products is suggested. The rate limiting constant, k, has been evaluated and its activation parameters are reported. The equilibrium constant K₁ for the formation of the substitution complex and its thermodynamic parameters are also reported.     

Chain conformation of carboxymethylated derivatives of (1 → 3)-β-d-glucan from Poria cocos sclerotium

A water-insoluble (1 → 3)-β-d-glucan (PCSG) isolated from the fresh sclerotium of Poria cocos was carboxymethylated to afford a water-soluble derivative coded as C-PCSG. The carboxymethylated (1 → 3)-β-d-glucan was fractionated to obtain eight fractions according to the nonsolvent addition method. The weight-average molecular mass (M_w), radius of gyration and intrinsic viscosity ([η]) of the fractions were determined by size-exclusion chromatography combined with laser light scattering (SEC-LLS) and viscometry in 0.2 M NaCl aqueous solution at 25 °C. The dependences of [η] and on M_w for C-PCSG were found to be , and (nm), respectively. Analysis of M_w and [η] in terms of the known theories for wormlike chain model yielded 633 nm⁻¹ for molar mass per unit contour length (M_L), 5.5 nm for persistence length (q), and 20.2 for characteristic ratio (C_∞). These results indicated that C-PCSG exists as a relatively extended flexible chain in 0.2 M NaCl aqueous solution. Therefore, the introduction of the carboxymethyl groups into the β-glucan improved significantly the water solubility and enhanced the stiffness of the chains.     

Using nonfluorescent Förster resonance energy transfer acceptors in protein binding studies

The purpose of this article is to highlight the versatility of nonfluorescent Förster resonance energy transfer (FRET) acceptors in determination of protein equilibrium dissociation constants and kinetic rates. Using a nonfluorescent acceptor eliminates the necessity to spectrally isolate the donor fluorescence when performing binding titrations covering a broad range of reagent concentrations. Moreover, random distribution of the donor and acceptor chromophores on the surface of proteins increases the probability of FRET occurring on their interaction. Three high-affinity antibodies are presented in this study as characteristic protein systems. Monoclonal antibody (mAb) 106.3 binds brain natriuretic peptide (BNP)5–13(C10A) and full-length BNP1–32 with the dissociation constants 0.26 ± 0.01 and 0.05 ± 0.02 nM, respectively, which was confirmed by kinetic measurements. For anti-hCG (human chorionic gonadotropin) mAb 8F11, studied at two incorporation ratios (IRs = 1.9 and 3.8) of the nonfluorescent FRET acceptor, K_D values of 0.04 ± 0.02 and , respectively, were obtained. Likewise, the binding of goat anti-hamster immunoglobulin G (IgG) antibody was not affected by conjugation and yielded K_D values of 1.26 ± 0.04, 1.25 ± 0.05, and 1.14 ± 0.04 nM at IRs of 1.7, 4.7, and 8.1, respectively. We conclude that this FRET-based method offers high sensitivity, practical simplicity, and versatility in protein binding studies.     

Physicochemical properties and antitumor activities for sulfated derivatives of lentinan

Five fractions of lentinan, a β-(1→3)-d-glucan bearing β-(1→6)-d-glucopyranosyl branches, were treated with chlorosulfonic acid for 90 min at 60 °C in pyridine medium to synthesize water-soluble sulfated derivatives having the substitution degree of 1.44–1.76. The ¹³C NMR spectra of the sulfated β-glucans indicated that the C-6 position was preferentially substituted by the sulfate groups. The values of the weight-average molecular weight (M_w), radius of gyration (), and intrinsic viscosity ([η]) of the sulfated lentinan fractions were determined by size-exclusion chromatography with multi-angle laser light scattering (SEC–MALLS) and viscometry in 0.15 M aq NaCl at 25 °C, respectively. The dependence of [η] on M_w for the sulfated lentinan was found to be [η] = 8.93 × 10⁻³ (mL/g) in 0.15 M aq NaCl (for M_w ranging from 14.6 × 10⁴ to 50.4 × 10⁴). On the basis of the Yamakawa–Fujii–Yoshizaki (YFY) theory, the conformational parameters of the sulfated lentinan were calculated as 950 nm⁻¹ for the molar mass per unit contour length (M_L), 4.8 nm for the persistence length (q), and 13.9 for the characteristic ratio (C_∞), indicating relatively extended single flexible chains in solution. The sulfated glucan fractions exhibited in vitro antiproliferative activities against sarcoma 180 (S-180) cells, and their inhibition ratios were lower than that of the triple-helix lentinan, but higher than that for the one with single random-coil lentinan chains.     

Further mechanistic orientation for the oxidation reaction between alkaline permanganate and poly galacturonate methyl ester. Novel spectrophotometric tracer of intrahypomanganate(V) – Intermediate

The kinetics of formation of the green manganate(VI) intermediate complex formed during the oxidation of pectin polysaccharide (poly galacturonate methyl ester) (PGME) by in alkaline solution at pH  12 have been studied. The rate law was suggested to be: rate = . The reaction was found to be base – catalyzed and fractional second – order in [PGME]. The activation parameters have been evaluated and discussed. A reaction mechanism was suggested according to the experimental data. A new blue hypomanganate(V) coordination polymer intermediate sol was spectrophotometrically detected for the first time for pectin (poly galacturonate methyl ester) oxidation reaction. A further mechanistic presentation was proposed.     

Alkyl dehydrogenation in iridium tri-cyclopentyl phosphines

The iridium cyclooctadiene complex incorporating a tricyclopentyl phosphine ligand (PCyp₃), Ir(η²:η²-C₈H₁₂)(PCyp₃)Cl, has been prepared. Removal of the chloride from this complex using in CH₂Cl₂/arene solvent results in dehydrogenation (C-H activation followed by β-H transfer) of one of the alkyl phosphine rings and formation of the complexes (X = H, F) which contain a hybrid phosphine-alkene ligand. These complexes are formed alongside another product (5-20% yield) that has been identified as , which can be prepared in high yield by an alternative, and slightly modified, route. This complex is with a minor isomer that has been tentatively identified as , which results from allylic C-H activation of cyclooctadiene. Addition of H₂ to and its isomer in arene solvent (C₆H₅X, X = F, H) forms the dihydrido η⁶-arene Ir(III) complexes . In contrast, hydrogenation in CH₂Cl₂ alone results in the formation of in which the anion is now acting as a ligand through one of its aryl rings. The fluorobenzene complex can be cleanly converted to by addition of the hydrogen acceptor tert-butylethene (tbe).     

Photocatalysis of chloroform degradation by μ-dichlorotetrachlorodipalladate(II)

Unlike other chlorometallate complexes that catalyze the photodecomposition of haloalkanes through photodissociation of a chlorine atom, both and catalyze chloroform decomposition through a process that appears to involve C-H bond breakage from an excited state association complex with chloroform. This would account for the greatly retarded rate of decomposition in CDCl₃ and for the generation of CCl₄ as a side product. In chloroform, and are in slow equilibrium with each other. The rate for the conversion of - in chloroform at 23 °C obeys the expression (0.03 M⁻¹ s⁻¹) [][Cl⁻]. The equilibrium constant, K = [][Cl⁻]²/[]², was estimated to be 3 × 10⁻³ M in CHCl₃.     

In situ spectroelectrochemical investigation of Pt(II/IV) oxidation in aqueous solution using X-ray absorption spectroscopy

The oxidation from to in HCl aq. was studied in situ by combining electrochemistry with XAFS spectroscopy. During the oxidation of , isosbestic points were observed in Pt L_III and L_II XANES spectra as a function of time, indicating that the Pt(II/IV) redox equilibrium is the only reaction in the system. The Pt L_III and L_II X-ray absorption edge energies of the initial Pt^IICl₄²⁻ are 11562.9 and 13271.8 eV, respectively, while those of the electrolyzed species are 11564.6 and 13273.7 eV which are identical with those of a reference sample. The coordination of the electrolyzed species was characterized by structural parameters derived from the EXAFS curve fit, and identified to .     

Ruthenium(II) complexes possessing the η-p-cymene ligand

Complexes possessing a soft donor η⁶-arene and hard donor acetylacetonate ligand, [(η⁶-p-cymene)Ru(κ²-O,O-acac-μ-CH)]₂[OTf]₂ (1) (OTf = trifluoromethanesulfonate; acac = acetylacetonate) and {Ar′ = 3,5-(CF₃)-C₆H₃}, were prepared and fully characterized. The lability of the μ-CH linkage for complex 1 and the THF ligand of 2 allow access to the unsaturated cation [(η⁶-p-cymene)Ru(κ²-O,O-acac)]⁺. The reaction of with KTp {Tp = hydridotris(pyrazolyl)borate} produces . The azide complex forms upon reaction of with N₃Ar (Ar = p-tolyl), and reaction of with CHCl₃ at 100 °C yields the chloride-bridged binuclear complex . The details of solid-state structures of [(η⁶-p-cymene)Ru(κ²-O,O-acac-μ-CH)]₂[OTf]₂ (1), and are disclosed.