Identification and Characterization of a Thermostable GH36 α-Galactosidase from Anoxybacillus vitaminiphilus WMF1 and Its Application in Synthesizing Isofloridoside by Reverse Hydrolysis

An α-galactosidase-producing strain named Anoxybacillus vitaminiphilus WMF1, which catalyzed the reverse hydrolysis of d-galactose and glycerol to produce isofloridoside, was isolated from soil. The α-galactosidase (galV) gene was cloned and expressed in Escherichia coli. The galV was classified into the GH36 family with a molecular mass of 80 kDa. The optimum pH and temperature of galV was pH 7.5 and 60 °C, respectively, and it was highly stable at alkaline pH (6.0–9.0) and temperature below 65 °C. The specificity for p-nitrophenyl α-d-galactopyranoside was 70 U/mg, much higher than that for raffinose and stachyose. Among the metals and reagents tested, galV showed tolerance in the presence of various organic solvents. The kinetic parameters of the enzyme towards p-nitrophenyl α-d-galactopyranoside were obtained as K_m (0.12 mM), V_max (1.10 × 10⁻³ mM s⁻¹), and K_cat/K_m (763.92 mM⁻¹ s⁻¹). During the reaction of reverse hydrolysis, the enzyme exhibited high specificity towards the glycosyl donor galactose and acceptors glycerol, ethanol and ethylene glycol. Finally, the isofloridoside was synthesized using galactose as the donor and glycerol as the acceptor with a 26.6% conversion rate of galactose. This study indicated that galV might provide a potential enzyme source in producing isofloridoside because of its high thermal stability and activity.     

Synthesis of 2-Acyloxycyclohexylsulfonamides and Evaluation on Their Fungicidal Activity

Eighteen N-substituted phenyl-2-acyloxycyclohexylsulfonamides (III) were designed and synthesized by the reaction of N-substituted phenyl-2-hydroxyl-cycloalkylsulfonamides (I, R¹) with acyl chloride (II, R²) in dichloromethane under the catalysis of TMEDA and molecular sieve. High fungicidal active compound N-(2,4,5-trichlorophenyl)-2-(2-ethoxyacetoxy) cyclohexylsulfonamide (III-18) was screened out. Mycelia growth assay against the Botrytis cinerea exhibited that EC₅₀ and EC₈₀ of compound III-18 were 4.17 and 17.15 μg mL⁻¹ respectively, which was better than the commercial fungicide procymidone (EC₅₀ = 4.46 μg mL⁻¹ and EC₈₀ = 35.02 μg mL⁻¹). For in vivo activity against B. cinerea in living leaf of cucumber, the control effect of compound III-18 was better than the fungicide cyprodinil. In addition, this new compound had broader fungicidal spectra than chlorothalonil.     

Enhancement of Phenol Biodegradation by Pseudochrobactrum sp. through Ultraviolet-Induced Mutation

The phenol-degrading efficiency of Pseudochrobactrum sp. was enhanced by ultraviolet (UV) irradiation. First, a bacterial strain, Pseudochrobactrum sp. XF1, was isolated from the activated sludge in a coking plant. It was subjected to mutation by UV radiation for 120 s and a mutant strain with higher phenol-degrading efficiency, Pseudochrobactrum sp. XF1-UV, was selected. The mutant strain XF1-UV was capable of degrading 1800 mg/L phenol completely within 48 h and had higher tolerance to hydrogen ion concentration and temperature variation than the wild type. Haldane’s kinetic model was used to fit the exponential growth data and the following kinetic parameters were obtained: μ_max = 0.092 h⁻¹, Ks = 22.517 mg/L, and Ki = 1126.725 mg/L for XF1, whereas μ_max = 0.110 h⁻¹, Ks = 23.934 mg/L, and Ki = 1579.134 mg/L for XF1-UV. Both XF1 and XF1-UV degraded phenol through the ortho-pathway; but the phenol hydroxylase activity of XF1-UV1 was higher than that of XF1, therefore, the mutant strain biodegraded phenol faster. Taken together, our results suggest that Pseudochrobactrum sp. XF1-UV could be a promising candidate for bioremediation of phenol-containing wastewaters.     

Preparation and Characterization of a Chloroperoxidase-like Catalytic Antibody

The small molecule, meso-tetra(α,α,α,α-o-phenylacetamidophenyl) porphyrin (Mr1147.0) was used as complete antigen to elicit MAb through the immunization and cell fusion techniques. The MAb 1F2 obtained was demonstrated to be very pure by MALDI/TOFMS. The subtype of MAb 1F2 is IgG2a, which has a relative molecular weight of 156,678.8 Da.No significant change in the intensity of absorption peaks in UV and CD spectra was observed over a pH range between 6 and 12. The high stability of the abzyme and the tight binding between Fe porphyrin and antibody were also demonstrated. V_max, K_m, κcat, κcat/K_m for abzyme are 5.18 × 10⁻⁸ Ms⁻¹, 1.50 × 10⁻⁸ M, 0.518 s⁻¹, 3.45 × 10⁷ M⁻¹s⁻¹, respectively. The data obtained indicate that catalytic antibody has high catalytic activity. The chloroperoxidase activity of MAb 1F2-Fe porphyrin complex is stable from 10 °C to 60 °C.     

Biosorption of Zn(II) from Seawater Solution by the Microalgal Biomass of Tetraselmis marina AC16-MESO

Biosorption refers to a physicochemical process where substances are removed from the solution by a biological material (live or dead) via adsorption processes governed by mechanisms such as surface complexation, ion exchange, and precipitation. This study aimed to evaluate the adsorption of Zn²⁺ in seawater using the microalgal biomass of Tetraselmis marina AC16-MESO “in vivo” and “not alive” at different concentrations of Zn²⁺ (0, 5, 10, and 20 mg L⁻¹) at 72 h. Analysis was carried out by using the Langmuir isotherms and by evaluating the autofluorescence from microalgae. The maximum adsorption of Zn²⁺ by the Langmuir model using the Q_max parameter in the living microalgal biomass (Q_max = 0.03051 mg g⁻¹) was more significant than the non-living microalgal biomass of T. marine AC16-MESO (Q_max = 0.02297 mg g⁻¹). Furthermore, a decrease in fluorescence was detected in cells from T. marina AC16-MESO, in the following order: Zn²⁺ (0 < 20 < 5 < 10) mg L⁻¹. Zn²⁺ was adsorbed quickly by living cells from T. marine AC16-MESO compared to the non-living microalgal biomass, with a decrease in photosystem II activities from 0 to 20 mg L⁻¹ Zn²⁺ in living cells.     

Unique Regulation of Intestinal Villus Epithelial Cl−/HCO3− Exchange by Cyclooxygenase Pathway Metabolites of Arachidonic Acid in a Mouse Model of Spontaneous Ileitis

Electrolytes (NaCl) and fluid malabsorption cause diarrhea in inflammatory bowel disease (IBD). Coupled NaCl absorption, mediated by Na⁺/H⁺ and Cl⁻/HCO₃⁻ exchanges on the intestinal villus cells brush border membrane (BBM), is inhibited in IBD. Arachidonic acid metabolites (AAMs) formed via cyclooxygenase (COX) or lipoxygenase (LOX) pathways are elevated in IBD. However, their effects on NaCl absorption are not known. We treated SAMP1/YitFc (SAMP1) mice, a model of spontaneous ileitis resembling human IBD, with Arachidonyl Trifluoro Methylketone (ATMK, AAM inhibitor), or with piroxicam or MK-886, to inhibit COX or LOX pathways, respectively. Cl⁻/HCO₃⁻ exchange, measured as DIDS-sensitive ³⁶Cl uptake, was significantly inhibited in villus cells and BBM vesicles of SAMP1 mice compared to AKR/J controls, an effect reversed by ATMK. Piroxicam, but not MK-886, also reversed the inhibition. Kinetic studies showed that inhibition was secondary to altered K_m with no effects on V_max. Whole cell or BBM protein levels of Down-Regulated in Adenoma (SLC26A3) and putative anion transporter-1 (SLC26A6), the two key BBM Cl⁻/HCO₃⁻ exchangers, were unaltered. Thus, inhibition of villus cell Cl⁻/HCO₃⁻ exchange by COX pathway AAMs, such as prostaglandins, via reducing the affinity of the exchanger for Cl⁻, and thereby causing NaCl malabsorption, could significantly contribute to IBD-associated diarrhea.     

A High Redox Potential Laccase from Pycnoporus sanguineus RP15: Potential Application for Dye Decolorization

Laccase production by Pycnoporus sanguineus RP15 grown in wheat bran and corncob under solid-state fermentation was optimized by response surface methodology using a Central Composite Rotational Design. A laccase (Lacps1) was purified and characterized and the potential of the pure Lacps1 and the crude culture extract for synthetic dye decolorization was evaluated. At optimal conditions (eight days, 26 °C, 18% (w/w) milled corncob, 0.8% (w/w) NH₄Cl and 50 mmol·L⁻¹ CuSO₄, initial moisture 4.1 mL·g⁻¹), the laccase activity reached 138.6 ± 13.2 U·g⁻¹. Lacps1 was a monomeric glycoprotein (67 kDa, 24% carbohydrate). Optimum pH and temperature for the oxidation of 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) were 4.4 and 74.4 °C, respectively. Lacps1 was stable at pH 3.0–8.0, and after two hours at 55–60 °C, presenting high redox potential (0.747 V vs. NHE). ABTS was oxidized with an apparent affinity constant of 147.0 ± 6.4 μmol·L⁻¹, maximum velocity of 413.4 ± 21.2 U·mg⁻¹ and catalytic efficiency of 3140.1 ± 149.6 L·mmol⁻¹·s⁻¹. The maximum decolorization percentages of bromophenol blue (BPB), remazol brilliant blue R and reactive blue 4 (RB4), at 25 or 40 °C without redox mediators, reached 90%, 80% and 60%, respectively, using either pure Lacps1 or the crude extract. This is the first study of the decolorization of BPB and RB4 by a P. sanguineus laccase. The data suggested good potential for treatment of industrial dye-containing effluents.     

Study of the oxygen vacancy influence on magnetic properties of Fe- and Co-doped SnO2 diluted alloys

Transition-metal (TM)-doped diluted magnetic oxides (DMOs) have attracted attention from both experimental and theoretical points of view due to their potential use in spintronics towards new nanostructured devices and new technologies. In the present work, we study the magnetic properties of Sn_0.96TM_0.04O₂ and Sn_0.96TM_0.04O_1.98(V_O)_0.02, where TM = Fe and Co, focusing in particular in the role played by the presence of O vacancies nearby the TM. The calculated total energy as a function of the total magnetic moment per cell shows a magnetic metastability, corresponding to a ground state, respectively, with 2 and 1 μ_B/cell, for Fe and Co. Two metastable states, with 0 and 4 μ_B/cell were found for Fe, and a single value, 3 μ_B/cell, for Co. The spin-crossover energies (E_S) were calculated. The values are E_S^0/2 = 107 meV and E_S^4/2 = 25 meV for Fe. For Co, E_S^3/1 = 36 meV. By creating O vacancies close to the TM site, we show that the metastablity and E_S change. For iron, a new state appears, and the state with zero magnetic moment disappears. The ground state is 4 μ_B/cell instead of 2 μ_B/cell, and the energy E_S^2/4 is 30 meV. For cobalt, the ground state is then found with 3 μ_B/cell and the metastable state with 1 μ_B/cell. The spin-crossover energy E_S^1/3 is 21 meV. Our results suggest that these materials may be used in devices for spintronic applications that require different magnetization states.     

Cloning,Expression and Characterization of a Thermostable Esterase HydS14 from Actinomadura sp. Strain S14 in Pichia pastoris

A thermostable esterase gene (hydS14) was cloned from an Actinomadura sp. S14 gene library. The gene is 777 bp in length and encodes a polypeptide of 258 amino acid residues with no signal peptide, no N-glycosylation site and a predicted molecular mass of 26,604 Da. The encoded protein contains the pentapeptide motif (GYSLG) and catalytic triad (Ser88-Asp208-His235) of the esterase/lipase superfamily. The HydS14 sequence shows 46%–64% identity to 23 sequences from actinomycetes (23 α/β-hydrolases), has three conserved regions, and contains the novel motif (GY(F)SLG), which distinguishes it from other clusters in the α/β-hydrolase structural superfamily. A plasmid containing the coding region (pPICZαA-hydS14) was used to express HydS14 in Pichia pastoris under the control of the AOXI promoter. The recombinant HydS14 collected from the supernatant had a molecular mass of ~30 kDa, which agrees with its predicted molecular mass without N-glycosylation. HydS14 had an optimum temperature of approximately 70 °C and an optimum pH of 8.0. HydS14 was stable at 50 and 60 °C for 120 min, with residual activities of above 80% and above 90%, respectively, as well as 50% activity at pH 6.0–8.0 and pH 9.0, respectively. The enzyme showed higher activity with p-nitrophenyl-C2 and C4. The K_m and V_max values for p-nitrophenyl-C4 were 0.21 ± 0.02 mM and 37.07 ± 1.04 μmol/min/mg, respectively. The enzyme was active toward short-chain p-nitrophenyl ester (C2–C6), displaying optimal activity with p-nitrophenyl-C4 (K_cat/K_m = 11.74 mM⁻¹·S⁻¹). In summary, HydS14 is a thermostable esterase from Actinomadura sp. S14 that has been cloned and expressed for the first time in Pichia pastoris.     

Isolation and Characterization of a Glycosyl Hydrolase Family 16 β-Agarase from a Mangrove Soil Metagenomic Library

A mangrove soil metagenomic library was constructed and a β-agarase gene designated as AgaML was isolated by functional screening. The gene encoded for a 659-amino-acids polypeptide with an estimated molecular mass of 71.6 kDa. The deduced polypeptide sequences of AgaML showed the highest identity of 73% with the glycoside hydrolase family 16 β-agarase from Microbulbifer agarilyticus in the GenBank database. AgaML was cloned and highly expressed in Escherichia coli BL21(DE3). The purified recombinant protein, AgaML, showed optimal activity at 50 °C and pH 7.0. The kinetic parameters of K_m and V_max values toward agarose were 4.6 mg·mL⁻¹ and 967.5 μM·min⁻¹·mg⁻¹, respectively. AgaML hydrolyzed the β-1,4-glycosidic linkages of agar to generate neoagarotetraose (NA4) and neoagarohexaose (NA6) as the main products. These characteristics suggest that AgaML has potential application in cosmetic, pharmaceuticals and food industries.     

New Mononuclear Cu(II) Complexes and 1D Chains with 4-Amino-4H-1,2,4-triazole

The crystal structures of two mononuclear Cu(II) NH₂trz complexes [Cu(NH₂trz)₄(H₂O)](AsF₆)₂ (I) and [Cu(NH₂trz)₄(H₂O)](PF₆)₂ (II) as well as two coordination polymers [Cu(μ₂-NH₂trz)₂Cl]Cl·H₂O (III) and [Cu(μ₂-NH₂trz)₂Cl] (SiF₆)_0.5·1.5H₂O (IV) are presented. Cationic 1D chains with bridging bis-monodentate μ₂-coordinated NH₂trz and bridging μ₂-coordinated chloride ligands are present in III and IV. In these coordination polymers, the Cu(II) ions are strongly antiferromagnetically coupled with J = −128.4 cm⁻¹ for III and J = −143 cm⁻¹ for IV (H = −J∑S_iS_i+1), due to the nature of the bridges between spin centers. Inter-chain interactions present in the crystal structures were taken into consideration, as well as g factors, which were determined experimentally, for the quantitative modeling of their magnetic properties.     

Adsorption and Release of Sulfamethizole from Mesoporous Silica Nanoparticles Functionalised with Triethylenetetramine

Mesoporous silica nanoparticles (MSN) were synthesised and functionalised with triethylenetetramine (MSN-TETA). The samples were fully characterised (transmission electron microscopy, small angle X-ray scattering, Fourier transform infrared spectroscopy, thermogravimetric analysis, zeta potential and nitrogen adsorption/desorption isotherms) and used as carriers for the adsorption of the antimicrobial drug sulphamethizole (SMZ). SMZ loading, quantified by UV–Vis spectroscopy, was higher on MSN-TETA (345.8 mg g⁻¹) compared with bare MSN (215.4 mg g⁻¹) even in the presence of a lower surface area (671 vs. 942 m² g⁻¹). The kinetics of SMZ adsorption on MSN and MSN-TETA followed a pseudo-second-order model. The adsorption isotherm is described better by a Langmuir model rather than a Temkin or Freundlich model. Release kinetics showed a burst release of SMZ from bare MSN samples (k₁ = 136 h⁻¹) in contrast to a slower release found with MSN-TETA (k₁ = 3.04 h⁻¹), suggesting attractive intermolecular interactions slow down SMZ release from MSN-TETA. In summary, the MSN surface area did not influence SMZ adsorption and release. On the contrary, the design of an effective drug delivery system must consider the intermolecular interactions between the adsorbent and the adsorbate.     

Spatio-kinetic variation of methane oxidizing bacteria in paddy soil at mid-tillering: effect of N-fertilizers

Laboratory experiments were conducted to evaluate the variation ofpopulation size of methanotrophs (MOB) and CH₄ oxidation pattern inflooded rice soils sampled at three spatial points (rhizosphere, bulk and baresoils). Rhizosphere soil had higher MOB population size (301.1 ×10⁵ cells g^–1 dry soil) than bulk(37.2× 10⁵ cells g^–1 dry soil) andbare soil (19.1 × 10⁵ cells g^–1dry soil). The population size of MOB followed a decreasing trend with respectto fertilizer (urea NH₄NO₃ NH₄Cl control). The result indicated that rhizosphere soil presented thestrongestCH₄ oxidation activities, as shown by the highest values of the twokinetic parameters (K _m(app) andV _max). K _m andV _max increased significantly from bare to bulkto rhizosphere soil in control and fertilized soil and ranged from 6.2 to 133.2g g^–1 dry soil and from 0.03 to 0.41g h^–1 g^–1 dry soil,respectively. The differences in K _m andV _max among the three soils (rhizosphere, bulkand bare) in this study could be due to differential species composition ofmethanotrophic community and/or to conditioning of MOB under different soilmicroenvironments. The present study has demonstrated a competitive inhibition effectof NH₄ ⁺-N on CH₄ oxidation.     

A Halomanganates(II) with P,P’-Diprotonated Bis(2-Diphenylphosphinophenyl)ether: Wavelength-Excitation Dependence of the Quantum Yield and Role of the Non-Covalent Interactions

A [H₂DPEphos][MnX₄] [X = Br, Cl] tetrahalomanganates(II) with P,P’-diprotonated bis[2-(diphenylphosphino)phenyl]ether cation has been designed and investigated in photophysics and EPR terms. The complexes exhibit a green luminescence resulted from the Mn(II) d–d transitions (⁴T₁→⁶A₁) with the wavelength-excitation dependence of the quantum yield. The solid [H₂DPEphos][MnBr₄] complex exhibits a bright green phosphorescence (λ_max = 515 nm) with the high luminescence quantum yield depending on the excitation energy whereas the solid [H₂DPEphos][MnCl₄] complex exhibits a very weak phosphorescence (λ_max = 523 nm). The unexpected shorter luminescence lifetime for the [H₂DPEphos][MnCl₄] than for the [H₂DPEphos][MnBr₄] at 300 K can be a result of the higher non-radiative relaxation contribution. On the one hand, the non-covalent PH…X(Mn) interactions quench the manganese(II) luminescence. On the other hand, the PH…X(Mn) interactions are a pathway of the excitation transfer from [H₂DPEphos]²⁺ to [MnX₄]²⁻.     

Study of iodide sorption to the argillite of Tournemire in alkaline media

Argillaceous rocks are considered potential host rocks for radioactive waste repositories. The concrete matrix that could be used as a barrier could react with the groundwater of the geological site, inducing a drastic change in its chemical composition and its pH (10–13). Consequently, the physicochemical properties of the rock in contact with this alkaline solution may be modified and, in turn, may induce modification on the behaviour of radioelements. This study, applied to the argillite of Tournemire, involves characterizing I⁻ sorption to an argillaceous rock in alkaline media in batch experiments under N₂-controlled conditions. I⁻ was added as a ¹²⁵I radiotracer and measured by γ spectrometry.Preliminary experiments were conducted with different solution/solid ratios (v/m=2.5, 5 and 20 ml g⁻¹) and contact times (1–14 days) in order to determine the optimal experimental conditions. The chosen v/m ratio was 5 ml g⁻¹ as the best compromise between a high K_d value and a low error of the measure. The chosen experiment duration was 1 day because I⁻ sorption was highest and to limit the effects of pyrite oxidation. One of the experiments, performed with a radio-sterilized sample to test possible effects from microorganisms, showed that they could enhance iodide retention, particularly during the first 2 contact days.The influence of pH on I⁻ sorption was tested using solutions between values of 8.3 and 12.8. The K_d values were independent of pH and very low (0.3 ml g⁻¹).Finally, the influence of the chemical composition of concrete fluids was also tested. Three solution compositions corresponding to different steps in the evolution of fluids in contact with altering concrete were used: fluid in contact with fresh concrete (pH 13.2), with moderately degraded concrete (pH 12.1) and with strongly degraded concrete (pH 11.5). Each solution contained variable amounts of sodium, potassium, calcium, silica and sulphate. I⁻ sorption was also very low (K_d0.2 ml g⁻¹). Additional experiments were conducted with alkaline solutions containing different amounts of SO₄²⁻ ions (10⁻³–10⁻² M) to test sulphate–iodide sorption competition. I⁻ retention was independent of the sulphate concentration.     

Novel Mechanism and Kinetics of Tetramethrin Degradation Using an Indigenous Gordonia cholesterolivorans A16

Tetramethrin is a pyrethroid insecticide that is commonly used worldwide. The toxicity of this insecticide into the living system is an important concern. In this study, a novel tetramethrin-degrading bacterial strain named A16 was isolated from the activated sludge and identified as Gordonia cholesterolivorans. Strain A16 exhibited superior tetramethrin degradation activity, and utilized tetramethrin as the sole carbon source for growth in a mineral salt medium (MSM). High-performance liquid chromatography (HPLC) analysis revealed that the A16 strain was able to completely degrade 25 mg·L⁻¹ of tetramethrin after 9 days of incubation. Strain A16 effectively degraded tetramethrin at temperature 20–40 °C, pH 5–9, and initial tetramethrin 25–800 mg·L⁻¹. The maximum specific degradation rate (q_max), half-saturation constant (K_s), and inhibition constant (K_i) were determined to be 0.4561 day⁻¹, 7.3 mg·L⁻¹, and 75.2 mg·L⁻¹, respectively. The Box–Behnken design was used to optimize degradation conditions, and maximum degradation was observed at pH 8.5 and a temperature of 38 °C. Five intermediate metabolites were identified after analyzing the degradation products through gas chromatography–mass spectrometry (GC-MS), which suggested that tetramethrin could be degraded first by cleavage of its carboxylester bond, followed by degradation of the five-carbon ring and its subsequent metabolism. This is the first report of a metabolic pathway of tetramethrin in a microorganism. Furthermore, bioaugmentation of tetramethrin-contaminated soils (50 mg·kg⁻¹) with strain A16 (1.0 × 10⁷ cells g⁻¹ of soil) significantly accelerated the degradation rate of tetramethrin, and 74.1% and 82.9% of tetramethrin was removed from sterile and non-sterile soils within 11 days, respectively. The strain A16 was also capable of efficiently degrading a broad spectrum of synthetic pyrethroids including D-cyphenothrin, chlorempenthrin, prallethrin, and allethrin, with a degradation efficiency of 68.3%, 60.7%, 91.6%, and 94.7%, respectively, after being cultured under the same conditions for 11 days. The results of the present study confirmed the bioremediation potential of strain A16 from a contaminated environment.     

Simultaneous Monitoring of pH and Chloride (Cl−) in Brain Slices of Transgenic Mice

Optosensorics is the direction of research possessing the possibility of non-invasive monitoring of the concentration of intracellular ions or activity of intracellular components using specific biosensors. In recent years, genetically encoded proteins have been used as effective optosensory means. These probes possess fluorophore groups capable of changing fluorescence when interacting with certain ions or molecules. For monitoring of intracellular concentrations of chloride ([Cl⁻]_i) and hydrogen ([H⁺] _i) the construct, called ClopHensor, which consists of a H⁺- and Cl⁻-sensitive variant of the enhanced green fluorescent protein (E²GFP) fused with a monomeric red fluorescent protein (mDsRed) has been proposed. We recently developed a line of transgenic mice expressing ClopHensor in neurons and obtained the map of its expression in different areas of the brain. The purpose of this study was to examine the effectiveness of transgenic mice expressing ClopHensor for estimation of [H⁺]_i and [Cl⁻]_i concentrations in neurons of brain slices. We performed simultaneous monitoring of [H⁺]_i and [Cl⁻]_i under different experimental conditions including changing of external concentrations of ions (Ca²⁺, Cl⁻, K⁺, Na⁺) and synaptic stimulation of Shaffer’s collaterals of hippocampal slices. The results obtained illuminate different pathways of regulation of Cl⁻ and pH equilibrium in neurons and demonstrate that transgenic mice expressing ClopHensor represent a reliable tool for non-invasive simultaneous monitoring of intracellular Cl⁻ and pH.     

Wild Soybean Oxalyl-CoA Synthetase Degrades Oxalate and Affects the Tolerance to Cadmium and Aluminum Stresses

Acyl activating enzyme 3 (AAE3) was identified as being involved in the acetylation pathway of oxalate degradation, which regulates the responses to biotic and abiotic stresses in various higher plants. Here, we investigated the role of Glycine soja AAE3 (GsAAE3) in Cadmium (Cd) and Aluminum (Al) tolerances. The recombinant GsAAE3 protein showed high activity toward oxalate, with a K_m of 105.10 ± 12.30 μM and V_max of 12.64 ± 0.34 μmol min⁻¹ mg⁻¹ protein, suggesting that it functions as an oxalyl–CoA synthetase. The expression of a GsAAE3–green fluorescent protein (GFP) fusion protein in tobacco leaves did not reveal a specific subcellular localization pattern of GsAAE3. An analysis of the GsAAE3 expression pattern revealed an increase in GsAAE3 expression in response to Cd and Al stresses, and it is mainly expressed in root tips. Furthermore, oxalate accumulation induced by Cd and Al contributes to the inhibition of root growth in wild soybean. Importantly, GsAAE3 overexpression increases Cd and Al tolerances in A. thaliana and soybean hairy roots, which is associated with a decrease in oxalate accumulation. Taken together, our data provide evidence that the GsAAE3-encoded protein plays an important role in coping with Cd and Al stresses.     

Cloning,Expression and Characterization of a Novel Thermophilic Polygalacturonase from Caldicellulosiruptor bescii DSM 6725

We cloned the gene {"type":"entrez-protein","attrs":{"text":"ACM61449","term_id":"222457187","term_text":"ACM61449"}}ACM61449 from anaerobic, thermophilic Caldicellulosiruptor bescii, and expressed it in Escherichia coli origami (DE3). After purification through thermal treatment and Ni-NTA agarose column extraction, we characterized the properties of the recombinant protein (CbPelA). The optimal temperature and pH of the protein were 72 °C and 5.2, respectively. CbPelA demonstrated high thermal-stability, with a half-life of 14 h at 70 °C. CbPelA also showed very high activity for polygalacturonic acid (PGA), and released monogalacturonic acid as its sole product. The V_max and K_m of CbPelA were 384.6 U·mg⁻¹ and 0.31 mg·mL⁻¹, respectively. CbPelA was also able to hydrolyze methylated pectin (48% and 10% relative activity on 20%–34% and 85% methylated pectin, respectively). The high thermo-activity and methylated pectin hydrolization activity of CbPelA suggest that it has potential applications in the food and textile industry.     

Influence of Green,Red and Blue Light Emitting Diodes on Multiprotein Complex Proteins and Photosynthetic Activity under Different Light Intensities in Lettuce Leaves (Lactuca sativa L.)

The objective of this study was to investigate the response of light emitting diodes (LEDs) at different light intensities (70 and 80 for green LEDs, 88 and 238 for red LEDs and 80 and 238 μmol m⁻² s⁻¹ for blue LEDs) at three wavelengths in lettuce leaves. Lettuce leaves were exposed to (522 nm), red (639 nm) and blue (470 nm) LEDs of different light intensities. Thylakoid multiprotein complex proteins and photosynthetic metabolism were then investigated. Biomass and photosynthetic parameters increased with an increasing light intensity under blue LED illumination and decreased when illuminated with red and green LEDs with decreased light intensity. The expression of multiprotein complex proteins including PSII-core dimer and PSII-core monomer using blue LEDs illumination was higher at higher light intensity (238 μmol m⁻² s⁻¹) and was lowered with decreased light intensity (70–80 μmol m⁻² s⁻¹). The responses of chloroplast sub-compartment proteins, including those active in stomatal opening and closing, and leaf physiological responses at different light intensities, indicated induced growth enhancement upon illumination with blue LEDs. High intensity blue LEDs promote plant growth by controlling the integrity of chloroplast proteins that optimize photosynthetic performance in the natural environment.