Design,synthesis and evaluation of N2,N4-diaminoquinazoline based inhibitors of phosphodiesterase type 5

We describe the design, synthesis and evaluation of a series of N²,N⁴-diaminoquinazoline analogs as PDE5 inhibitors. Twenty compounds were prepared and these were assessed in terms of their PDE5 and PDE6 activity, ex-vivo vasodilation response, mammalian cytotoxicity and aqueous solubility. Molecular docking was used to determine the binding mode of the series and this was demonstrated to be consistent with the observed SAR. Compound 15 was the most active PDE5 inhibitor (IC₅₀?=?0.072?±?0.008?µM) and exhibited 4.6-fold selectivity over PDE6. Ex-vivo assessment of 15 and 22 in a rat pulmonary artery vasodilation model demonstrated EC₅₀s of 1.63?±?0.72?µM and 2.28?±?0.74?µM respectively.     

The biotechnological potential of subtilisin‐like fibrinolytic enzyme from a newly isolated Lactobacillus plantarum KSK‐II in blood destaining and antimicrobials

An antimicrobial oxidative‐ and SDS‐stable fibrinolytic alkaline protease designated as KSK‐II was produced by Lactobacillus plantarum KSK‐II isolated from kishk, a traditional Egyptian food. Maximum enzyme productivity was obtained in medium containing 1% lactose and 0.5% soybean flour as carbon and nitrogen sources, respectively. Purification of enzyme increased its specific activity to 1,140‐fold with a recovery of 33% and molecular weight of 43.6 kDa. Enzyme activity was totally lost in the presence of ethylenediaminetetraacetic acid and was restored after addition of Fe²⁺ suggesting that KSK‐II is a metalloprotease and Fe²⁺ acts as cofactor. Enzyme hydrolyzed not only the natural proteins but also synthetic substrates, particularly Suc‐Ala‐Ala‐Pro‐Phe‐pNA. KSK‐II can hydrolyze the Lys‐X easier than Arg‐X; thus, it was considered as a subtilisin‐family protease. Its apparent K_m, V_max, and K_cat were 0.41 mM, 6.4 µmol mg^?1 min^?1, and 28.0 s^?1, respectively. KSK‐II is industrially important from the perspectives of its maximal activity at 50°C (stable up to 70°C), ability to function at alkaline pH (10.0), stability at broad pH ranges (7.5–12.0) in addition to its stability toward SDS, H₂O₂, organic solvents, and detergents. We emphasize for the first time the potential of fibrinolytic activity for alkaline proteases used in detergents especially in blood destaining. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:316–324, 2015     

Interaction of elongation factor EF-Tu with γ-amides of GTP and β-amides of GDP bearing the azidoaryl group or the chloroethylaminoaryl group placed at the terminal phosphate

New types of azidoaryl analogs of GTP: γ-(4-azido)anilide of GTP (I), γ-(N-(4-azidobenzyl)-N-methyl)amide of GTP (II) and of GDP: β-(4-azido)anilide of GDP (III), β-(N-(4-azidobenzyl)-N-methyl)amide of GDP (IV) have been synthesized by treatment of the nucleotide in aqueous solution with N-cyclohexyl-N′-β-(4-methylmorpholinium)- ethylcarbodiimidep-toluene sulfonate and the respective amine. The analog of GTP bearing at the γ-phosphate an alkylating 2-chloroethylamino group: γ-(4-N-(2-chloroethyl)-N-methylaminobenzyl)amide of GTP (V) was prepared by the method described previously for the preparation of the analog of ATP (Knorre, D.G., Kurbatov, V.A. and Samukov, V.V. (1976) FEBS Lett. 70, 105–108). Azidoaryl analogs of GTP and GDP as well as the chloroethylaminoaryl analog of GTP compete with GDP in the formation of the binary complex EF-Tu·GDP with the respective K_i values 3.9·10^?7 M (I), 2.9·10^?8 M (II), 6.9·10^?7 M (III), 5.0·10^?7 M (IV) and 3.8·10^?8 M (V) relative to GDP. constants of the complexes of the radioactively-labeled GTP analogs I, II and V with elongation factor Tu were calculated to be 8.5·10^?6 M, 3.4·10^?7 M and 4.6·10^?8 M, respectively, or approx. 1740-, 70- and 9-times greater than that of GDP. GTP analogs I, II and V were found to substitute GTP in the stimulation of EF-Tu-dependent binding of aminoacyl-tRNA to the ribosome-mRNA complex.     

Comparative Effects of Hydrogen Sulfide-Releasing Compounds on [<Superscript>3</Superscript>H]D-Aspartate Release from Bovine Isolated Retinae

We investigated the pharmacological actions of a slow-releasing H₂S donor, GYY 4137; a substrate for the biosynthesis of H₂S, l-cysteine and its precursor, N-acetylcysteine on potassium (K⁺; 50 mM)-evoked [³H]D-aspartate release from bovine isolated retinae using the Superfusion Method. GYY 4137 (10 nM–10 µM), l-cysteine (100 nM–10 µM) and N-acetylcysteine (10 µM–1 mM) elicited a concentration-dependent decrease in K⁺-evoked [³H]D-aspartate release from isolated bovine retinae without affecting basal tritium efflux. At equimolar concentration of 10 µM, the rank order of activity was as follows: l-cysteine?>?GYY 4137?>?N-acetylcysteine. A dual inhibitor of the biosynthetic enzymes for H₂S, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), amino-oxyacetic acid (AOA; 3 mM) reversed the inhibitory responses caused by GYY 4137, l-cysteine and N-acetylcysteine on K⁺-evoked [³H]D-aspartate release. Glibenclamide (300 µM), an inhibitor of K_ATP channels blocked the inhibitory action of GYY 4137 and l-cysteine but not that elicited by N-acetylcysteine on K⁺-induced [³H]D-aspartate release. The inhibitory effect of GYY 4137 and l-cysteine on K⁺-evoked [³H]D-aspartate release was reversed by the non-specific inhibitor of nitric oxide synthase (NOS), l-NAME (300 µM). Furthermore, a specific inhibitor of inducible NOS (iNOS), aminoguanidine (10 µM) blocked the inhibitory action of l-cysteine on K⁺-evoked [³H]D-aspartate release. We conclude that both donors and substrates for H₂S production can inhibit amino acid neurotransmission in bovine isolated retinae, an effect that is dependent, at least in part, upon the intramural biosynthesis of this gas, and on the activity of K_ATP channels and NO synthase.     

The nitrite reductase activity of horse heart carboxymethylated-cytochrome <Emphasis Type="Italic">c</Emphasis> is modulated by cardiolipin

Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO₂ ^?-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k _on = (7.3 ± 0.7) × 10^?2 M^?1 s^?1; at pH 7.4], whereas the value of k _on for NO₂ ^? reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M^?1 s^?1 (at pH 7.4). CL facilitates the NO₂ ^?-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k _on for the NO₂ ^?-mediated conversion of CL–CM-cytc-Fe(II) to CL–CM-cytc-Fe(II)-NO (5.6 ± 0.6 M^?1 s^?1; at pH 7.4) being slightly higher than that for the NO₂ ^?-mediated conversion of CL–cytc-Fe(II) to CL–cytc-Fe(II)-NO (2.6 ± 0.3 M^?1 s^?1; at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10^?6 M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k _on versus pH are ?1.05 ± 0.07 and ?1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH^?. These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL–CM-cytc.     

Purification and characterization of two distinct acidic phytases with broad pH stability from Aspergillus niger NCIM 563

Aspergillus niger NCIM 563 produced two different extracellular phytases (Phy I and Phy II) under submerged fermentation conditions at 30°C in medium containing dextrin-glucose-sodium nitrate-salts. Both the enzymes were purified to homogeneity using Rotavapor concentration, Phenyl-Sepharose column chromatography and Sephacryl S-200 gel filtration. The molecular mass of Phy I and II as determined by SDS–PAGE and gel filtration were 66, 264, 150 and 148 kDa respectively, indicating that Phy I consists of four identical subunits and Phy II is a monomer. The pI values of Phy I and II were 3.55 and 3.91, respectively. Phy I was highly acidic with optimum pH of 2.5 and was stable over a broad pH range (1.5–9.0) while Phy II showed a pH optimum of 5.0 with stability in the range of pH 3.5–9.0. Phy I exhibited very broad substrate specificity while Phy II was more specific for sodium phytate. Similarly Phy II was strongly inhibited by Ag⁺, Hg²⁺ (1 mM) metal ions and Phy I was partially inhibited. Peptide analysis by Mass Spectrometry (MS) MALDI-TOF also indicated that both the proteins were totally different. The K _m for Phy I and II for sodium phytate was 2.01 and 0.145 mM while V _max was 5,018 and 1,671 μmol min^?1 mg^?1, respectively. The N-terminal amino acid sequences of Phy I and Phy II were FSYGAAIPQQ and GVDERFPYTG, respectively. Phy II showed no homology with Phy I and any other known phytases from the literature suggesting its unique nature. This, according to us, is the first report of two distinct novel phytases from Aspergillus niger.     

Sulphydryl groups of (Na+ + K+)-ATPase from rectal glands of Squalus acanthias: Detection of ligand-induced conformational changes

1. Modification of the Class II sulphydryl groups on the (Na⁺ + K⁺)-ATPase from rectal glands of Squalus acanthias with N-ethylmaleimide has been used to detect conformational changes in the protein. The rates of inactivation of the enzyme and the incorporation of N-ethylmaleimide depend on the ligands present in the incubation medium. With 150 mM K⁺ the rate of inactivation is largest (k₁ = 1.73 mM^?1 · min^?1) and four SH groups per α-subunit are modified. The rate of inactivation in the presence of 150 mM Na⁺ is smaller (k₁ = 1.08 mM^?1 · min^-1) but the incorporation of N-ethylmaleimide is the same as with K⁺. 2. ATP in micromolar concentrations protects the Class II groups in the presence of Na⁺ (k₁ = 0.08 mM^?1 · min^?1 at saturating ATP) and the incorporation id drastically reduced. ATP in millimolar concentrations protects the Class II groups partially in the presence of K⁺ (k₁ = 1.08 mM^?1 · min^?1) and three SH groups are labelled per α subunit. 3. The K⁺ -dependent phosphatase is inhibited in parallel to the (Na⁺ + K⁺)-ATPase under all conditions, and the ligand-dependent incorporation of N-ethylmaleimide was on the α-subunit only. 4. It is shown that the difference between the Na⁺ and K⁺ conformations sensed with N-ethylmaleimide depends on the pH of the incubation medium. At pH 6 there is a very small difference between the rates of inactivation in the presence of Na⁺ and K⁺, but at higher pH the difference increases. It is also shown that the rate of inactivation has a minimum at pH 6.9, which suggests that the conformation of the enzyme changes with pH. 5. Modification of the Class III groups with N-ethylmaleimide-whereby the enzyme activity is reduced from about 16% to zero-shows that these groups are also sensitive to conformational changes. As with the Class II groups, ATP in micromolar concentrations protects in the presence of Na⁺ relative to Na⁺ or K⁺ alone. ATP in millimolar concentrations with K⁺ present increases the rate of inactivation relative to K⁺ alone, in contrast to the effect on the Class II groups. 6. Modification of the Class II groups with a maleimide spin label shows a difference between Class II groups labelled in the presence of Na⁺ (or K⁺) and Class II groups labelled in the presence of K + ATP, in agreement with the difference in incorporation of N-ethylmaleimide. The spectra suggest that the SH group protected by ATP in the presence of K⁺ is buried in the protein. 7. The results suggest that at least four different conformations of the (Na⁺ + K⁺)-ATPase can be sensed with N-ethylmaleimide: (i) a Na⁺ form of the enzyme with ATP bound to a high-affinity site (E₁-Na-ATP); (ii) a Na⁺ form without ATP bound (E₁-Na); (iii) a K⁺ form without ATP bound (E₂-K); and (iv) an enzyme form with ATP bound to a low-affinity site in the presence of K⁺, probably and E₁-K-ATP form.     

Synthesis,FT-IR and 1H NMR studies of alkali and alkaline earth metal complexes with guanosine

The synthesis of complexes of Li(I), K(I), Mg(II), Ca(II) and Ba(II) with guanosine in basic non aqueous solutions is described. The complexes were of two types: (1) complexes having the general formula, M(Guo)_nX_m·YH₂O·ZC₂H₅OH, where M = Mg(II), Ca(II), Ba(II) and Li(I), n = 1,2,4, X = Cl^?, Br^?, NO₃^?, ClO₄^? and OH^?, m = 1,2, Y = 0?6 and X = 0?2, and (2) complexes with the general formula, M(GuoH-1)(OH)_n^?1·YH₂O, where M = K(I), Ca(Il) and Ba(II), GuoH-1 =Ionized guanosine at N₁, n = 1,2 and Y = 1?3. The complexes are characterized by their proton nuclear magnetic resonance (¹H NMR) and Fourier transform infrared (FT-IR) spectra. The FT-IR and ¹H NMR data of the non ionized nucleoside complexes suggest that the metal binding is through the N₇-site of guanine and that the anion (X) is hydrogen bonded to N₁H and NH₂ groups. In the N₁-ionized guanosine complexes the metal binding is via the O₆^? of guanine. All the complexes formed exhibited a transition of the sugar conformation from C₂′-endo/anti in the free nucleoside to C₃′-endo/anti in the metal complexes.     

Cardiolipin modulates allosterically the nitrite reductase activity of horse heart cytochrome c

Upon cardiolipin (CL) liposomes binding, horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential, binds CO and NO with high affinity, displays peroxidase activity, and facilitates peroxynitrite isomerization. Here, the effect of CL liposomes on the nitrite reductase activity of ferrous cytc (cytc-Fe(II)) is reported. In the absence of CL liposomes, hexa-coordinated cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO₂ ^?-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO (k _on = (7.3 ± 0.7) × 10^?2 M^?1 s^?1; at pH 7.4 and 20.0 °C). However, CL liposomes facilitate the NO₂ ^?-mediated nitrosylation of cytc-Fe(II) in a dose-dependent manner inducing the penta-coordination of the heme-Fe(II) atom. The value of k _on for the NO₂ ^?-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO is 2.6 ± 0.3 M^?1 s^?1 (at pH 7.4 and 20.0 °C). Values of the apparent dissociation equilibrium constant for CL liposomes binding to cytc-Fe(II) are (2.2 ± 0.2) × 10^?6 M, (1.8 ± 0.2) × 10^?6 M, and (1.4 ± 0.2) × 10^?6 M at pH 6.5, 7.4, and 8.1, respectively, and 20.0 °C. These results suggest that the NO₂ ^?-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO could play anti-apoptotic effects impairing lipid peroxidation and therefore the initiation of the cell death program by the release of pro-apoptotic factors (including cytc) in the cytoplasm.     

Phenoloxidase Activity of Helix aspersa Maxima (Garden Snail, Gastropod) Hemocyanin

The oxygen-transporting protein, hemocyanin (Hc), of the garden snail Helix aspersa maxima (HaH) was isolated and kinetically characterized. Kinetic parameters of the reaction of catalytic oxidation of catechol to quinone, catalyzed by native HaH were determined: the V _max value amounted to 22 nmol min^?1 mg^?1, k _cat to 1.1 min^?1. Data were compared to those reported for other molluscan Hcs and phenoloxidases (POs). The o-diphenoloxidase activity of the native HaH is about five times higher than the activity determined for the Hcs of the terrestrial snail Helix pomatia and of the marine snail Rapana thomasiana (k _cat values of 0.22 and 0.25 min^?1, respectively). The K _m values obtained for molluscan Hcs from different species are comparable to those for true POs, but the low catalytic efficiency of Hcs is probably related to inaccessibility of the active sites to potential substrates. Upon treatment of HaH with subtilisin DY, the enzyme activity against substrate catechol was considerably increased. The relatively high proteolytically induced o-diPO activity of HaH allowed using it for preparation of a biosensor for detection of catechol.     

Soil N and C trace gas fluxes and microbial soil N turnover in a sessile oak (Quercus petraea (Matt.) Liebl.) forest in Hungary

During two intensive field campaigns in summer and autumn 2004 nitrogen (N₂O, NO/NO₂) and carbon (CO₂, CH₄) trace gas exchange between soil and the atmosphere was measured in a sessile oak (Quercus petraea (Matt.) Liebl.) forest in Hungary. The climate can be described as continental temperate. Fluxes were measured with a fully automatic measuring system allowing for high temporal resolution. Mean N₂O emission rates were 1.5 μg N m⁻² h⁻¹ in summer and 3.4 μg N m⁻² h⁻¹ in autumn, respectively. Also mean NO emission rates were higher in autumn (8.4 μg N m⁻² h⁻¹) as compared to summer (6.0 μg N m⁻² h⁻¹). However, as NO₂ deposition rates continuously exceeded NO emission rates (−9.7 μg N m⁻² h⁻¹ in summer and −18.3 μg N m⁻² h⁻¹ in autumn), the forest soil always acted as a net NO _x sink. The mean value of CO₂ fluxes showed only little seasonal differences between summer (81.1 mg C m⁻² h⁻¹) and autumn (74.2 mg C m⁻² h⁻¹) measurements, likewise CH₄uptake (summer: −52.6 μg C m⁻² h⁻¹; autumn: −56.5 μg C m⁻² h⁻¹). In addition, the microbial soil processes net/gross N mineralization, net/gross nitrification and heterotrophic soil respiration as well as inorganic soil nitrogen concentrations and N₂O/CH₄ soil air concentrations in different soil depths were determined. The respiratory quotient (ΔCO_{2 resp} ΔO_{2 resp}⁻¹) for the uppermost mineral soil, which is needed for the calculation of gross nitrification via the Barometric Process Separation (BaPS) technique, was 0.8978 ± 0.008. The mean value of gross nitrification rates showed only little seasonal differences between summer (0.99 μg N kg⁻¹ SDW d⁻¹) and autumn measurements (0.89 μg N kg⁻¹ SDW d⁻¹). Gross rates of N mineralization were highest in the organic layer (20.1–137.9 μg N kg⁻¹ SDW d⁻¹) and significantly lower in the uppermost mineral layer (1.3–2.9 μg N kg⁻¹ SDW d⁻¹). Only for the organic layer seasonality in gross N mineralization rates could be demonstrated, with highest mean values in autumn, most likely caused by fresh litter decomposition. Gross mineralization rates of the organic layer were positively correlated with N₂O emissions and negatively correlated with CH₄ uptake, whereas soil CO₂ emissions were positively correlated with heterotrophic respiration in the uppermost mineral soil layer. The most important abiotic factor influencing C and N trace gas fluxes was soil moisture, while the influence of soil temperature on trace gas exchange rates was high only in autumn.     

Rhizotoxicity of aluminate and polycationic aluminium at high pH

Although monomeric Al species are often toxic in acidic soils, the effects of the aluminate ion (Al(OH) ₄ ⁻ ) on roots grown in alkaline media are still unclear. Dilute, alkaline (pH 9.5) nutrient solutions were used to investigate the effects of Al(OH) ₄ ⁻ on root growth of mungbean (Vigna radiata L.). Root growth was reduced by 13% after 3 d growth in solutions with an Al(OH) ₄ ⁻ activity of 16 μM and no detectable polycationic Al (Al₁₃). This decrease in root growth was associated with the formation of lesions on the root tips (due to the rupturing of the epidermal and outer cortical cells) and a slight limitation to root hair growth (particularly on the lateral roots). When roots displaying these symptoms were transferred to fresh Al(OH) ₄ ⁻ solutions for a further 12 h, no root tip lesions were observed and root hair growth on the lateral roots improved. The symptoms were similar to those induced by Al₁₃ at concentrations as low as 0.50 μM Al which are below the detection limit of the ferron method. Thus, Al(OH) ₄ ⁻ is considered to be non-toxic, with the observed reduction in root growth in solutions containing Al(OH) ₄ ⁻ due to the gradual formation of toxic Al₁₃ in the bulk nutrient solution resulting from the acidification of the alkaline nutrient solution by the plant roots.     

Inactivation of the Pore-Forming Toxin Sticholysin I by Peroxynitrite: Protection by Cys Groups Incorporated in the Toxin

Sea anemones synthesize a variety of toxic peptides and proteins of biological interest. The Caribbean Sea anemone Stichodactyla helianthus, produces two pore-forming toxins, Sticholysin I (St I) and Stichloysin II (St II), with the ability to form oligomeric pores in cell and lipid bilayers characteristically lacking cysteine in their amino acid sequences. Recently, two mutants of a recombinant variant of Sticholysin I (rSt I) have been obtained with a Cys residue in functionally relevant regions for the pore-forming activity of the toxin: r St I F15C (in the amino terminal sequence) and r St I R52C (in the binding site). Aiming at characterizing the effects of oxidants in toxins devoid (r St I) or containing –SH moieties (r St I F15C and r St I R52C), we measured their hemolytic activity and pore forming capacity prior and after their incubation with peroxynitrite (ONOO^?). At low ONOO^?/Toxin ratios, nearly 0.8 Trp groups are modified by each added peroxynitrite molecule, and the toxin activity is reduced in ca. 20 %. On the other hand, in –SH bearing mutants only 0.5 Trp groups are modified by each peroxynitrite molecule and the toxin activity is only reduced in 10 %. The results indicated that Cys is the initial target of the oxidative damage and that Trp residues in Cys-containing toxins were less damaged than those in r St I. This relative protection of Trp groups correlates with a smaller loss of hemolytic activity and permeabilization ability in liposomes and emphasizes the relevance of Trp groups in the pore forming capacity of the toxins.     

Biological evaluation and structure activity relationship of 9-methyl-1-phenyl-9H-pyrido[3,4-b]indole derivatives as anti-leishmanial agents

A series of piperazinyl-β-carboline-3-carboxamide derivatives were designed through a molecular hybridization approach. Designed analogues were synthesized, characterized and evaluated for anti-leishmanial activity against Leishmania infantum and Leishmania donovani. In L. infantum inhibition assay, compounds 7d, 7g and 7c displayed potent inhibition of promastigotes (EC₅₀ 1.59, 1.47 and 3.73 µM respectively) and amastigotes (EC₅₀ 1.4, 1.9 and 2.6 µM respectively). SAR studies revealed that, para substitution of methoxy, chloro groups and methyl group on ortho position favored anti-leishmanial activity against L. infantum. Among these analogues 7d, 7h, 7n and 7g exhibited potent inhibition against L. donovani promastigotes (EC₅₀ 0.91, 4.0, 4.57 and 5.02 µM respectively), axenic amastigotes (EC₅₀ 0.9, 3.5, 2.2 and 3.8 µM respectively) and intracellular amastigotes (EC₅₀ 1.3, 7.8, 5.6 and 6.3 µM respectively). SAR studies suggested that, para substitution of methoxy group, para and meta substitution of chloro groups and benzyl replacement recommended for significant anti-leishmanial against L. donovani.     

Computer-augmented modeling studies of Pb(II) and Cd(II) complexes with maleic acid in ethylene glycol–water mixture

Chemical speciation of binary complexes of Pb(II) and Cd(II) ions with maleic acid have been studied pH metrically in the concentration range of 0–50% v/v ethylene glycol (EG)–water mixtures maintaining an ionic strength of 0.16 molL^?1 at 303 K. Alkalimetric titrations were carried out in different relative concentrations of metal and maleic acid. Stability constants of various models of binary complexes were refined with MINIQUAD75. The best-fit chemical models were selected based on statistical parameters and residual analysis. The species detected are ML₂, ML₃, and ML₂H for Pb(II) and Cd(II). The chemical speciation, metal bioavailability, and transportation are explained based on the distribution diagrams.     

Baseline sensitivity and efficacy of thifluzamide in Rhizoctonia solani

Thifluzamide is a SDHI (succinate dehydrogenase inhibitor) fungicide, which interferes with succinate ubiquinone reductase in the mitochondrial electron transport chain of fungi. Presently, jinggangmycin is the major fungicide extensively used for the control of rice sheath blight caused by Rhizoctonia solani and resistance to jinggangmycin was first reported to occur in China. A total of 128 isolates of R. solani from Anhui Province of China were characterised for the baseline sensitivity to thifluzamide. The isolates were very sensitive to thifluzamide and the baseline sensitivity curve was unimodal with an average EC₅₀ value of 0.058 ± 0.012 µg mL^?1. However, EC₅₀ values of boscalid (another SDHI fungicide) for inhibition of mycelial growth of 22 arbitrarily selected R. solani isolates ranged from 1.89 to 2.68 µg mL^?1. Thifluzamide applied at 110 µg mL^?1 exhibited excellent protective and curative activity against rice sheath blight and provided 81.1–91.0% protective or curative control efficacy. In field trials in 2010 and 2011, control efficacies of thifluzamide at 82 g.a.i ha^?1 15 and 30 days after second application were 84.2% and 86.7%, respectively, suggesting excellent activity against sheath blight. There was a statistically significant difference in the efficacy between thifluzamide and boscalid or jinggangmycin. These results suggested that thifluzamide should be a good alternative fungicide to jinggangmycin for the control of rice sheath blight.     

The oxidation of ferrocytochrome c by Br−2, (SCN)−2, N3 and OH radicals studied by pulsed-electron and γ-ray radiolysis

The reactions of ferrocytochrome c with Br^?₂, (SCN)^?₂, N₃ and OH radicals were followed by measuring the change in the optical spectra of cytochrome c on γ-irradiation as well as the rate of change of absorbance upon pulse irradiation.Ferrocytochrome c is oxidized to ferricytochrome c by Br^?₂, (SCN)^?₂ or N₃ radical with an efficiency of about 100% through a second-order process in which no intermediates were observed. The rate constants in neutral solutions at I = 0.073 are 9.7 · 10⁸ M^?1 · s^?1, 7.9 · 10⁸ M^?1 · s^?1, 1.3 · 10⁹ M^?1 · s^?1 for the oxidation by Br^?₂, (SCN)^?₂ and N₃ radicals, respectively. The rate constants do not vary appreciably in alkaline solutions (pH 8.9). The ionic strength dependence was observed for the rate constants of the oxidation by Br^?₂ and (SCN)^?₂. Those rate constants estimated on the assumption that the radicals react only with the amino acid residues with the characteristic steric correction factors were less than one-tenth of the observed ones. These results suggest that the partially exposed region of the heme is the probable site of electron transfer from ferrocytochrome c to the radical.Hydroxyl radicals also oxidize ferrocytochrome c with a high rate constant (k > 1 · 10¹⁰ M^?1 · s^?1), but with a very small efficiency (5%).     

Cu2+ Inhibits Photosystem II Activities but Enhances Photosystem I Quantum Yield of Microcystis aeruginosa

Responses of photosystem I and II activities of Microcystis aeruginosa to various concentrations of Cu²⁺ were simultaneously examined using a Dual-PAM-100 fluorometer. Cell growth and contents of chlorophyll a were significantly inhibited by Cu²⁺. Photosystem II activity [Y(II)] and electron transport [rETR_max(II)] were significantly altered by Cu²⁺. The quantum yield of photosystem II [Y(II)] decreased by 29 % at 100 μg L^?1 Cu²⁺ compared to control. On the contrary, photosystem I was stable under Cu²⁺ stress and showed an obvious increase of quantum yield [Y(I)] and electron transport [rETR_max(I)] due to activation of cyclic electron flow (CEF). Yield of cyclic electron flow [Y(CEF)] was enhanced by 17 % at 100 μg L^?1 Cu²⁺ compared to control. The contribution of linear electron flow to photosystem I [Y(II)/Y(I)] decreased with increasing Cu²⁺ concentration. Yield of cyclic electron flow [Y(CEF)] was negatively correlated with the maximal photosystem II photochemical efficiency (F _v/F _m). In summary, photosystem II was the major target sites of toxicity of Cu²⁺, while photosystem I activity was enhanced under Cu²⁺ stress.     

UFEIII, a fibrinolytic protease from the marine invertebrate, Urechis unicinctus

A new serine protease with fibrinolytic activity from a marine invertebrate, Urechis unicinctus, was purified to electrophoretic homogeneity using column chromatography. SDS-PAGE of the purified enzyme showed a single polypeptide chain with MW ~20.8 kDa. Its N-terminal sequence was IIGGSQAAITSY. The purified enzyme, UFEIII, was stable at pH 6–10 below 60 °C with an optimum pH of 8.5 at approx. 55 °C. The enzyme activity was significantly inhibited by PMSF and SBTI suggesting that it was a serine protease. In fibrin plate assays, UFEIII was contained 1.46 × 10³ U (urokinase units) mg^?1 total fibrinolytic activity, which consisted of 692 U mg^?1 direct fibrinolytic activity and 769 U mg^?1 plasminogen-activator activity. K_m and V_max values for azocasein were 1 mg ml^?1 and 43 μg min^?1 ml^?1, respectively.     

Nitrous oxide and nitric oxide emissions from an irrigated cotton field in Northern China

Cotton is one of the major crops worldwide and delivers fibers to textile industries across the globe. Its cultivation requires high nitrogen (N) input and additionally irrigation, and the combination of both has the potential to trigger high emissions of nitrous oxide (N₂O) and nitric oxide (NO), thereby contributing to rising levels of greenhouse gases in the atmosphere. Using an automated static chamber measuring system, we monitored in high temporal resolution N₂O and NO fluxes in an irrigated cotton field in Northern China, between January 1st and December 31st 2008. Mean daily fluxes varied between 5.8 to 373.0 µg N₂O-N m^?2?h^?1 and ?3.7 to 135.7 µg NO-N m^?2?h^?1, corresponding to an annual emission of 2.6 and 0.8 kg N ha^?1?yr^?1 for N₂O and NO, respectively. The highest emissions of both gases were observed directly after the N fertilization and lasted approximately 1 month. During this time period, the emission was 0.85 and 0.22 kg N ha^?1 for N₂O and NO, respectively, and was responsible for 32.3% and 29.0% of the annual total N₂O and NO loss. Soil temperature, moisture and mineral N content significantly affected the emissions of both gases (p?<?0.01). Direct emission factors were estimated to be 0.95% (N₂O) and 0.24% (NO). We also analyzed the effects of sampling time and frequency on the estimations of annual cumulative N₂O and NO emissions and found that low frequency measurements produced annual estimates which differed widely from those that were based on continuous measurements.