Enhancement of Lanthanum (III) on Sodium Currents in Acutely Isolated Hippocampal CA1 Neurons of Rat

The effects of lanthanum (III) (La³⁺) on voltage-gated sodium channel currents (I _Na) in freshly dissociated rat hippocampal CA1 neurons were studied using the whole-cell patch clamp techniques. La³⁺ reversibly enhanced I _Na in a concentration- and voltage-dependent manner. The 50% enhancement concentration (EC₅₀) of La³⁺ on I _Na was 9.93 μM. In addition, 10 μM La³⁺ shifted the steady state activation curve of I _Na towards positive potential and the steady state inactivation curve towards negative potential without changing the slope factor. These results indicated that La³⁺ could increase the amplitudes of I _Na and change the activation and inactivation courses of I _Na even in very low concentration.     

Characterization of a novel β-thioglucosidase CpTGG1 in Carica papaya and its substrate-dependent and ascorbic acid-independent O-β-glucosidase activity

Plant thioglucosidases are the only known S-glycosidases in the large superfamily of glycosidases.These enzymes evolved more recently and are distributed mainly in Brassicales.Thioglucosidase research has focused mainly on the cruciferous crops due to their economic importance and cancer preventive benefits.In this study,we cloned a novel myrosinase gene,CpTGG1,from Carica papaya Linnaeus.and showed that it was expressed in the aboveground tissues in planta.The recombinant CpTGG1 expressed in Pichia pastoris catalyzed the hydrolysis of both sinigrin and glucotropaeolin(the only thioglucoside present in papaya),showing that CpTGG1 was indeed a functional myrosinase gene.Sequence alignment analysis indicated that CpTGG1 contained all the motifs conserved in functional myrosinases from crucifers,except for two aglycon-binding motifs,suggesting substrate priority variation of the non-cruciferous myrosinases.Using sinigrin as substrate,the apparent Km and Vmax values of recombinant CpTGG1 were 2.82 mM and 59.9 μmol min-1 mg protein-1,respectively.The Kcat IKm value was 23 s-1 mM-1.O-β-glucosidase activity towards a variety of substrates were tested,CpTGG1 displayed substrate-dependent and ascorbic acid-independent O-β-glucosidase activity towards 2-nitrophenyl-βD-glucopyranoside and 4-nitrophenyl-β-D-glucopyranoside,but was inactive towards glucovanillin and n-octyl-β-D-glucopyranoside.Phylogenetic analysis indicated CpTGG1 belongs to the MYR II subfamily of myrosinases.     

Effects of 1-aminocyclopropane-1-carboxylic acid and oxygen concentrations on in vivo and in vitro activity of ACC oxidase of sunflower hypocotyl segments

In vivo ethylene production by hypocotyl segments of sunflower seedlings and in vitro activity of 1-aminocyclopropane-1-carboxylic acid oxidase (formerly ethylene-forming enzyme) extacted from the same tissues increase with increasing concentrations of 1-aminocyclopropane-1-carboxylic acid (ACC) and oxygen. ACC oxidase activity follows Michaelis-Menten kinetics. The apparent Km values of the enzyme towards ACC, estimated in vivo and in vitro, are respectively 219 M and 20.6 M. Both Km values towards O₂ are similar, ca 10.6–11.4%. A decrease in concentration in one of the substrates (ACC or O₂) results in an increase in in vivo apparent Km of ACC oxidase for the other substrate. On the contrary, Km values of the enzyme towards ACC or O₂ estimated in vitro are not dependent upon the concentration of the other substrate (ACC or O₂).Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - MACC malonylate 1-aminocyclopropane-1-carboxylic acid - SD standard deviation     

OXA-162, a novel variant of OXA-48 displays extended hydrolytic activity towards imipenem,meropenem and doripenem

Context: Isolation and characterization of OXA-162, a novel variant of OXA-48.

Objectives: Klebsiella pneumoniae isolate with decreased susceptibility to carbapenems was recovered from a Turkish patient. This study aimed at characterizing the carbapenem resistance determinants of this isolate.

Materials and methods: Antibiotic susceptibility tests, analytic isoelectric focusing (IEF), cloning and sequencing were performed. Cloned β-lactamase was purified by means of preparative gel electrophoresis and the kinetic constants were determined under initial rate conditions.

Results: The identified bla_OXA-162 gene was located on a ca. 45-kb plasmid carrying a transposon consisted of two IS1999-2 elements. OXA-162 differed from OXA-48 by a single amino acid substitution (Thr213Ala) which increased the catalytic efficiency (k_cat/K_M) of OXA-162 towards imipenem and meropenem. Also this substitution caused a gain of hydrolysis ability towards doripenem. Analysis of OXA-162 model implied that the amino acid change might generate an extension in the opening of the substrate entry site and might cause extended hydrolytic activity towards imipenem, meropenem and doripenem.

Discussion and conclusion: OXA-162, a derivative of OXA-48 has enhanced catalytic properties.     

Synthesis,carbonic anhydrase I and II inhibition studies of the 1,3,5-trisubstituted-pyrazolines

4-(3-(4-Substituted-phenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl) benzenesulfonamides (9–16) were successfully synthesized and their chemical structures were confirmed by ¹H NMR, ¹³C NMR, and HRMS spectra. Carbonic anhydrase I and II inhibitory effects of the compounds were investigated. K_i values of the compounds were in the range of 316.7?±?9.6–533.1?±?187.8?nM towards hCA I and 412.5?±?115.4–624.6?±?168.2?nM towards hCA II isoenzymes. While K_i values of the reference compound Acetazolamide were 278.8?±?44.3?nM and 293.4?±?46.4?nM towards hCA I and hCA II izoenzymes, respectively. Compound 14 with bromine and compound 13 with fluorine substituents can be considered as the leader compounds of the series because of the lowest K_i values in series to make further detailed carbonic anhydrase inhibiton studies.     

Mathematically combined half-cell reduction potentials of low-molecular-weight thiols as markers of seed ageing

Abstract

The half-cell reduction potential of the glutathione disulphide (GSSG)/glutathione (GSH) redox couple appears to correlate with cell viability and has been proposed to be a marker of seed viability and ageing. This study investigated the relationship between seed viability and the individual half-cell reduction potentials (E_is) of four low-molecular-weight (LMW) thiols in Lathyrus pratensis seeds subjected to artificial ageing: GSH, cysteine (Cys), cysteinyl-glycine (Cys-Gly) and γ-glutamyl-cysteine (γ-Glu-Cys). The standard redox potential of γ-Glu-Cys was previously unknown and was experimentally determined. The E_is were mathematically combined to define a LMW thiol-disulphide based redox environment (E_{thiol-disulphide}). Loss of seed viability correlated with a shift in E_{thiol-disulphide} towards more positive values, with a LD₅₀ value of ?0.90 ± 0.093 mV M (mean ± SD). The mathematical definition of E_{thiol-disulphide} is envisaged as a step towards the definition of the overall cellular redox environment, which will need to include all known redox-couples.     

Cytology of F1 hybrids and chromosome number of F2 and BC1 progeny of the cross Bromus riparius x B. inermis

Summary Hybrids between B. inermis Leyss (2n=8x=56) and B. riparius Rehm. (2n=10x=70) were easily made. The F₁ hybrids had a fertility of 20%–50% under open pollination and backcrossing to B. inermis. Chromosome pairing in B. riparius was predominantly as bivalents (29.04–33.85 per cell for plant means). Bivalents also predominated in the F₁ hybrid (2n=9x=63) and there was a high level of pairing with no reduction in chiasma frequency. It was impossible to estimate the frequency of auto-versus allosyndetic pairing. Chromosome pairing in a hybrid between B. arvensis (2n=2x=14) and B. riparius confirmed that the B. riparius complement is capable of complete autosyndetic pairing. Chromosome numbers in the F₂ progeny ranged from 2n=56 to 72 but they were skewed towards 2n=63 to 70. Backcrosses ranged from 2n=56 to 63, as expected, with the distribution skewed towards 2n=56. Selection towards the 2n=56 level would be difficult in the F₂. Empirical observation suggested that cytoplasm had a major influence on morphology in the backcrosses. Additional studies are required to determine the best breeding scheme to introgress germ plasm between B. inermis and B. riparius.     

Eine verbesserte Extraktionsmethode für den elektronenmikroskopischen Nachweis von Pflanzenviren: (Kurze Mitteilung)

Spring barley cultivar ‘Carina’ was classed as tolerant, cv. ‘Ultra’ as vulnerable towards infestation with Rhopalosiphum padi. These differences in tolerance did, however, not exsist towards infestation with Metopolophium dirhodum. Faba bean cv. ‘Apollo’ was classed as tolerant and ‘Albatross’ as vulnerable towards Aphis fabae.

Tolerance was correlated with lower assimilate deprivation by Rhopalosiphum padi and less decreased CO₂‐assimilation rates. After infestation with Metopolophium dirhodum CO₂‐assimilation rates were not influenced in cultivar specific ways at equal rates of assimilate deprivations.

Also, Aphis fabae deprivated more assimilates on the vulnerable cv. ‘Albatross’ in comparison to the tolerant cv. ‘Apollo’. Simultaneously, for the tolerant cultivar the supply of inflorescences with assimilates after the infestation was even better than for none infested one. This surely contributed to a stabilization of yield in the tolerant cultivar.     

Redox chemistry of low-pH forms of tetrahemic cytochrome c3

Desulfovibrio vulgaris Hildenborough cytochrome c₃ contains four hemes in a low-spin state with bis-histidinyl coordination. High-spin forms of cytochrome c₃ can be generated by protonation of the axial ligands in order to probe spin equilibrium (low-spin/high-spin). The spin alterations occurring at acid pH, the associated changes in redox potentials, as well as the reactivity towards external ligands were followed by the conjunction of square wave voltammetry and UV–visible, CD, NMR and EPR spectroscopies. These processes may be used for modelling the action of enzymes that use spin equilibrium to promote enzyme activity and reactivity towards small molecules.     

Partial purification and some properties of lipoxygenase from Saccharomyces cerevisiae

A partially purified lipoxygenase extract was obtained from the yeast Saccharomyces cerevisiae by precipitation with solid (NH₄)SO₄ at 20% to 80% saturation. The enzyme had two pH optima, at pH 8.0 and 10.0, with respective apparent K _m values of 13 and 9.5 m. At both pH optima, the lipoxygenase demonstrated highest substrate specificity towards linoleic acid, followed by linolenic acid; although the enzyme had less specificity towards mono-linolein than di-linolein at pH 8.0, the reverse was true at pH 10.0.     

Kinetic study of monophenol and o-diphenol binding to oxytyrosinase

The complex reaction mechanism of tyrosinase involves three enzymatic forms, two overlapping catalytic cycles and a dead-end complex. The deoxytyrosinase form binds oxygen with a high degree of affinity, μM. The mettyrosinase and oxytyrosinase forms bind monophenols and o-diphenols, although the former is inactive on monophenols. Analytical expressions for the catalytic and Michaelis constants of tyrosinase towards phenols and o-diphenols have been derived. Thus, the Michaelis constant of tyrosinase towards monophenols and o-diphenols are related with the catalytic constants for monophenols and o-diphenols , respectively, and with the binding rate constants of the oxytyrosinase form with these substrates (k₊₄ and k₊₆, respectively), by means of the expressions and . From these expressions, we calculate the values of the binding rate constant of oxytyrosinase to the substrates (monophenols and o-diphenols) for tyrosinases from different biological sources, and reveal that the o-diphenols bind more rapidly to oxytyrosinase than the monophenols. In addition, a new kinetic constant (the Michaelis constant for o-diphenol in the monophenolase activity), is derived and determined. Thus, it has been shown that tyrosinase has apparently higher affinity towards o-diphenols in its monophenolase than in its diphenolase activity.     

Kinetic Modeling of Amino Acid Oxidation Catalyzed by a New D‐Amino Acid Oxidase from Arthrobacter protophormiae

Amino acid oxidases, which enantiospecifically catalyze the oxidative deamination of either D‐ or L‐amino acids, belong to the class of oxidoreductases functioning with a tightly bound cofactor. This cofactor favors industrial applications of D‐amino acid oxidases (D‐AAO). Hence, the enzyme is very important for the industrial application in the purification and determination of certain amino acids. In developing the enzyme‐catalyzed reaction for large‐scale production, modeling of the reaction kinetics plays an important role. Therefore, the subject of this study was the kinetics of the oxidative deamination, a very complex reaction system, which is catalyzed by D‐AAO from Arthrobacter protophormiae using its natural substrate D‐methionine and the aromatic amino acid 3,4‐dihydroxyphenyl‐D‐alanine (D‐DOPA). The kinetic parameters determined by the measurement of the initial rate and nonlinear regression were verified in batch reactor experiments by comparing calculated and experimental concentration‐time curves. It was found that the enzyme is highly specific towards D‐methionine (K_m = 0.24 mM) and not as specific to D‐DOPA as a substrate (K_m = 9.33 mM). The enzyme activity towards D‐methionine ( = 3.01 U/mL) was approx. seven times higher than towards D‐DOPA ( = 20.01 U/mL). The enzyme exhibited no activity towards L‐methionine and L‐DOPA. Batch and repetitive batch experiments were performed with both substrates in the presence and in the absence of catalase for hydrogen peroxide decomposition. Their comparison made it possible to conclude that hydrogen peroxide has no negative influence on the enzyme activity.     

Purification and characterization of the plasmodial phosphatase that hydrolyses the phosphorylated light chain of Physarum myosin II from Physarum polycephalum

A phosphatase was purified through a combination of ion‐exchange and hydrophobic chromatography followed by native PAGE from Physarum plasmodia. Recently, we demonstrated that this phosphatase isoform has a hydrolytic activity towards the PMLC (phosphorylated light chain of Physarum myosin II) at pH 7.6. The apparent molecular mass of the purified enzyme was estimated at approximately 50 kDa by means of analytical gel filtration. The enzyme was purified 340‐fold to a final phosphatase activity of 400 pkat/mg of protein. Among the phosphorylated compounds tested for hydrolytic activity at pH 7.6, the enzyme showed no activity towards nucleotides. At pH 7.6, hydrolytic activity of the enzyme against PMLC was detected; at pH 5.0, however, no hydrolytic activity towards PMLC was observed. The K _m of the enzyme for PMLC was 10 μM, and the V _max was 1.17 nkat/mg of protein. Ca²⁺ (10 μM) inhibited the activity of the enzyme, and Mg²⁺ (8.5 μM) activated the dephosphorylation of PMLC. Mn²⁺ (1.6 μM) highly stimulated the enzyme's activity. Based on these results, we concluded that the enzyme is likely to be a phosphatase with hydrolytic activity towards PMLC.     

Interactions between Abscisic Acid, Gibberellins and Cytokinins in Grand Rapids Lettuce Seeds Germination

Experiments with Grand Rapids lettuce seeds (Lactuca sativa L.) maintained in darkness or irradiated with red light have shown that the inhibition of germination induced by low concentrations of ABA (2, 4, 6 μM) could be overcome by gibberellins (GA₃ or GA₄). The same results were obtained, although to a lesser extent, under the influence of two out of the four cytokinins tested (K and BAP) for seeds maintained in darkness. To suppress the block induced by higher concentrations of ABA (for example 8 μM), it was necessary to apply a cytokinin (K, BAP, Z or 2iP) and a gibberellin (GA₄ or GA₃) simultaneously, or a cytokinin following a red light treatment. Experiments conducted in darkness in which ABA (8 μM) was applied together with a cytokinin (BAP) and a gibberellin (GA₄) showed that the gibberellin and the cytokinin played similar roles towards each other and towards ABA.     

Effect of lactoferrin on oxidative features of ceruloplasmin

In our previous report we first described a complex between lactoferrin (Lf) and ceruloplasmin (Cp) with K _d ~ 1.8 μM. The presence of this complex in colostrum that never contains more than 0.3 μM Cp questions the reliability of K _d value. We carefully studied Lf binding to Cp and investigated the enzymatic activity of the latter in the presence of Lf, which allowed obtaining a new value for K _d of Cp–Lf complex. Lf interacting with Cp changes its oxidizing activity with various substrates, such as Fe²⁺, o-dianisidine (o-DA), p-phenylenediamine (p-PD) and dihydroxyphenylalanine (DOPA). The presence of at least two binding sites for Lf in Cp molecule is deduced from comparison of substrates’ oxidation kinetics with and without Lf. When Lf binds to the first site affinity of Cp to Fe²⁺ and to o-DA increases, but it decreases towards DOPA and remains unchanged towards p-PD. Oxidation rate of Fe²⁺ grows, while that of o-DA, p-PD and DOPA goes down. Subsequent Lf binding to the second center has no effect on iron oxidation, hampers DOPA and o-DA oxidation, and reduces affinity towards p-PD. Scatchard plot for Lf sorbing to Cp-Sepharose allowed estimating K _d for Lf binding to high-affinity (~13.4 nM) and low-affinity (~211 nM) sites. The observed effect of Lf on ferroxidase activity of Cp is likely to have physiological implications.     

Production and characterization of a cold-active and n-hexane activated lipase from a newly isolated Serratia grimesii RB06-22

Abstract

A lipase-producing bacterium isolated from raw milk was identified as Serratia grimesii based on 16S rRNA sequence analysis. The extracellular lipase was partially purified by ammonium sulfate precipitation and ultrafiltration. Maximal activity was observed at 10°C, the optimum pH was 8.0 and the enzyme was stable at 5–30°C for 1 h. The K_m and V_max values were 1.7 mM and 0.3 mM/min respectively. It was found that the lipase had the highest hydrolytic activity towards sunflower oil and soybean oil. CaCl₂ had a stimulatory effect on lipase activity, while EDTA and iodoacetic acid slightly inhibited the lipase activity and the enzyme was strongly inhibited by PMSF. The enzyme was compatible with various non-ionic surfactants as well as sodium cholate and saponin. In addition, the enzyme was relatively stable towards oxidizing agents. This lipase exhibited maximum activity in 35% n-hexane retaining about 2191% activity for 1 h.     

Effect of some Cardiac and Respiratory Drugs on Succinate-Cytochrome c Reductase

Succinate-cytochrome c reductase was inhibited in vitro and in vivo by phenobarbitone, aminophylline and neostigmine using both 2,6-dichlorophenolindophenol (DCIP) and cytochrome c (cyt c) as substrates. The enzyme was also activated by gallamine towards both substrates. In vitro, phenobarbitone and aminophylline inhibited the enzyme with respect to the reduction of DCIP and cyt c in a non-competitive manner with K_i values of 1.5 × 10^?5 and 5.7 × 10^?5 M, respectively. Moreover, neostigmine competitively inhibited the enzyme towards both substrates with K_i values of 1.36 × 10^?5 and 1.50 × 10^?5 M, respectively.     

Genetic composition of a population of natural common bream Abramis brama × roach Rutilus rutilus hybrids and their morphological characteristics in comparison with parent species

Common bream Abramis brama , roach Rutilus rutilus and their hybrids were collected in the Dobczyce Reservoir in southern Poland in 2006–2013 to study whether it is better for a hybrid individual to resemble and compete with one of its parents, or to minimize competition by having a distinctive phenotype. All hybrids were F₁ crosses and originated predominantly (93·2%) from matings between female A. brama and male R. rutilus parents. In morphometric analyses, a newly defined coefficient, L ₃ = 2·5 (body mass) (L _S × body depth)^?1, which enables forms with similar length–depth proportions but different length–mass relationships to be distinguished was used. Morphometric and meristic characteristics of the hybrids were intermediate in comparison with the parental species, with small but significant deviation towards R. rutilus in longitudinal body dimensions (trunk and tail length) and towards A. brama in body cross‐sectional shape (body depth and L ₃ coefficient). This may result in a more R. rutilus like propulsion in hybrids and a more A. brama like ability to manoeuvre.