Heterologous expression,refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53 °C ± 0.53 °C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34 °C ± 0.25 °C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.     

Characterization of an antifreeze protein from the polar diatom Fragilariopsis cylindrus and its relevance in sea ice

Antifreeze proteins (AFPs), characterized by their ability to separate the melting and growth temperatures of ice and to inhibit ice recrystallization, play an important role in cold adaptation of several polar and cold-tolerant organisms. Recently, a multigene family of AFP genes was found in the diatom Fragilariopsis cylindrus, a dominant species within polar sea ice assemblages. This study presents the AFP from F. cylindrus set in a molecular and crystallographic frame. Differential protein expression after exposure of the diatoms to environmentally relevant conditions underlined the importance of certain AFP isoforms in response to cold. Analyses of the recombinant AFP showed freezing point depression comparable to the activity of other moderate AFPs and further enhanced by salt (up to 0.9 °C in low salinity buffer, 2.5 °C at high salinity). However, unlike other moderate AFPs, its fastest growth direction is perpendicular to the c-axis. The protein also caused strong inhibition of recrystallization at concentrations of 1.2 and 0.12 μM at low and high salinity, respectively. Observations of crystal habit modifications and pitting activity suggested binding of AFPs to multiple faces of the ice crystals. Further analyses showed striations caused by AFPs, interpreted as inclusion in the ice. We suggest that the influence on ice microstructure is the main characteristic of these AFPs in sea ice.     

Combined effect of temperature and zinc on Caenorhabditis elegans wild type and daf-21 mutant strains

Heavy metal pollution in aquatic ecosystems is a far reaching environmental problem. The possible influences of heavy metal exposure and the potential harm to organisms when combined with other environmental stressors such as temperature have been largely unexplored. An aquatic toxicity test of Caenorhabditis elegans was performed to estimate the 24 h median lethal concentration (LC₅₀) of different zinc concentrations at different temperatures (15 °C, 20 °C, 25 °C, and 30 °C). We also examined the time course thermotolerance on wild type (N2) and daf-21 null (JT6130) adults exposed to 6.1 mM zinc at 37 °C. Hsp90 protein expression level in response to the combined effect of temperature and zinc toxicity was also investigated by both Western blots and ELISA. Our results show that C. elegans wild type nematodes exhibit severe lethal toxicity after a 24 h exposure to zinc at higher temperatures. In addition, the expression level of Hsp90 was highly inhibited in adult worms subjected to zinc stress. This toxicity assay at different temperatures provides insight into organism response to combined effects of temperature and zinc toxicity.     

Effect of the timing of ice slurry ingestion for precooling on endurance exercise capacity in a warm environment

It has been demonstrated that precooling with ice slurry ingestion enhances endurance exercise capacity in the heat. However, no studies have yet evaluated the optimal timing of ice slurry ingestion for precooling. This study aimed to investigate the effects of varying the timing of ice slurry ingestion for precooling on endurance exercise capacity in a warm environment. Ten active male participants completed 3 experimental cycling trials to exhaustion at 55% peak power output (PPO) after 15 min of warm-up at 30% PPO at 30 °C and 80% relative humidity. Three experimental conditions were set: no ice slurry ingestion (CON), pre-warm-up ice slurry ingestion (−1 °C; 7.5 g kg⁻¹) (PRE), and post-warm-up ice slurry ingestion (POST). Rectal and mean skin temperatures at the beginning of exercise in the POST condition (37.1±0.2 °C, 33.8±0.9 °C, respectively) were lower than those in the CON (37.5±0.3 °C; P<0.001, 34.8±0.8 °C; P<0.01, respectively) and PRE (37.4±0.2 °C; P<0.01, 34.6±0.7 °C; P<0.01, respectively) conditions. These reductions increased heat storage capacity and resulted in improved exercise capacity in the POST condition (60.2±8.7 min) compared to that in the CON (52.0±11.9 min; effect size [ES]=0.78) and PRE (56.9±10.4 min; ES=0.34) conditions. Ice slurry ingestion after warm-up effectively reduced both rectal and skin temperatures and increased cycling time to exhaustion in a warm environment. Timing ice slurry ingestion to occur after warm-up may be effective for precooling in a warm environment.     

High-level production of erythritol by mutants of osmophilic Moniliella sp.

An erythritol-producing osmophilic yeast-like fungus, Moniliella sp. 440, was isolated from honey and then successively mutated with iterative rounds of N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment and selection. Six generations of mutants, named N12115-6, N21105-6, N31074-3, N42208-2, N53199-9, and N61188-12, were selected for and produced erythritol at 151.0, 157.2, 177.8, 191.4, 196.6, and 237.8 g/L, respectively, while the wild type strain produced 113.0 g/L erythritol in media containing 40% glucose and 1% yeast extract. The mutant cells were found to have a short rod-like shape, while the wild type cells have a long rod-like shape. The most efficient erythritol producer, N61188-12, assimilated myo-inositol and weakly assimilated erythritol. However, the wild type strain did not assimilate myo-inositol and assimilated erythritol well. In 250-L and 2000-L pilot-scale fermentors, the erythritol production by N61188-12 was 151.4 g/L and 152.4 g/L, respectively. A simple fed-batch culture of strain N61188-12 in a 2000-L fermentor increased erythritol production to 189.4 g/L after 10 days fermentation.     

Relationship of Prostate -Specific Antigen to Age and Testosterone in Men With Type 2 Diabetes Mellitus

ObjectiveTo determine whether prostate-specific antigen (PSA) concentrations in type 2 diabetic men with hypogonadotrophic hypogonadism are lower than those in eugonadal men with type 2 diabetes and whether PSA concentrations are related to plasma testosterone concentrations.MethodsIn this cross-sectional study, we measured serum total testosterone, sex hormone–binding globulin, free testosterone, PSA, hematocrit, and hemoglobin A_1c in consecutive type 2 diabetic men who presented to 2 endocrinology referral centers between January 2006 and January 2007. We collected other clinical and demographic data including age, height, weight, and ethnicity.ResultsOf 400 eligible patients, 280 men met inclusion criteria. Plasma PSA concentrations were lower in type 2 diabetic men with low free testosterone concentrations than in those with normal free testosterone concentrations (25.65 ± 2.02 ng/dL vs 31.70 ± 2.31 ng/dL, P = .011). PSA concentrations were positively related to age (r = 0.34, P < .001), total testosterone (r = 0.29, P < .001), free testosterone (r = 0.17, P = .02), and sex hormone– binding globulin (r = 0.22, P < .001) and negatively related to body mass index (r = –0.28, P < .001). In stepwise backward regression analysis, PSA concentration was predicted by age (P < .001) and free testosterone (P < .001), but not by body mass index or sex hormone–binding globulin.ConclusionsPlasma PSA concentrations are lower in type 2 diabetic men with hypogonadism than in eugonadal men with type 2 diabetes, and plasma PSA is related to age, plasma total testosterone concentrations, and free testosterone concentrations in patients with type 2 diabetes. (Endocr Pract. 2008;14:1000-1005)     

Heme interacts with histidine- and tyrosine-based protein motifs and inhibits enzymatic activity of chloramphenicol acetyltransferase from Escherichia coli

BackgroundThe occurrence of free organismal heme can either contribute to serious diseases or beneficially regulate important physiological processes. Research on transient binding to heme-regulatory motifs (HRMs) in proteins resulted in the discovery of numerous Cys-based, especially Cys-Pro (CP)-based motifs. However, the number of His- and Tyr-based protein representatives is comparatively low so far, which is in part caused by a lack of information regarding recognition and binding requirements.MethodsTo understand transient heme association with such motifs on the molecular level, we analyzed a set of 44 His- and Tyr-based peptides using UV–vis, resonance Raman, cw-EPR and 2D NMR spectroscopy.ResultsWe observed similarities with Cys-based sequences with respect to their spectral behavior and complex geometries. However, significant differences regarding heme-binding affinities and sequence requirements were also found. Compared to Cys-based peptides and proteins all sequences investigated structurally display increased flexibility already in the free-state, which is also maintained upon heme association. The acquired knowledge allowed for identification and prediction of a His-based HRM in chloramphenicol acetyltransferase from Escherichia coli as potential heme-regulated protein. The enzyme's heme-interacting capability was studied, and revealed an inhibitory effect of heme on the protein activity with an IC₅₀ value of 57.69 ± 4.37 μM.ConclusionsIt was found that heme inhibits a bacterial protein carrying a potential His-based HRM. This finding brings microbial proteins more into focus of regulation by free heme.General significanceUnderstanding transient binding and regulatory action of heme with bacterial proteins, being crucial for survival, might promote new strategies for the treatment of bacterial infections.     

A new strategy for strain improvement of Aurantiochytrium sp. based on heavy-ions mutagenesis and synergistic effects of cold stress and inhibitors of enoyl-ACP reductase

Developing a strain with high docosahexaenoic acid (DHA) yield and stable fermenting-performance is an imperative way to improve DHA production using Aurantiochytrium sp., a microorganism with two fatty acid synthesis pathways: polyketide synthase (PKS) pathway and Type I fatty acid synthase (FAS) pathway. This study investigated the growth and metabolism response of Aurantiochytrium sp. CGMCC 6208 to two inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan), and proposed a method of screening high DHA yield Aurantiochytrium sp. strains with heavy ion mutagenesis and pre-selection by synergistic usage of cold stress (4 °C) and FAS inhibitors (triclosan and isoniazid). Results showed that (1) isoniazid and triclosan have positive effects on improving DHA level of cells; (2) mutants from irradiation dosage of 120 Gy yielded more DHA compared with cells from 40 Gy, 80 Gy treatment and wild type; (3) DHA contents of mutants pre-selected by inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan)at 4 °C, were significantly higher than that of wild type; (4) compared to the wild type, the DHA productivity and yield of a mutant (T-99) obtained from Aurantiochytrium sp. CGMCC 6208 by the proposed method increased by 50% from 0.18 to 0.27 g/Lh and 30% from 21 to 27 g/L, respectively. In conclusion, this study developed a feasible method to screen Aurantiochytrium sp. with high DHA yield by a combination of heavy-ion mutagenesis and mutant-preselection by FAS inhibitors and cold stress.     

Hyperuricemia influences tryptophan metabolism via inhibition of multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP)

Hyperuricemia is related to a variety of pathologies, including chronic kidney disease (CKD). However, the pathophysiological mechanisms underlying disease development are not yet fully elucidated. Here, we studied the effect of hyperuricemia on tryptophan metabolism and the potential role herein of two important uric acid efflux transporters, multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP). Hyperuricemia was induced in mice by treatment with the uricase inhibitor oxonic acid, confirmed by the presence of urate crystals in the urine of treated animals. A transport assay, using membrane vesicles of cells overexpressing the transporters, revealed that uric acid inhibited substrate-specific transport by BCRP at clinically relevant concentrations (calculated IC₅₀ value: 365 ± 13 μM), as was previously reported for MRP4. Moreover, we identified kynurenic acid as a novel substrate for MRP4 and BCRP. This finding was corroborated by increased plasma levels of kynurenic acid observed in Mrp4^?/? (107 ± 19 nM; P = 0.145) and Bcrp^?/? mice (133 ± 10 nM; P = 0.0007) compared to wild type animals (71 ± 11 nM). Hyperuricemia was associated with > 1.5 fold increase in plasma kynurenine levels in all strains. Moreover, hyperuricemia led to elevated plasma kynurenic acid levels (128 ± 13 nM, P = 0.005) in wild type mice but did not further increase kynurenic acid levels in knockout mice. Based on our results, we postulate that elevated uric acid levels hamper MRP4 and BCRP functioning, thereby promoting the retention of other potentially toxic substrates, including kynurenic acid, which could contribute to the development of CKD.     

Stabilization of Escherichia coli uridine phosphorylase by evolution and immobilization

Mutation and immobilization techniques were applied to uridine phosphorylase (UP) from Escherichia coli in order to enhance its thermal stability and hence productivity in a biocatalytic reaction. UP was evolved by iterative saturation mutagenesis. Compared to the wild type enzyme, which had a temperature optimum of 40 °C and a half-life of 9.89 h at 60 °C, the selected mutant had a temperature optimum of 60 °C and a half-life of 17.3 h at 60 °C. Self-immobilization of the native UP as a Spherezyme showed a 3.3 fold increase in thermostability while immobilized mutant enzyme showed a 4.4 fold increase in thermostability when compared to native UP. Combining UP with the purine nucleoside phosphorylase from Bacillus halodurans allows for synthesis of 5-methyluridine (a pharmaceutical intermediate) from guanosine and thymine in a one-pot transglycosylation reaction. Replacing the wild type UP with the mutant allowed for an increase in reaction temperature to 65 °C and increased the reaction productivity from 10 to 31 g l⁻¹ h⁻¹.     

Impaired trafficking of the very low density lipoprotein receptor caused by missense mutations associated with dysequilibrium syndrome

Dysequilibrium syndrome (DES, OMIM 224050) is a genetically heterogeneous condition that combines autosomal recessive non-progressive cerebellar ataxia with mental retardation. The subclass dysequilibrium syndrome type 1 (CAMRQ1) has been attributed to mutations in the VLDLR gene encoding the very low density lipoprotein receptor (VLDLR). This receptor is involved in the Reelin signaling pathway that guides neuronal migration in the cerebral cortex and cerebellum. Three missense mutations (c.1459G > T; p.D487Y, c.1561G > C; p.D521H and c.2117G > T; p.C706F) have been previously identified in VLDLR gene in patients with DES. However, the functional implications of those mutations are not known and therefore we undertook detailed functional analysis to elucidate the cellular mechanisms underlying their pathogenicity. The mutations have been generated by site-directed mutagenesis and then expressed in cultured cell lines. Confocal microscopy and biochemical analysis have been employed to examine the subcellular localization and functional activities of the mutated proteins relative to wild type. Our results indicate that the three missense mutations lead to defective intracellular trafficking and ER retention of the mutant VLDLR protein. This trafficking impairment prevents the mutants from reaching the plasma membrane and binding exogenous Reelin, the initiating event in Reelin signaling. Collectively, our results provide evidence that ER quality control is involved in the functional inactivation and underlying pathogenicity of these DES-associated mutations in the VLDLR.     

Preliminary investigations into triazole derived androgen receptor antagonists

A range of 1,4-substituted-1,2,3-N-phenyltriazoles were synthesized and evaluated as non-steroidal androgen receptor (AR) antagonists. The motivation for this study was to replace the N-phenyl amide portion of small molecule antiandrogens with a 1,2,3-triazole and determine effects, if any, on biological activity. The synthetic methodology presented herein is robust, high yielding and extremely rapid. Using this methodology a series of 17 N-aryl triazoles were synthesized from commercially available starting materials in less than 3 h. After preliminary biological screening at 20 and 40 μM, the most promising three compounds were found to display IC₅₀ values of 40–50 μM against androgen dependent (LNCaP) cells and serve as a starting point for further structure–activity investigations. All compounds in this work were the focus of an in silico study to dock the compounds into the human androgen receptor ligand binding domain (hARLBD) and compare their predicted binding affinity with known antiandrogens. A comparison of receptor–ligand interactions for the wild type and T877A mutant AR revealed two novel polar interactions. One with Q738 of the wild type site and the second with the mutated A877 residue.     

Control of wild oat (Avena fatua) using some phenolic compounds I – Germination and some growth parameters

The percentage of germination of wild oat was significantly inhibited by increasing the concentrations of phenolic compounds. Ferulic acid was the most effective compound which completely inhibited germination at a concentration of 3.0 mM. At the same time, wheat and barley were slightly affected with different concentrations of the four phenolic compounds. The percentage of germination of wheat significantly decreased with increasing of ferulic acid reaching a maximum inhibition at 3.0 mM concentration. On the other hand, the germination of wheat was not affected with the other three phenolic compounds. The percentage of germination of barley was not affected with all phenolic compounds except for hydroxy phenolic acetic acid which has significant effect at a concentration of 3.0 mM. Salicylic acid significantly inhibited the growth parameters gradually in wild oat, wheat and barley. The shoot/root ratio was decreased in wild oat and barley, while the ratio increased in wheat. The growth parameters were completely inhibited at 3.0 mM of ferulic acid for both wild oat and wheat but slightly inhibited for barley. The shoot/root ratio was increased in all concentrations of ferulic acid except at 3.0 mM which was completely inhibited for both wild oat and wheat, while the ratio was increased in all treatments of ferulic acid in the case of barley. The growth parameters were highly significant and decreased in wild oat, wheat and barley with increasing the concentrations of hydroxybenzoic acid and hydroxyphenyl acetic acid. The shoot/root ratio was not changed in all concentrations except at 3.0 mM in the case of wild oat, the ratio was decreased at 2.0 and 3.0 mM in the case of wheat, while the ratio increased in most of hydroxybenzoic acid concentrations in the case of barley. The shoot/root ratio was increased with increasing of the hydroxyphenyl acetic acid concentrations.     

Overexpression of Escherichia coli nucleotide excision repair genes after cisplatin-induced damage

Cisplatin is currently used in tumor chemotherapy to induce the death of malignant cells through blockage of DNA replication. It is a commonly used chemotherapeutic agent binding mono- or bifunctionally to guanines in DNA. Escherichia coli K12 mutant strains deficient in nucleotide excision repair (NER) were submitted to increasing concentrations of cisplatin, and the results revealed that uvrA and uvrB mutants are sensitive to this agent, while uvrC and cho mutants remain as the wild type strain. The time required for both gene expression turn-off and return to normal weight DNA in wild-type E. coli was not accomplished even after 4 h post-treatment with cisplatin, while the same process takes place within 1.5 h after ultraviolet radiation (UV). Besides, a heavily damaging action of cisplatin can be seen not only by persistent nicks on genomic DNA, but also by NER gene expression exceeding manifold that seen after equivalent lethal doses of UV. Moreover, cisplatin caused an increase in uvrB gene expression from its putative upstream promoter P3 in an SOS-independent manner.     

Synthesis and Characterization of Time-resolved Fluorescence Probes for Evaluation of Competitive Binding to Melanocortin Receptors

Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining K_d values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited K_d values of 27 ± 3.9 nM and 4.2 ± 0.48 nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The K_i values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the K_i values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported.     

Response of native grasses and Cicer arietinum to soil polluted with mining wastes: Implications for the management of land adjacent to mine sites

Mine tailings are an environmental problem in Southern Spain because wind and water erosion of bare surfaces results in the dispersal of toxic metals over nearby urban or agricultural areas. Revegetation with tolerant native species may reduce this risk. We grew two grasses, Lygeum spartum and Piptatherum miliaceum, and the crop species Cicer arietinum (chickpea) under controlled conditions in pots containing a mine tailings mixed into non-polluted soil to give treatments of 0%, 25%, 50%, 75% and 100% mine tailings. We tested a neutral (pH 7.4) mine tailings which contained high concentrations of Cd, Cu, Pb and Zn. Water-extractable metal concentrations increased in proportion to the amount of tailings added. The biomass of the two grasses decreased in proportion to the rate of neutral mine-tailing addition, while the biomass of C. arietinum only decreased in relation to the control treatment. Neutron radiography revealed that root development of C. arietinum was perturbed in soil amended with the neutral tailings compared to those of the control treatment, despite a lack of toxicity symptoms in the shoots. In all treatments and for all metals, the plants accumulated higher concentrations in the roots than in shoots. The highest concentrations occurred in the roots of P. miliaceum (2500 mg kg^?1 Pb, 146 mg kg^?1 Cd, 185 mg kg^?1 Cu, 2700 mg kg^?1 Zn). C. arietinum seeds had normal concentrations of Zn (70–90 mg kg^?1) and Cu (6–9 mg kg^?1). However, the Cd concentration in this species was ～1 mg kg^?1 in the seeds and 14.5 mg kg^?1 in shoots. Consumption of these plant species by cattle and wild fauna may present a risk of toxic metals entering the food chain.     

The PprA protein is required for accurate cell division of γ-irradiated Deinococcus radiodurans bacteria

Deinococcus radiodurans, one of the most radioresistant organisms known to date is able to reconstruct an intact genome from hundreds of DNA fragments. Here, we investigate the in vivo role of PprA, a radiation-induced Deinococcus specific protein. We report that DNA double strand break repair in cells devoid of PprA and exposed to 3800 Gy γ-irradiation takes place efficiently with a delay of only 1 h as compared to the wild type, whereas massive DNA synthesis begins 90 min after irradiation as in the wild type, a phenotype insufficient to explain the severe radiosensitivity of the ΔpprA mutant. We show that the slow kinetics of reassembly of DNA fragments in a ΔpprA ΔrecA double mutant was the same as that observed in a ΔrecA single mutant demonstrating that PprA does not play a major role in DNA repair through RecA-independent pathways. Using a tagged PprA protein and immunofluorescence microscopy, we show that PprA is recruited onto the nucleoid after γ-irradiation before DNA double strand break repair completion, and then is found as a thread across the septum in dividing cells. Moreover, whereas untreated cells devoid of PprA displayed a wild type morphology, they showed a characteristic cell division abnormality after irradiation not found in other radiosensitive mutants committed to die, as DNA is present equally in the two daughter cells but not separated at the division septum. We propose that PprA may play a crucial role in the control of DNA segregation and/or cell division after DNA double strand break repair.     

Calorimetric and spectroscopic investigations of the thermal denaturation of wild type nitrite reductase

Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type 1 is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes faecalis S-6. The temperature-induced changes of the copper centres are characterized by optical spectroscopy and electron paramagnetic resonance spectroscopy, and by establishing the thermal stability by differential scanning calorimetry. The calorimetric profile of the enzyme shows a single endothermic peak with maximum heat absorption at T_m ≈ 100 °C, revealing an exceptional thermal stability. The thermal transition is irreversible and the scan rate dependence of the calorimetric trace indicates that the denaturation of NiR is kinetically controlled. The divergence of the activation energy values determined by different methods is used as a criterion for the inapplicability of the one-step irreversible model. The best fit of the DSC profiles is obtained when the classical Lumry–Eyring model, N ? U ? F, is considered. The simulation results indicate that the irreversible step prevails on the reversible one. Moreover, it is found that the conformational changes within the type-1 copper environments precede the denaturation of the whole protein. No evidence of protein dissociation within the temperature range investigated was observed.     

Fluoroquinolones stimulate the DNA cleavage activity of topoisomerase IV by promoting the binding of Mg2 + to the second metal binding site

BackgroundFluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase–DNA covalent complex as a topoisomerase–fluoroquinolone–DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction.MethodsWe conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg^2 +-, Mn^2 +-, or Ca^2 +-supported DNA cleavage activity of Escherichia coli Topo IV.ResultsIn the absence of any drug, 20–30 mM Mg^2 + was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1 mM of either Mn^2 + or Ca^2 + was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg^2 + concentrations where Topo IV alone could not efficiently cleave DNA.Conclusions and general significanceAt low Mg^2 + concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg^2 + binding to metal binding site B through the structural distortion in DNA. As Mg^2 + concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg^2 + at site B or inhibition the binding of Mg^2 + to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg^2 + binding.     

Insecticidal activity and feeding behavior of the green peach aphid,Myzus persicae,after treatment with nano types of pyrifluquinazon

Pyrifluquinazon modifies insect behavior by rapidly stopping feeding so that insects starve to death. In the present study, the effects of two nano types of pyrifluquinazon and a non-nano type of pyrifluquinazon on mortality of the green peach aphid, Myzus persicae, were investigated using three different concentrations (25, 50, and 100 ppm). The nano types of pyrifluquinazon were formulated with a different molecular weight and density than that of chitosan (CS 30,000, 0.1% and CS 3000, 0.3%) for stability of unsteady core materials. According to the bioassay and the increased rate of aphids, both types of pyrifluquinazon at all concentrations were effective for controlling M. persicae. The CS 3000 0.3% nano type pyrifluquinazon exhibited a more effective controlled-release feature than that of the other type. The two nano types and the non-nano type pyrifluquinazon were bioassayed against M. persicae, and feeding behaviors were compared using the electrical penetration graph technique. The non-nano type appeared to have the best lethal efficiency at 2 days after treatment at 50 and 25 ppm according to the non-penetration time; however, both CS 30,000 0.1% and CS 3000 0.3% were effective at 14 days after treatment. Furthermore, reaction time slowed from 14 to 30 days after treatment in the CS 3000 0.3% treated aphids. As a result, CS 3000 0.3% had more effective controlled release at both concentrations.