Identification of a radical formed in the reaction mixtures of oxidized phosphatidylcholine with ferrous ions using HPLC–ESR and HPLC–ESR–MS

Identification of free radicals was performed for the reaction mixtures of autoxidized 1,2-dilinoleoylphosphatidylcholine (DLPC) with ferrous ions (or DLPC hydroperoxide with ferrous ions) and of DLPC with soybean lipoxygenase using electron spin resonance (ESR), high performance liquid chromatography (HPLC)–ESR and HPLC–ESR–mass spectrometry (MS) combined use of spin trapping technique. ESR measurements of the reaction mixtures showed prominent signals with hyperfine coupling constants (a^N=1.58?mT and a^Hβ=0.26?mT). Outstanding peaks with almost same retention times (autoxidized DLPC, 36.9?min; DLPC hydroperoxide, 35.0?min; DLPC with soybean lipoxygenase, 37.1?min) were observed on the elution profile of the HPLC–ESR analyses of the reaction mixtures. HPLC–ESR–MS analyses of the reaction mixtures gave two ions at m/z 266 and 179, suggesting that 4-POBN/pentyl radical adduct forms in these reaction mixtures.     

GroEL-induced topological dislocation of a substrate protein β-sheet core: a solution EPR spin–spin distance study

The Hsp60-type chaperonin GroEL assists in the folding of the enzyme human carbonic anhydrase II (HCA II) and protects it from aggregation. This study was aimed to monitor conformational rearrangement of the substrate protein during the initial GroEL capture (in the absence of ATP) of the thermally unfolded HCA II molten-globule. Single- and double-cysteine mutants were specifically spin-labeled at a topological breakpoint in the β-sheet rich core of HCA II, where the dominating antiparallel β-sheet is broken and β-strands 6 and 7 are parallel. Electron paramagnetic resonance (EPR) was used to monitor the GroEL-induced structural changes in this region of HCA II during thermal denaturation. Both qualitative analysis of the EPR spectra and refined inter-residue distance calculations based on magnetic dipolar interaction show that the spin-labeled positions F147C and K213C are in proximity in the native state of HCA II at 20 °C (as close as ～8 Å), and that this local structure is virtually intact in the thermally induced molten-globule state that binds to GroEL. In the absence of GroEL, the molten globule of HCA II irreversibly aggregates. In contrast, a substantial increase in spin–spin distance (up to >20 Å) was observed within minutes, upon interaction with GroEL (at 50 and 60 °C), which demonstrates a GroEL-induced conformational change in HCA II. The GroEL binding-induced disentanglement of the substrate protein core at the topological break-point is likely a key event for rearrangement of this potent aggregation initiation site, and hence, this conformational change averts HCA II misfolding.     

Estimation of binding parameters for the protein–protein interaction using a site-directed spin labeling and EPR spectroscopy

Sensitivity of the electron paramagnetic resonance (CW EPR) to molecular tumbling provides potential means for studying processes of molecular association. It uses spin-labeled macromolecules, whose CW EPR spectra may change upon binding to other macromolecules. When a spin-labeled molecule is mixed with its liganding partner, the EPR spectrum constitutes a linear combination of spectra of the bound and unbound ligand (as seen in our example of spin-labeled cytochrome c ₂ interacting with cytochrome bc ₁ complex). In principle, the fraction of each state can be extracted by the numerical decomposition of the spectrum; however, the accuracy of such decomposition may often be compromised by the lack of the spectrum of the fully bound ligand, imposed by the equilibrium nature of molecular association. To understand how this may affect the final estimation of the binding parameters, such as stoichiometry and affinity of the binding, a series of virtual titration experiments was conducted. Our non-linear regression analysis considered a case in which only a single class of binding sites exists, and a case in which classes of both specific and non-specific binding sites co-exist. The results indicate that in both models, the error due to the unknown admixture of the unbound ligand component in the EPR spectrum causes an overestimation of the bound fraction leading to the bias in the dissociation constant. At the same time, the stoichiometry of the binding remains relatively unaffected, which overall makes the decomposition of the EPR spectrum an attractive method for studying protein–protein interactions in equilibrium. Our theoretical treatment appears to be valid for any spectroscopic techniques dealing with overlapping spectra of free and bound component.     

Timing of butterfly parasitization of a plant–ant–scale symbiosis

In the Southeast Asian tropics, Arhopala lycaenid butterflies feed on Macaranga ant-plants inhabited by Crematogaster (subgenus Decacrema) ants tending Coccus-scale insects. A recent phylogenetic study showed that (1) the plants and ants have been codiversifying for the past 20–16 million years (Myr), and that (2) the tripartite symbiosis was formed 9–7 Myr ago, when the scale insects became involved in the plant–ant mutualism. To determine when the lycaenids first parasitized the Macaranga tripartite symbiosis, we constructed a molecular phylogeny of the lycaenids that feed on Macaranga by using mitochondrial and nuclear DNA sequence data and estimated their divergence times based on the cytochrome oxidase I molecular clock. The minimum age of the lycaenids was estimated by the time-calibrated phylogeny to be 2.05 Myr, about one-tenth the age of the plant–ant association, suggesting that the lycaenids are latecomers that associated themselves with the pre-existing symbiosis of plant, ant, and scale insects.     

Structural characterization of a carbon monoxide adduct of a heme–DNA complex

Abstract  

The structure of a carbon monoxide (CO) adduct of a complex between heme and a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), has been characterized using ¹H and ¹³C NMR spectroscopy and density function theory calculations. The study revealed that the heme binds to the 3′-terminal G-quartet of the DNA though a π–π stacking interaction between the porphyrin moiety of the heme and the G-quartet. The π–π stacking interaction between the pseudo-C ₂-symmetric heme and the C ₄-symmetric G-quartet in the complex resulted in the formation of two isomers possessing heme orientations differing by 180° rotation about the pseudo-C ₂ axis with respect to the DNA. These two slowly interconverting heme orientational isomers were formed in a ratio of approximately 1:1, reflecting that their thermodynamic stabilities are identical. Exogenous CO is coordinated to heme Fe on the side of the heme opposite the G-quartet in the complex, and the nature of the Fe–CO bond in the complex is similar to that of the Fe–CO bonds in hemoproteins. These findings provide novel insights for the design of novel DNA enzymes possessing metalloporphyrins as prosthetic groups.     

Synthesis of a Bioactive Composition of Chitosan–Selenium Nanoparticles

Applied Biochemistry and Microbiology - A biologically active composition of chitosan-selenium nanoparticles has been developed. Selenium nanoparticles are characterized by a clear bimodal size...     

Kinetics of demetallation of a zinc–salophen complex into liposomes

A Zn–salophen complex has been incorporated into POPC large unilamellar liposomes (LUV) obtained in phosphate buffer at pH 7.4. Fluorescence optical microscopy and anisotropy measurements show that the complex is located at the liposomal surface, close to the polar headgroups. The interaction of the POPC phosphate group with Zn^2 + slowly leads to demetallation of the complex. The process follows first order kinetics and rate constants have been measured fluorimetrically in pure water and in buffered aqueous solution.The coordination of the phosphate group of monomeric POPC with salophen zinc also occurs in chloroform as detected by ESI-MS measurements.The effect of the Zn–salophen complex on the stability of POPC LUV has been evaluated at 25 °C by measuring the rate of release of entrapped 5(6)-carboxyfluorescein (CF) in the presence and in the absence of Triton X-100 as the perturbing agent. It turns out that the inclusion of the complex significantly increases the stability of POPC LUV.     

Investigation of Lysozyme–Antilysozyme Interactions in a Model Tetrahymena–EscherichiaCommunity

Lysozyme and antilysozyme activities present in a wide range of microorganisms determine the so-called lysozyme–antilysozyme system of hydrobionts, which greatly contribute to the formation of aquatic biocenoses. However, the mechanism of the functioning of this system in natural freshwater communities remains obscure. The experimental investigation of lysozyme–antilysozyme interactions in a model Tetrahymena–Escherichiacommunity showed that the antilysozyme activity of Escherichia colileads to incomplete phagocytosis, thus enhancing bacterial survival in a mixed culture with infusoria. The selection and reproduction of bacterial cells resistant to grazing by infusoria determine the character of host–parasite interactions and allow bacteria to survive. It was demonstrated that the antilysozyme activity of microorganisms, which is responsible for bacterial persistency in natural biocenoses, is involved in the maintenance of protozoa–bacteria communities in bodies of water.     

EPR analysis of multiple forms of [4Fe–4S]3+ clusters in HiPIPs

The electron paramagnetic resonance (EPR) spectrum from the [4Fe–4S]³⁺ cluster in several high-potential iron–sulfur proteins (HiPIPs) is complex: it is not the pattern of a single, isolated S=1/2 system. Multifrequency EPR from 9 to 130 GHz reveals that the apparent peak positions (g values) are frequency-independent: the spectrum is dominated by the Zeeman interaction plus g-strain broadening. The spectra taken at frequencies above the X-band are increasingly sensitive to rapid-passage effects; therefore, the X-band data, which are slightly additionally broadened by dipolar interaction, were used for quantitative spectral analysis. For a single geometrical [4Fe–4S]³⁺ structure the (Fe–Fe)⁵⁺ mixed-valence dimer can be assigned in six different ways to a pair of iron ions, and this defines six valence isomers. Systematic multicomponent g-strain simulation shows that the [4Fe–4S]³⁺ paramagnets in seven HiPIPs from different bacteria each consist of three to four discernible species, and these are assigned to valence isomers of the clusters. This interpretation builds on previous EPR analyzes of [4Fe–4S]³⁺ model compounds, and it constitutes a high-resolution extension of the current literature model, proposed from paramagnetic NMR studies.     

Analysis of a competitive prey–predator system with a prey refuge

Gauss's competitive exclusive principle states that two competing species having analogous environment cannot usually occupy the same space at a time but in order to exploit their common environment in a different manner, they can co-exist only when they are active in different times. On the other hand, several studies on predators in various natural and laboratory situations have shown that competitive coexistence can result from predation in a way by resisting any one prey species from becoming sufficiently abundant to outcompete other species such that the predator makes the coexistence possible. It has also been shown that the use of refuges by a fraction of the prey population exerts a stabilizing effect in the interacting population dynamics. Further, the field surveys in the Sundarban mangrove ecosystem reveal that two detritivorous fishes, viz. Liza parsia and Liza tade (prey population) coexist in nature with the presence of the predator fish population, viz. Lates calcarifer by using refuges.     

Evolution of conditional dispersal: a reaction–diffusion–advection model

To study evolution of conditional dispersal, a Lotka-Volterra reaction-diffusion-advection model for two competing species in a heterogeneous environment is proposed and investigated. The two species are assumed to be identical except their dispersal strategies: both species disperse by random diffusion and advection along environmental gradients, but one species has stronger biased movement (i.e., advection along the environmental gradients) than the other one. It is shown that at least two scenarios can occur: if only one species has a strong tendency to move upward the environmental gradients, the two species can coexist since one species mainly pursues resources at places of locally most favorable environments while the other relies on resources from other parts of the habitat; if both species have such strong biased movements, it can lead to overcrowding of the whole population at places of locally most favorable environments, which causes the extinction of the species with stronger biased movement. These results provide a new mechanism for the coexistence of competing species, and they also imply that selection is against excessive advection along environmental gradients, and an intermediate biased movement rate may evolve.     

Synthesis of a cyclic isostere of α-methyl homoserine by a stereoselective acylation–alkylation sequence of a chiral γ-lactam

Starting from a chiral 4-hydroxymethyl pyrrolidin-2-one, an isostere of α-methyl homoserine tethered on a γ-lactam ring was prepared exploiting a stereoselective acylation–methylation sequence, followed by Curtius rearrangement, and structural assignment was confirmed by n.O.e. experiments. By reverting the sequence, the 3-carboxy-3-methyl derivative having the opposite configuration at C-3 was obtained with total stereoselection, but Curtius rearrangement invariably afforded only inseparable mixtures of decomposition products.     

Characterization of a HAP–phytase from a thermophilic mould Sporotrichum thermophile

The phytase of Sporotrichum thermophile was purified to homogeneity using acetone precipitation followed by ion-exchange and gel-filtration column chromatography. The purified phytase is a homopentamer with a molecular mass of ～456 kDa and pI of 4.9. It is a glycoprotein with about 14% carbohydrate, and optimally active at pH 5.0 and 60 °C with a T_1/2 of 16 h at 60 °C and 1.5 h at 80 °C. The activation energy of the enzyme reaction is 48.6 KJ mol^?1 with a temperature quotient of 1.66, and it displayed broad substrate specificity. Mg²⁺ exhibited a slight stimulatory effect on the enzyme activity, while it was markedly inhibited by 2,3-butanedione suggesting a possible role of arginine in its catalysis. The chaotropic agents such as guanidinium hydrochloride, urea and potassium iodide strongly inhibited phytase activity. Inorganic phosphate inhibited enzyme activity beyond 3 mM. The maximum hydrolysis rate (V_max) and apparent Michaelis–Menten constant (K_m) for sodium phytate were 83 nmol mg^?1 s^?1 and 0.156 mM, respectively. The catalytic turnover number (K_cat) and catalytic efficiency (K_cat/K_m) of phytase were 37.8 s^?1 and 2.4 × 10⁵ M^?1 s^?1, respectively. Based on the N-terminal and MALDI–LC–MS/MS identified amino acid sequences of the peptides, the enzyme did not show a significant homology with the known phytases.     

Biodegradation of ketoprofen using a microalgal–bacterial consortium

Objective

To test the toxicity of ketoprofen (a commonly-used NSAIDs) using two microalgal strains and Artemia sp. following the isolation of bacterial and microalgal strains and testing their ability to biodegrade and tolerate ketoprofen.

Results

Chlorella sp. was the most resistant to ketoprofen. A defined bacterial consortium (K₂) degraded 5 mM ketoprofen as a sole carbon source both in the dark or continuous illumination. Ketoprofen did not undergo photodegradation. In the dark, biodegradation was faster with a lag phase of 10 h, 41% COD removal and 82 % reduction in toxicity. The consortium degraded up to 16 mM ketoprofen. The consortium was composed of four bacterial isolates that were identified. MS/MS analysis suggested a ketoprofen biodegradation pathway that has not been previously reported. Combining Chlorella sp. and the K₂ consortium, ketoprofen was degraded within 7 days under a diurnal cycle of 12 h light/12 h dark.

Conclusion

The feasibility of using a microalgal–bacterial system to treat pharmaceutical wastewater is promising for the reduction of the process cost and providing a safer technology for pharmaceutical wastewater treatment.

     

Coevolution of dispersal in a parasitoid–host system

Interspecific interactions and the evolution of dispersal are both of interest when considering the potential impact of habitat fragmentation on community ecology, but the interaction between these processes is not well studied. We address this by considering the coevolution of dispersal strategies in a host–parasitoid system. An individual-based host–parasitoid metapopulation model was constructed for a patchy environment, allowing for evolution in dispersal rates of both species. Highly rarefied environments with few suitable patches selected against dispersal in both species, as did relatively static environments. Provided that parasitoids persist, all the variables studied led to stable equilibria in dispersal rates for both species. There was a tendency toward higher dispersal rates in parasitoids because of the asymmetric relationships of the two species to the patches: vacant patches are most valuable for hosts, but unsuitable for parasitoids, which require an established host population to reproduce. High host dispersal rate was favoured by high host population growth rate, and in the parasitoid by high growth rates in both species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis and cellular studies of polyamine conjugates of a mercaptomethyl–carboranylporphyrin

Seven polyamine conjugates of a tri(p-carboranylmethylthio)tetrafluorophenylporphyrin were prepared in high yields by sequential substitution of the p-phenyl fluoride of tetrakis(pentafluorophenyl)porphyrin (TPPF), and investigated as boron delivery agents for boron neutron capture therapy (BNCT). The polyamines used were derivatives of the natural-occurring spermine with different lengths of the carbon chains, terminal primary amine groups and, in two of the conjugates, additional aminoethyl moieties. A tri(polyethylene glycol) conjugate was also synthesized for comparison purposes. The polyamine conjugates showed low dark cytotoxicity (IC₅₀ >400 μM) and low phototoxicity (IC₅₀ >40 μM at 1.5 J/cm²). All polyamine conjugates, with one exception, showed higher uptake into human glioma T98G cells (up to 12-fold) than the PEG conjugate, and localized preferentially in the cell ER, Golgi and the lysosomes. Our results show that spermine derivatives can serve as effective carriers of boronated porphyrins for the BNCT of tumors.     

Dermatophyte–host relationship of a murine model of experimental invasive dermatophytosis

Recognizing the invasive potential of the dermatophytes and understanding the mechanisms involved in this process will help with disease diagnosis and with developing an appropriate treatment plan. In this report, we present the histopathological, microbiological and immunological features of a model of invasive dermatophytosis that is induced by subcutaneous infection of Trichophyton mentagrophytes in healthy adult Swiss mice. Using this model, we observed that the fungus rapidly spreads to the popliteal lymph nodes, spleen, liver and kidneys. Similar to the human disease, the lymph nodes were the most severely affected sites. The fungal infection evoked acute inflammation followed by a granulomatous reaction in the mice, which is similar to what is observed in patients. The mice were able to mount a Th1-polarized immune response and displayed IL-10-mediated immune regulation. We believe that the model described here will provide valuable information regarding the dermatophyte–host relationship and will yield new perspective for a better understanding of the immunological and pathological aspects of invasive dermatophytosis.     

Parametric Analysis of a Predator–prey System Stabilized by a Top Predator

We present a complete parametric analysis of a predator–prey system influenced by a top predator. We study ecosystems with abundant nutrient supply for the prey where the prey multiplication can be considered as proportional to its density. The main questions we examine are the following: (1) Can the top predator stabilize such a system at low densities of prey? (2) What possible dynamic behaviors can occur? (3) Under which conditions can the top predation result in the system stabilization? We use a system of two nonlinear ordinary differential equations with the density of the top predator as a parameter. The model is investigated with methods of qualitative theory of ODEs and the theory of bifurcations. The existence of 12 qualitatively different types of dynamics and complex structure of the parametric space are demonstrated. Our studies of phase portraits and parametric diagrams show that a top predator can be an important factor leading to stabilization of the predator-prey system with abundant nutrient supply. Although the model here is applied to the plankton communities with fish (or carnivorous zooplankton) as the top trophic level, the general form of the equations allows applications of our results to other ecological systems.