Distribution of mitochondrial NADH fluorescence lifetimes: steady-state kinetics of matrix NADH interactions

The lifetimes of fluorescent components of matrix NADH in isolated porcine heart mitochondria were investigated using time-resolved fluorescence spectroscopy. Three distinct lifetimes of fluorescence were resolved: 0.4 (63%), 1.8 (30%), and 5.7 (7%) ns (% total NADH). The 0.4 ns lifetime and the emission wavelength of the short component were consistent with free NADH. In addition to their longer lifetimes, the remaining pools also had a blue-shifted emission spectrum consistent with immobilized NADH. On the basis of emission frequency and lifetime data, the immobilized pools contributed >80% of NADH fluorescence. The steady-state kinetics of NADH entering the immobilized pools was measured in intact mitochondria and in isolated mitochondrial membranes. The apparent binding constants (K(D)s) for NADH in intact mitochondria, 2.8 mM (1.9 ns pool) and >3 mM (5.7 ns pool), were on the order of the estimated matrix [NADH] (approximately 3.5 mM). The affinities and fluorescence lifetimes resulted in an essentially linear relationship between matrix [NADH] and NADH fluorescence intensity. Mitochondrial membranes had shorter emission lifetimes in the immobilized poo1s [1 ns (34%) and 4.1 ns (8%)] with much higher apparent K(D)s of 100 microM and 20 microM, respectively. The source of the stronger NADH binding affinity in membranes is unknown but could be related to high order structure or other cofactors that are diluted out in the membrane preparation. In both preparations, the rate of NADH oxidation was proportional to the amount of NADH in the long lifetime pools, suggesting that a significant fraction of the bound NADH might be associated with oxidative phosphorylation, potentially in complex 1.     

Activation of murine macrophages by hydrogen peroxide

Hydrogen peroxide at concentrations from 0.1 to 20 μM enhances phagocytosis and oxidative burst of murine peritoneal macrophages. The activation of these macrophage functions is paralled by prolonged hyperpolarization and a transient increase in cytoplasmic free calcium concentration. All the effects are dose- and time-dependent. The results obtained for H₂O₂ are compared with those for a natural activator, peptide N-formyl-methionyl-leucly-phenylalanine. The data demonstrate the ability of small doses of hydrogen peroxide to stimulate macrophages through the intracellular mechanisms of ion transduction.     

The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.     

Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes

Oomycete plant pathogens deliver effector proteins inside host cells to modulate plant defense circuitry and to enable parasitic colonization. These effectors are defined by a conserved motif, termed RXLR (for Arg, any amino acid, Leu, Arg), that is located downstream of the signal peptide and that has been implicated in host translocation. Because the phenotypes of RXLR effectors extend to plant cells, their genes are expected to be the direct target of the evolutionary forces that drive the antagonistic interplay between pathogen and host. We used the draft genome sequences of three oomycete plant pathogens, Phytophthora sojae, Phytophthora ramorum, and Hyaloperonospora parasitica, to generate genome-wide catalogs of RXLR effector genes and determine the extent to which these genes are under positive selection. These analyses revealed that the RXLR sequence is overrepresented and positionally constrained in the secretome of Phytophthora relative to other eukaryotes. The three examined plant pathogenic oomycetes carry complex and diverse sets of RXLR effector genes that have undergone relatively rapid birth and death evolution. We obtained robust evidence of positive selection in more than two-thirds of the examined paralog families of RXLR effectors. Positive selection has acted for the most part on the C-terminal region, consistent with the view that RXLR effectors are modular, with the N terminus involved in secretion and host translocation and the C-terminal domain dedicated to modulating host defenses inside plant cells.     

Alginate/chitosan hydrogels as perspective transport systems for cefotaxime

This work reports synthesis of pH-responsive alginate/chitosan hydrogel spheres with the average diameter of 2.0 ± 0.05 mm, which contain cefotaxime that is an antibiotic of the cefalosporine group. The spheres provided the cefotaxime encapsulation efficiency of 95 ± 1%. An in vitro release of cefotaxime from the spheres in the media that simulate human biological fluids in peroral delivery conditions was found to be a pH-dependent process. The analysis of cefotaxime release kinetics by the Korsmeyer–Peppas model revealed a non-Fickian mechanism of its diffusion, which may be related to intermolecular interactions occurring between the antibiotic and chitosan. Conductometry, UV spectroscopy, and IR spectroscopy were used to study complexation of chitosan with cefotaxime in aqueous media with varied pH, characterize the composition of the complexes, and calculate their stability constants. The composition of the cefotaxime–chitosan complexes was found to correspond to the 1.0:4.0 and 1.0:2.0 molar ratios of the components at pH 2.0 and 5.6, respectively. Quantum chemical modeling was used to evaluate energy characteristics of chitosan–cefotaxime complexation considering the influence of a solvent.     

Occurrence of aminoglycoside transpherase genes in methicillin-resistant strains of Staphylococcus aureus

Fifty MRSA strains originated from clinical specimens were examined by the PCR method, for the presence of three genes: aacA-aphD, aadD oraz aph(3")-IIIa. The obtained results were correlated with the susceptibility of the strains to gentamicin, tobramicin, kanamicin, neomicin, amikacin and netilmicin. The susceptibility results were interpreted according with CLSI and EUCAST guidelines. aacA-aphD gene was found in 34 strains, aadD in 27 strains and aph(3")-IIIa was present in 22 strains. In 19 strains (38%) was present one of the investigated genes, in 29 (58%) strains two genes and in two strains (4%) all three genes were found. The most frequent variant was combination of aacA-aphD and aadD genes.     

Methylotrophic autotrophy in Beijerinckia mobilis

Representatives of the genus Beijerinckia are known as heterotrophic, dinitrogen-fixing bacteria which utilize a wide range of multicarbon compounds. Here we show that at least one of the currently known species of this genus, i.e., Beijerinckia mobilis, is also capable of methylotrophic metabolism coupled with the ribulose bisphosphate (RuBP) pathway of C1 assimilation. A complete suite of dehydrogenases commonly involved in the sequential oxidation of methanol via formaldehyde and formate to CO2 was detected in cell extracts of B. mobilis grown on CH3OH. Carbon dioxide produced by oxidation of methanol was further assimilated via the RuBP pathway as evidenced by reasonably high activities of phosphoribulokinase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Detection and partial sequence analysis of genes encoding the large subunits of methanol dehydrogenase (mxaF) and form I RubisCO (cbbL) provided genotypic evidence for methylotrophic autotrophy in B. mobilis.     

Effects of SPLAT® GM sprayable pheromone formulation on gypsy moth mating success

Several integrated pest management programs rely on the use of mating disruption tactics to control insect pests. Some programs specifically target non‐native species, such as the gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae). We evaluated SPLAT^® GM, a new sprayable formulation of the gypsy moth sex pheromone disparlure, for its ability to disrupt gypsy moth mating. The study was conducted in 2006, 2007, and 2008 in forested areas in Virginia, USA. Mating success of gypsy moth females was reduced by >99% and male moth catches in pheromone‐baited traps by >90%, in plots treated with SPLAT^® GM at dosages ranging from 15 to 75 g of active ingredient (a.i.) ha^?1. Dosage‐response tests conducted in 2008 indicated that SPLAT^® GM applied at a dosage of 7.5 g a.i. ha^?1 was as effective as a 15 g a.i. ha^?1 dosage.     

Potential of S-containing and P-containing complexones in improving phytoextraction of mercury by Trifolium repens L.

Mercury is a global pollutant in the modern world. There is a large number of areas in the world where mercury is present in soils in significant quantities. Remediation methods which have traditionally been proposed may pose a risk of secondary mercury contamination and/or adverse health effects for cleaners. Phytoextraction of heavy metals from the soil environment is currently considered one of the promising non-invasive methods of remediation. But this approach has limited effectiveness. Chemically induced phytoextraction can increase the efficiency of this process both by converting less bioavailable mercury compounds to bioavailable fractions in the soil and by increasing the rate of transfer of metals in plants. This paper presents the results of a screening study of various chemical amendments to enhance the phytoextraction of mercury by Trifolium repens L. The results showed good potential for the induction of phytoextraction of phosphorus(P) and sulfur (S)-containing chelates. With this study, for the first time for the phytoextraction of mercury, the monoethanolamine salt of 2,2′-(ethylenedithio) diacetic acid was used as the S-containing chelate, and the disubstituted potassium salt of 1-hydroxy ethylidene-1,1-diphosphonic acid was used as the P-containing chelate. Further attention is given to study the effect that exogenous application of phytohormones and plant growth regulators has on the efficiency of mercury absorption and physiological status of plants, which performed well in combination with a P-containing chelate.