Structural basis of the interspecies interaction between the chaperone DnaK(Hsp70) and the co-chaperone GrpE of archaea and bacteria

Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I' band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.     

Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases

Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione-GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.     

Habitat requirements of the <Emphasis Type="Italic">Charetum intermediae</Emphasis> phytocoenoses in lakes of western Poland

The study presents habitat and phytosociological analyses of the Chara intermedia phytocenoses, rare described in Europe. 16 physico-chemical water parameters were analysed, coming from the samples taken in 20 phytocenoses of 13 lakes located in western Poland. The analysed community appeared in naturally shallow lakes representing last stages of the disappearance of glacial water basins. The study attempts to estimate the bioindicative value of the charophyta meadow Charetum intermediae in relation to its habitat. A particular attention has been paid to the determination of the habitat trophic condition, and to the concentration of elements connected with the hardness of water and the content of humic substances. The study shows crucial habitat gradients of the C. intermediae association, taking into account also the species composition of phytocenoses.     

Temperature affects the supramolecular structures resulting from alpha-lactalbumin-lysozyme interaction

The interaction between alpha-lactalbumin and lysozyme, two globular proteins with highly homologous tertiary structures but opposite electric charges, was investigated. As assessed by isothermal titration calorimetry, lysozyme did not bind to the native form of alpha-lactalbumin but did interact with calcium-depleted alpha-lactalbumin (apo alpha-LA). This interaction leads to the formation of different supramolecular structures depending on the temperature. Heterogeneous, amorphous aggregates are formed at 5 degrees C, while droplets, coacervate-like structures, exist at 45 degrees C. The coacervates are formed by equimolar quantities of the two proteins, but their size and number depend on the initial protein molar ratio. These supramolecular structures are found to be stable when the temperature is decreased to 5 degrees C, while prolonged heating at 45 degrees C induces the formation of larger coacervates through a coalescence phenomenon. Surprisingly, interplay occurs between aggregates and coacervates when the temperature is increased from 5 to 45 degrees C. We discuss the results in terms of subtle heat-induced conformational changes in apo alpha-LA. In conclusion, our results show an association between globular proteins that leads to the formation of a variety of supramolecular structures in a temperature-dependent manner and confirm the primordial role of certain alpha-lactalbumin unfolding intermediates in protein-driven assembly.     

Chemoenzymatic formation of novel aminocoumarin antibiotics by the enzymes CouN1 and CouN7

The aminocoumarin antibiotics novobiocin, clorobiocin, and coumermycin A1 are highly potent inhibitors of the bacterial type II topoisomerase DNA gyrase. The key pharmacophore of both clorobiocin and coumermycin A1, the 5-methyl-2-pyrrolylcarbonyl moiety, targets the ATP-binding site of GyrB. The 5-methyl-2-pyrrolylcarbonyl group is transferred by the acyltransferases Clo/CouN7 from the carrier proteins Clo/CouN1 to the 3'-hydroxyl of the l-noviosyl scaffold during the late steps of clorobiocin and coumermycin A1 biosynthesis. We first examined the substrate specificity of the purified thiolation domain protein CouN1 in becoming primed by the phosphopantetheinyltransferase Sfp using a variety of synthetic CoA analogues of the 5-methyl-2-pyrrolylcarbonyl moiety. The acyl-S-CouN1 thioesters were then assayed as donors to the 3'-OH group of descarbamoylnovobiocin by the acyltransferase CouN7, resulting in 21 novel variants with heterocyclic acyl groups installed on the noviosyl moiety of the aminocoumarin scaffold. Scaleup of a 5-methylthiophene derivative yielded a compound with activity against both Gram-negative and Gram-positive bacteria. The minimal inhibitory concentration found for the Gram-positive bacteria was comparable to that of novobiocin.     

Trehalose accumulation by tropical yeast strains submitted to stress conditions

Trehalose, a non-reducing disaccharide that accumulates in Saccharomyces cerevisiae, has been implicated in survival under various stress conditions by acting as membrane protectant, as a supplementary compatible solute or as a reserve carbohydrate which may be mobilized during stress. However, most of these studies have been done with strains isolated from European or Asian habitats of temperate climate. In this study, yeasts living in tropical environments, isolated from different microhabitats in Southeastern Brazil, were used to evaluate whether trehalose contributes to survival under osmotic, ethanol and heat stress. The survival under severe stress was compared to a well-characterized laboratorial wild-type strain (D273-10B). Most of the Saccharomyces cerevisiae strains isolated from Drosophila in Tropical Rain Forest were able to accumulate trehalose after a preconditioning treatment at 40 °C for 1 h. The amount of intracellular trehalose levels was better correlated with survival during a challenging heat shock at 50.5 °C for 8 min. Saccharomyces cerevisiae and Candida guilliermondii were observed to be thermotolerant as well as osmotolerant. No clear correlation between intracellular trehalose levels and survival could be derived during ethanol stress. In some cases, the amount of trehalose accumulated before the ethanol stress seemed to play an important role for the survival of these strains.     

The “Window <Emphasis Type="Italic">t</Emphasis> test”: a simple and powerful approach to detect differentially expressed genes in microarray datasets

This work focuses on differential expression analysis of microarray datasets. One way to improve such statistical analyses is to integrate biological information in the design of these analyses. In this paper, we will use the relationship between the level of gene expression and variability. Using this biological information, we propose to integrate the information from multiple genes to get a better estimate of individual gene variance, when a small number of replicates are available, to increase the power of the statistical analysis. We describe a strategy named the “Window t test” that uses multiple genes which share a similar expression level to compute the variance which is then incorporated a classic t test. The performances of this new method are evaluated by comparison with classic and widely-used methods for differential expression analysis (the classic Student t test, the Regularized t test (reg t test), SAM, Limma, LPE and Shrinkage t). In each case tested, the results obtained were at least equivalent to the best performing method and, in most cases, outperformed it. Moreover, the Window t test relies on a very simple procedure requiring small computing power compared with other methods designed for microarray differential expression analysis. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

Physical mapping in large genomes: accelerating anchoring of BAC contigs to genetic maps through in silico analysis

Anchored physical maps represent essential frameworks for map-based cloning, comparative genomics studies, and genome sequencing projects. High throughput anchoring can be achieved by polymerase chain reaction (PCR) screening of bacterial artificial chromosome (BAC) library pools with molecular markers. However, for large genomes such as wheat, the development of high dimension pools and the number of reactions that need to be performed can be extremely large making the screening laborious and costly. To improve the cost efficiency of anchoring in such large genomes, we have developed a new software named Elephant (electronic physical map anchoring tool) that combines BAC contig information generated by FingerPrinted Contig with results of BAC library pools screening to identify BAC addresses with a minimal amount of PCR reactions. Elephant was evaluated during the construction of a physical map of chromosome 3B of hexaploid wheat. Results show that a one dimensional pool screening can be sufficient to anchor a BAC contig while reducing the number of PCR by 384-fold thereby demonstrating that Elephant is an efficient and cost-effective tool to support physical mapping in large genomes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. E. Paux and F. Legeai contributed equally to this work.     

Co-localization of apoptosis-regulating proteins in mouse mammary epithelial HC11 cells exposed to TGF-beta1

TGF-beta1 is an apoptogenic agent for mammary epithelial cells (MEC). The molecular mechanism of the TGF-beta1-induced apoptosis remains, however, obscure. In the present study we used laser scanning cytometry, confocal microscopy and immunogold electron microscopy to analyze the expression, aggregation and co-localization of caspase-8, Bid, Bax and VDAC-1. These proteins are regarded as the most important factors involved in the regulatory phase of TGF-beta1-induced apoptosis. Apoptosis in HC11 mouse MEC manifested with a simultaneous increase in expression and subcellular aggregation of caspase-8, Bid, Bax and VDAC-1. Confocal microscopy revealed a strong pattern of co-localization of examined proteins during both early and late apoptosis. Experiments with double- and triple-staining immunoelectron microscopy showed a co-localization of Bax/Bid, caspase-8/Bax/Bid, and Bax/VDAC-1, on the membranes of mitochondria, Golgi apparatus, rough endoplasmic reticulum, nuclear envelope, nuclear pore, and within the nucleus. In conclusion, the observed pattern of changes in aggregation and subcellular localization of caspase-8, Bid, Bax and VDAC-1 during TGF-beta1-induced apoptosis in HC11 mouse MEC suggests an interaction between these proteins and formation of multimeric complexes on organellar membranes, thus controlling their permeability for intracellular mediators of apoptosis.     

Nitrous oxide-induced hypothermia in the rat

Exposure of rats to high levels of nitrous oxide (N2O) in oxygen (O2) reduced body temperature in a concentration-related manner. The hypothermia was partly reversed by pretreatment with naloxone but not naltrexone. But in rats rendered tolerant to morphine by pellet implantation, exposure to 75% N2O/25% O2 evoked a marked hypothermia similar to that observed in morphine-naive animals. In another experiment, the hypothermic effect of chloral hydrate was also sensitive to antagonism by pretreatment with naloxone but not naltrexone. These observations lead us to suspect that N2O-induced hypothermia in rats is possibly not mediated by opiate receptors. The thermotropic activity of N2O may result from some non-opioid action of N2O. Its selective antagonism by naloxone (but not naltrexone) may be due to a unique non-opioid analeptic action of naloxone.