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Genetic and epigenetic variation in a cosmopolitan grass <i>Poa annua</i> from Antarctic and Polish populations
Get PDFPoa annua L. is the only non−native vascular plant that was successfully estab−
lished in the maritime Antarctic. This project aimed to determine the amount of genetic and
epigenetic variation within and between two populations of P. annua, one from South Shet−
land Is. (Antarctic) and the other one from Central Europe. We applied two AFLP marker
systems, using endonucleases that recognised the same restriction site but differed in their
sensitivity towards methylation. The Antarctic population differed from the Polish one both
at the genetic and epigenetic levels. Genetic variability in the Antarctic population was
lower than in the Polish one. Some loci in the Antarctic population showed signs of selec−
tion. The difference between Polish and Antarctic populations might be due to a weak bot−
tleneck effect followed by population expansion. Using only epigenetic markers, the Ant−
arctic population exhibited increased variation level compared to the Polish one. These may
have resulted from plastic responses to environmental factors and could be associated with
survival in extreme conditions
The role of CRISPR/cas system in the development of bacteriophage resistance
No full textAcquisition of foreign DNA can be of advantage or disadvantage to the host cell. New DNAs can increase the fitness of an organism to certain environmental conditions or on the other hand, replication and maintenance of incorporated nucleotide sequences can be a burden for the host cell. These circumstances have resulted in development of certain cellular mechanisms limiting horizontal gene transfer, including the immune system of vertebrates or RNAi mechanisms in Eukaryotes. Also in Prokaryotes specific systems have been characterized, which are aimed especially at limiting invasion of bacteriophage DNA, e.g. adsorption inhibition, injection blocking, restriction/modification, abortive infection. Quite recently, another distinct mechanism limiting horizontal transfer of genetic elements has been identified in Prokaryotes and shown to protect microbial cells against exogenous nucleic acids of phage or plasmid origin. This system has been termed CRISPR/cas and consists of two main components: (i) the CRISPR (clustered, regularly interspaced short palindromic regions) locus and (ii) cas genes, encoding CRISPR-associated (Cas) proteins. In simplest words, the mechanism of CRISPR/cas activity is based on active integration of small fragments (proto-spacers) of the invading DNAs (phage or plasmids) into microbial genomes, which are subsequently transcribed into short RNAs that direct degradation of foreign invading DNA elements. By this way, the host organism acquires immunity toward mobile elements carrying matching sequences. The CRISPR/cas system is regarded as one of the earliest defense system that has evolved in prokaryotic organisms. It is inheritable, but at the same time unstable when regarding the evolutionary scale. Comparative sequence analyses indicate that CRISPR/cas systems play an important role in the evolution of microbial genomes and their predators, bacteriophages
Analysis of immunogenicity of different protein groups from malaria parasite Plasmodium falciparum
Get PDFIt was observed that pressure of host immune system leads to diversifying selection (which can be measured in terms of pN/pS ratio). In this research we checked whether Plasmodium falciparum proteins containing experimentally evident epitopes from the IEDB database are subject to diversifying selection. We also investigated which life stage of this parasite and which proteins are subject to the strongest immune pressure. To answer these questions we used information about experimentally evident epitopes from Plasmodium, that interact with human immune system and sequences of different isolates of P. falciparum.
We confirmed the expectations that proteins containing IEDB epitopes are subject to stronger diversifying selection which is evidenced by higher pN/pS ratio. A stage characterized by the highest average pN/pS ratio is that of the sporozoite. The greatest fraction of putative antigens is also present at this stage. We also found that the sporozoite stage is particularly interesting for further analysis as it potentially contains the highest number of unidentified epitopes
The C-terminal domain of the Salmonella enterica WbaP (UDP-galactose:Und-P galactose-1-phosphate transferase) is sufficient for catalytic activity and specificity for undecaprenyl monophosphate
Get PDFTwo families of membrane enzymes catalyze the initiation of
the synthesis of O-antigen lipopolysaccharide. The
Salmonella enterica Typhimurium WbaP is a prototypic
member of one of these families. We report here the purification
and biochemical characterization of the WbaP
C-terminal (WbaPCT) domain harboring one putative
transmembrane helix and a large cytoplasmic tail. An Nterminal
thioredoxin fusion greatly improved solubility and
stability of WbaPCT allowing us to obtain highly purified
protein. We demonstrate that WbaPCT is sufficient to catalyze
the in vitro transfer of galactose (Gal)-1-phosphate from
uridine monophosphate (UDP)-Gal to the lipid carrier undecaprenyl
monophosphate (Und-P). We optimized the in vitro
assay to determine steady-state kinetic parameters with the
substrates UDP-Gal and Und-P. Using various purified polyisoprenyl
phosphates of increasing length and variable saturation
of the isoprene units, we also demonstrate that the
purified enzyme functions highly efficiently with Und-P,
suggesting that theWbaPCT domain contains all the essential
motifs to catalyze the synthesis of the Und-P-P-Gal molecule
that primes the biosynthesis of bacterial surface glycans
Voltammetric Detection of a Specific DNA Sequence of Avian Influenza Virus H5N1 Using HS-ssDNA Probe Deposited onto Gold Electrode
Get PDFThe working principle of genosensor relay on the mechanism of ion–channel mimetic sensor. The analytical signals generated upon hybridization processes were recorded by redox active marker [Fe(CN)6]3-/4- present in the sample solution using voltammetric techniques. The developed genosensor was suitable for determination of 20-mer complementary oligonucleotide sequence, and also of PCR products containing the complementary 20-mer sequence in various positions, with detection limits in 10 pM range. Non-complementary 20-mer oligonucleotide sequence as well as PCR product without complementary region generated very weak response. The good discrimination of the position of complementary part in the PCR products was observe
Isomeric Mono-, Di-, and Tri-Bromobenzo-1H-Triazoles as Inhibitors of Human Protein Kinase CK2α.
No full textTo further clarify the role of the individual bromine atoms of 4,5,6,7-tetrabromotriazole (TBBt), a relatively selective inhibitor of protein kinase CK2, we have examined the inhibition (IC(50)) of human CK2α by the two mono-, the four di-, and the two tri- bromobenzotriazoles relative to that of TBBt. Halogenation of the central vicinal C(5)/C(6) atoms proved to be a key factor in enhancing inhibitory activity, in that 5,6-di-Br(2)Bt and 4,5,6-Br(3)Bt were almost as effective inhibitors as TBBt, notwithstanding their marked differences in pK(a) for dissociation of the triazole proton. The decrease in pK(a) on halogenation of the peripheral C(4)/C(7) atoms virtually nullifies the gain due to hydrophobic interactions, and does not lead to a decrease in IC(50). Molecular modeling of structures of complexes of the ligands with the enzyme, as well as QSAR analysis, pointed to a balance of hydrophobic and electrostatic interactions as a discriminator of inhibitory activity. The role of halogen bonding remains debatable, as originally noted for the crystal structure of TBBt with CK2α (pdb1j91). Finally we direct attention to the promising applicability of our series of well-defined halogenated benzotriazoles to studies on inhibition of kinases other than CK2
Synthesis and Physico-Chemical Properties in Aqueous Medium of All Possible Isomeric Bromo Analogues of Benzo-1H-Triazole, Potential Inhibitors of Protein Kinases.
Get PDFIn ongoing studies on the role of the individual bromine atoms of 4,5,6,7-tetrabromobenzotriazole (TBBt) in its relatively selective inhibition of protein kinase CK2α, we have prepared all the possible two mono-, four di-, and two tri- bromobenzotriazoles, and determined their physico-chemical properties in aqueous medium. They exhibited a general trend of a decrease in solubility with an increase in the number of bromines on the benzene ring, significantly modulated by the pattern of substitution. For a given number of attached bromines, this was directly related to the electronic effects resulting from different sites of substitution, leading to marked variations of pKa values for dissociation of the triazole proton. Experimental data (pKa, solubility) and ab initio calculations demonstrated that hydration of halogenated benzotriazoles is driven by a subtle balance of hydrophobic and polar interactions. The combination of QM-derived free energies for solvation and proton dissociations was found to be a reasonably good predictor of inhibitory activity of halogenated benzotriazoles vs CK2α. Since the pattern of halogenation of the benzene ring of benzotriazole has also been shown to be one of the determinants of inhibitory potency vs some viruses and viral enzymes, the present comprehensive description of their physico-chemical properties should prove helpful in efforts to elucidate reaction mechanisms, including possible halogen bonding, and the search for more selective and potent inhibitors
Guidelines for the use and interpretation of assays for monitoring autophagy
Get PDFIn 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued
to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been
expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have
described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable
methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a
difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or
autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete
process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result
in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within,
lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is
especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily
equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a
concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity.
Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine
macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are
focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part
on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most
appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we
consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by
discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field
Acute Cytotoxicity Evoked by Tetrabromobisphenol A in Primary Cultures of Rat Cerebellar Granule Cells Outweighs the Effects of Polychlorinated Biphenyls
No full textThe aim of this study was to identify, among selected environmental toxins, the substance with the highest in vitro toxicity to neurons combined with the most marked induction of calcium imbalance, oxidative stress and mitochondrial dysfunction. Exposure of primary cultures of rat cerebellar granule cells for 30 min to polychlorinated biphenyls (PCBs) or brominated flame retardants (BFRs) at concentrations of 10-50 �M identified tetrabromobisphenol A as the compound with the highest toxicity. At a concentration of 25 �M, apart from the moderate activation of 45Ca uptake, this BFR induced the most pronounced increase in intracellular Ca2+ concentration, depolarization of mitochondria, and activation of ROS productio
Two high-resolution crystal structures of potato 1,3-β-glucanase reveal subdomain flexibility with implications for substrate binding
Get PDFEndo-1,3-β-glucanases are widely distributed among bacteria, fungi and higher plants. They are responsible for the hydrolysis of the glycosidic bond in specific polysaccharides with tracts of unsubsituted -1,3-linked glucosyl residues. The plant enzymes belong to the glycoside hydrolase family 17 (GH17) and are also members of class 2 of pathogenesis-related (PR) proteins. For endo-1,3-β-glucanase from Solanum tuberosum (potato, cultivar Désirée), X-ray diffraction data were collected to 1.40 and 1.26 Å resolution for two crystals which, despite a similar packing framework, represent two separate crystal forms. In particular, they differ in the Matthews coefficient and are consequently referred to as higher density (HD, 1.40 Å) and lower density (LD, 1.26 Å) forms. The general fold of the protein resembles that of other known plant endo-1,3--glucanases and is defined by a (β/α)8 barrel with an additional subdomain built around the C-terminal half of the barrel. The structures reveal high flexibility of the subdomain, which forms part of the catalytic cleft. Comparison with other GH17 endo-1,3-beta-glucanase structures reveals differences in the arrangement of the secondary structure elements in this region, which can be correlated with sequence variability and may suggest distinct substrate binding patterns. The crystal structures reveal an unusual packing mode, clearly visible in the LD structure, caused by the presence of the C-terminal His6-tag, which extends from the compact fold of the enzyme molecule and docks in the catalytic cleft of a neighboring molecule. In this way, an infinite chain of His-tag-linked protein molecules is formed along the c direction