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AlkB dioxygenase preferentially repairs protonated substrates: specificity against exocyclic adducts and molecular mechanism of action.
Get PDFEfficient repair by AlkB dioxygenase of exocyclic DNA adducts: 3,N(4)-ethenocytosine, 1,N(6)-ethenoadenine, 3,N(4)- α-hydroxyethanocytosine and, reported here for the first time, 3,N(4)- α-hydroxypropanocytosine, requires higher Fe(II) concentration than the reference 3-methylcytosine. The pH optimum for the repair follows the order of pK(a) values for protonation of the adduct suggesting that positively charged substrates favorably interact with the negatively charged carboxylic group of Asp135 side-chain in the enzyme active centre. This interaction is supported by molecular modeling indicating that 1,N(6)-ethenoadenine and 3,N(4)-ethenocytosine are bound to AlkB more favorably in their protonated cationic forms. An analysis of the pattern of intermolecular interactions that stabilize the location of the ligand points to a role of Asp135 in recognition of the adduct in its protonated form. Moreover, also ab initio calculations underline the role of substrate protonation in lowering the free energy barrier of the transition state of epoxidation of the ethenoadducts studied. The observed time-courses of repair of racemic mixtures of 3,N(4)- α-hydroxyethanocytosine or 3,N(4)- α-hydroxypropanocytosine are unequivocally two-exponential, indicating that the respective stereoisomers are repaired by AlkB with different efficiencies. Molecular modeling of these adducts bound by AlkB allowed evaluation of the participation of their possible conformational states in the enzymatic reaction
The quantum casimir effect may be a universal force organizing the bilayer structure of the cell membrane.
Get PDFA mathematic-physical model of the interaction between cell membrane bilayer leaflets is proposed based on the Casimir effect in dielectrics. This model explains why the layers of a lipid membrane gently slide one past another rather than penetrate each other. The presented model reveals the dependence of variations in the free energy of the system on the membrane thickness. This function is characterized by the two close minima corresponding to the different levels of interdigitation of the lipids from neighbor layers. The energy barrier of the compressing transition between the predicted minima is estimated to be 5.7 kT/lipid, and the return energy is estimated to be 3.1 kT/lipid. The proposed model enables estimation of the value of the membrane elastic thickness modulus of compressibility, which is 1.7 × 10(9) N/m(2), and the value of the interlayer friction coefficient, which is 1.9 × 10(8) Ns/m(3)
Cohesin Irr1/Scc3 is likely to influence transcription in Saccharomyces cerevisiae via interaction with Mediator complex.
Get PDFThe evolutionarily conserved proteins forming sister chromatid cohesion complex are also involved in the regulation of gene transcription. The participation of SA2p (mammalian ortholog of yeast Irr1p, associated with the core of the complex) in the regulation of transcription is already described. Here we analyzed microarray profiles of gene expression of a Saccharomyces cerevisiae irr1-1/IRR1 heterozygous diploid strain. We report that expression of 33 genes is affected by the presence of the mutated Irr1-1p and identify those genes. This supports the suggested role of Irr1p in the regulation of transcription. We also indicate that Irr1p may interact with elements of transcriptional coactivator Mediator
Diaspores of the Introduced Species Poa annua L. in Soil Samples from King George Island (South Shetlands, Antarctica)
Get PDFThe soil seed bank and seed germination capacity of Poa annua in the vicinity of the Polish Antarctic Station (South Shetlands, Antarctica) were investigated. It was documented that annual bluegrass can reproduce sexually and produce a functional seed bank of close to 5000 seeds/m2 under maritime Antarctic conditions. Comparison of germination between Poa annua and two native plant species revealed that Poa annua seeds can germinate as fast or even faster than native species, and are more vigorous. Our studies show that in the Antarctic Poa annua can successfully reproduce sexually and produce fully developed, viable caryopses that are able to survive the maritime Antarctic winter, not only in a soil bank, but also directly in the previous year’s inflorescences
Self-adjuvanting influenza candidate vaccine presenting epitopes forcell-mediated immunity on a proteinaceous multivalentnanoplatform
Get PDFWe exploit the features of a virus-like particle, adenoviral dodecahedron (Ad Dd), for engineering amultivalent vaccination platform carrying influenza epitopes for cell-mediated immunity. The deliveryplatform, Ad Dd, is a proteinaceous, polyvalent, and biodegradable nanoparticle endowed with remark-able endocytosis activity that can be engineered to carry 60 copies of a peptide. Influenza M1 is the mostabundant influenza internal protein with the conserved primary structure. Two different M1 immuno-dominant epitopes were separately inserted in Dd external positions without destroying the particles’dodecahedric structure. Both kinds of DdFluM1 obtained through expression in baculovirus system wereproperly presented by human dendritic cells triggering efficient activation of antigen-specific T cellsresponses. Importantly, the candidate vaccine was able to induce cellular immunity in vivo in chickens.These results warrant further investigation of Dd as a platform for candidate vaccine, able to stimulatecellular immune responses
Single Electrode Genosensor for Simultaneous Determination of Sequences Encoding Hemagglutinin and Neuraminidase of Avian Influenza Virus Type H5N1
No full textThe duo-genosensor consisting of two different oligonucleotide probes immobilized covalently on the surface of one gold electrode via Au–S bond formation was used for simultaneous determination of two different oligonucleotide targets. One of the probes, decorated on its 5′-end with ferrocene (SH-ssDNA-Fc), is complementary to the cDNA representing a sequence encoding part of H5 hemagglutinin from H5N1 virus. The second probe, decorated on its 5′-end with methylene blue (SH-ssDNA-MB), is complementary to cDNA representing the fragment of N1 neuraminidase from the same virus. The presence of both probes on the surface of gold electrodes was confirmed with Osteryoung square-wave voltammetry (OSWV). The changes in redox activity of both redox active complexes before and after the hybridization process were used as analytical signal. The peak at +400 ± 2 mV was observed in the presence of 40 nM ssDNA used as a target for SH-ssDNA-Fc probe. This peak increased with the increase of concentration of target ssDNA. It indicates the “signal on” mode of analytical signal generation. The peak at −250 ± 4 mV, characteristic for SH-ssDNA-MB probe, was decreasing with the increase of the concentration of the complementary ssDNA target starting from 8 to 100 nM. This indicates the generation of electrochemical signal according to the “signal off” mode. The proposed duo-genosensor is capable of simultaneous, specific, and good sensitivity probing for the sequences derived from genes encoding two main markers of the influenza virus, hemagglutinin and neuraminidase
Revised Coordination Model and Stability Constants of Cu(II) Complexes of Tris Buffer
No full text2-Amino-2-hydroxymethyl-propane-1,3-diol, or tris(hydroxymethyl)aminomethane (Tris), is probably the most common biochemical buffer used alone or in combination with other buffers because it is stable, unreactive, and compatible with most proteins and other biomolecules. Being nontoxic, it has even found applications in medicine. Tris is known, however, to coordinate transition metal ions, Cu(II) among them. Although often ignored, this feature affects interactions of Cu(II) ions with biomolecules, as Tris is usually used in high molar excess. Therefore, it is important to have precise knowledge on the stoichiometry, stability, and reactivity of cupric Tris complexes. The literature data are incoherent in this respect. We reinvestigated the complex formation in the Tris-Cu(II) system by potentiometry, UV-vis, ESI-MS, and EPR at a broad range of concentrations and ratios. We found, contrary to several previous papers, that the maximum stoichiometry of Tris to Cu(II) is 2 and at neutral pH, dimeric complexes are formed. The apparent affinity of Tris buffer for Cu(II), determined by the competitivity index (CI) approach [Krężel, A.; Wójcik, J.; Maciejczyk, M.; Bal, W. Chem. Commun. 2003, 6, 704-705] at pH 7.4 varies between 2 × 10(6) and 4 × 10(4) M(-1), depending on the Tris and Cu(II) concentrations and molar ratio
Vertical microzonation of ciliates in cryoconite holes in Ecology Glacier, King George Island
Get PDFThere are hardly any data concerning the vertical micro−distribution of protozoa in
water column in cryoconite holes on the glacier surface. Such comparisons can provide in−
sights into the ecology of protozoa. The present research wasmade on Ecology Glacier (South
Shetland Islands, Antarctic); vertical microzonation of ciliates in relation to physical and
chemical parameters in cryoconite holes was studied. The density and biomass of protozoans
significantly differed between the studied stations (cryoconite holes), with the lowest num−
bers in the surface water and the highest in the bottom water. The surface waters were domi−
nated by mixotrophic and omnivorous taxa,whereas the deepest sampling level has shown the
increase of the proportion of bacterivore species. Ordination analysis indicated that TN and
P−PO4 can strongly regulate the abundance and species composition of protozoa. The redun−
dancy analyses (RDA) showed that the ciliate communities can be separated into two groups.
The first group included species associated with surface water: Halteria grandinella and
Codonella sp. The second group included species that are associated with bottom water:
Prorodon sp., Holosticha pullaster, Stylonychia mytilus−complex and small scuticociliates
Global transcriptional regulator KorC coordinates expression of three backbone modules of the broad-host-range RA3 plasmid from IncU incompatibility group.
Get PDFThe broad-host-range conjugative RA3 plasmid from IncU incompatibility group has been
isolated from the fish pathogen Aeromonas hydrophila. DNA sequencing has revealed a
mosaic modular structure of RA3 with the stabilization module showing some similarity
to IncP-1 genes and the conjugative transfer module highly similar to that from PromA
plasmids. The integrity of the mosaic plasmid genome seems to be specified by its regulatory
network. In this paper the transcriptional regulator KorC was analyzed. KorCRA3 (98
amino acids) is encoded in the stabilization region and represses four strong promoters
by binding to a conserved palindrome sequence, designated OC on the basis of homology
to the KorC operator sequences in IncP-1 plasmids. Two of the KorCRA3-regulated promoters
precede the first two cistrons in the stabilization module, one fires towards replication
module, remaining one controls a tricistronic operon, whose products are involved in the
conjugative transfer process. Despite the similarity between the binding sites in IncU
and IncP-1 plasmids, no cross-reactivity between their KorC proteins has been detected.
KorC emerges as a global regulator of RA3, coordinating all its backbone functions: replication,
stable maintenance and conjugative transfer