1461 research outputs found
Sort by
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
SNF1-related protein kinases type 2 are involved in plant responses to cadmium stress
Get PDFCadmium ions are notorious environmental pollutants. In order to adapt to cadmium-induced deleterious effects plants have developed sophisticated defense mechanisms. However, the signaling pathways underlying the plant response to cadmium are still elusive. Our data demonstrate that SNF1-related protein kinases 2 (SnRK2s) are transiently activated during cadmium exposure and are involved in the regulation of plant response to this stress. Analysis of Nicotiana tabacum Osmotic Stress-Activated Protein Kinase (NtOSAK) activity in tobacco BY-2 cells indicates that reactive oxygen species (ROS) and nitric oxide, produced mainly via an L-arginine-dependent process, contribute to the kinase activation in response to cadmium. SnRK2.4 is the closest homologue of NtOSAK in Arabidopsis thaliana. Comparative analysis of seedling growth of snrk2.4 knockout mutants versus wild type Arabidopsis suggests that SnRK2.4 is involved in the inhibition of root growth triggered by cadmium; the mutants were more tolerant to the stress. Measurements of the level of three major species of phytochelatins in roots of plants exposed to Cd2+ showed a similar (PC2, PC4) or lower (PC3) concentration in snrk2.4 mutants in comparison to wild type plants. These results indicate that the enhanced tolerance of the mutants does not result from a difference in the phytochelatins level. Additionally, we have analyzed ROS accumulation in roots subjected to Cd2+ treatment. Our data show significantly lower Cd2+-induced ROS accumulation in the mutants’ roots. Concluding, the obtained results indicate that SnRK2s play a role in the regulation of plant tolerance to cadmium, most probably by controlling ROS accumulation triggered by cadmium ions
Molecular factors involved in the development of diabetic foot syndrome
Get PDFDiabetes is one of the major challenges of modern medicine,
as it is considered a global epidemic of the XXI
century. The disease often leads to the development of
serious, health threatening complications. Diabetic foot
syndrome is a characteristic set of anatomical and molecular
changes. At the macroscopic level, major symptoms
are neuropathy, ischemia and chronic ulceration
of the lower limb. In every third patient, the neuropathy
develops into Charcot neuroarthropathy characterized
by bone and joints deformation. Interestingly, all these
complications are a result of impaired healing processes
and are characteristic for diabetes. The specificity of
these symptoms comes from impaired molecular mechanisms
observed in type 1 and type 2 diabetes. Decreased
wound and fracture healing reflect gene expression, cellular
response, cell functioning and general metabolism.
Here we present a comprehensive literature update on
the molecular factors contributing to diabetic foot syndrome
On the aromatic stabilization of benzenoid hydrocarbons
Get PDFA general scheme for estimation of aromatic stabilization energies of benzenoid hydrocarbons based on selected topological features has been presented. The reactions have been applied to benzene, naphthalene, anthracene, phenanthrene, pyrene, tetracene, benz[a]anthracene, chrysene, [4]-helicene, anthanthrene and coronene
Crystal structure and mechanism of action of the N6-methyladenine-dependent type IIM restriction endonuclease R.DpnI.
Get PDFDNA methylation-dependent restriction enzymes have many applications in genetic engineering and in the analysis of the epigenetic state of eukaryotic genomes. Nevertheless, high-resolution structures have not yet been reported, and therefore mechanisms of DNA methylation-dependent cleavage are not understood. Here, we present a biochemical analysis and high-resolution DNA co-crystal structure of the N(6)-methyladenine (m6A)-dependent restriction enzyme R.DpnI. Our data show that R.DpnI consists of an N-terminal catalytic PD-(D/E)XK domain and a C-terminal winged helix (wH) domain. Surprisingly, both domains bind DNA in a sequence- and methylation-sensitive manner. The crystal contains R.DpnI with fully methylated target DNA bound to the wH domain, but distant from the catalytic domain. Independent readout of DNA sequence and methylation by the two domains might contribute to R.DpnI specificity or could help the monomeric enzyme to cut the second strand after introducing a nick
Phage lambda—New Insights into Regulatory Circuits
Get PDFBacteriophage l, rediscovered in the early 1950s, has served as a model in molecular biology studies for decades. Although currently more complex organisms and more complicated biological systems can be studied, this phage is still an excellent model to investigate principles of biological processes occurring at the molecular level. In fact, very few other biological models provide possibilities to examine regulations of biological mechanisms as detailed as performed with l. In this chapter, recent advances in our understanding of mechanisms of bacteriophage l development are summarized and discussed. Particularly, studies on (i) phage DNA
injection, (ii) molecular bases of the lysis-versus-lysogenization decision and the lysogenization process itself, (iii) prophage maintenance and induction, (iv), l DNA replication, (v) phage-encoded recombination systems, (vi) transcription antitermination, (vii) formation
of the virion structure, and (viii) lysis of the host cell, as published during several past years, will be presented
Damage of DNA and proteins by major lipid peroxidation products in genome stability
Get PDFOxidative stress and lipid peroxidation (LPO) accompanying infections and chronic inflammation may induce several human cancers. LPO products are characterized by carbohydrate chains of different length, reactive aldehyde groups and double bonds, which make these molecules reactive to nucleic acids, proteins and cellular thiols. LPO-derived adducts to DNA bases form etheno-type and propano-type exocyclic rings, which have profound mutagenic potential, and are elevated in several cancer-prone diseases. Adducts of long chain LPO products to DNA bases inhibits transcription. Elimination from DNA of LPO-induced lesions is executed by several repair systems: base excision repair (BER), direct reversal by AlkB family proteins, nucleotide excision repair (NER) and recombination. Modifications of proteins with LPO products may regulate cellular processes like apoptosis, cell signaling and senescence. This review summarizes consequences of LPO products presence in cell, particularly 4-hydroxy-2-nonenal in terms of genomic stability
Thermodynamic Study of Cu2+-Binding to the DAHK and GHK Peptides by Isothermal Titration Calorimetry (ITC) with the Weaker Competitor Glycine
Get PDFThe peptides Asp-Ala-His-Lys (DAHK) and Gly-His-Lys (GHK) are naturally occurring copper(II)-chelating motives in human serum and cerebrospinal fluid. Here the sensitive thermodynamic technique isothermal titration calorimetry (ITC) has been used to study the energetics of copper(II) binding to DAHK and GHK peptides in the presence of the weaker ligand glycine as a competitor. DAHK and GHK bind Cu(II) predominantly in a 1:1 stoichiometry with conditional dissociation constants (i.e. at pH 7.4, in the absence of the competing chelators glycine and HEPES buffer) of 2.6 +/- 0.4 x 10-14 M and of 7.0 +/- 1.0 x 10-14 M, respectively. Furthermore, the apparent ΔH values were measured and the number of protons released upon Cu(II) binding was determined by performing experiments in different buffers. This allowed us to determine the conditional ΔG, ΔH, and ΔS, i.e. corrected for the contributions of the weaker ligand glycine and the buffer at pH 7.4. We found that the entropic and enthalpic contributions to the Cu(II)-binding to GHK and DAHK are distinct, with a higher enthalpic contribution for GHK. The obtained thermodynamic parameters correspond well to those in the literature obtained by other techniques, suggesting that the use of the weaker ligand glycine as a competitor in ITC provides accurate data for Cu(II)-binding to high affinity peptides, which cannot be accurately determined without the use of a competitor ligand
The contribution of microbial mats to the arsenic geochemistry of an ancient gold mine
Get PDFThe ancient Zloty Stok (SW Poland) gold mine is such an environment, where different
microbial communities, able to utilize inorganic arsenic species As(III) and As(V), are found.
The purpose of the present study was to (i) estimate the general diversity of microbial mats in
bottom sediments of this gold mine, (ii) identify microorganisms that can metabolize arsenic, and
(iii) estimate their potential role in the arsenic geochemistry of the mine and in the environment.
The oxidation/reduction experiments showed that the microbial mat community may
significantly contribute to arsenic contamination in groundwater. The presence of both arsenite
oxidizing and dissimilatory arsenate reducing bacteria in the mat was confirmed by the detection
of arsenite oxidase and dissimilatory arsenate reductase genes, respectively. This work also
demonstrated that microorganisms utilizing other compounds that naturally co-occur with
arsenic are present within the microbial mat community and may contribute to the arsenic
geochemistry in the environment
Gene expression-targeted isoflavone therapy
Get PDFLysosomal storage diseases (LSD) form a group of inherited
metabolic disorders caused by dysfunction of one of the lysosomal proteins, resulting in the accumulation of certain
compounds. Although these disorders are among first genetic
diseases for which specific treatments were proposed, there
are still serious unsolved problems that require development
of novel therapeutic procedures. An example is neuronopathy,
which develops in most of LSD and cannot be treated efficiently by currently approved therapies. Recently, a new
potential therapy, called gene expression-targeted isoflavone therapy (GET IT), has been proposed for a group of LSD named mucopolysaccharidoses (MPS), in which storage of incompletely degraded glycosaminoglycans (GAGs) results in severe symptoms of virtually all tissues and organs, including central nervous system. The idea of this therapy is to inhibit synthesis of GAGs by modulating expression of genes coding for enzymes involved in synthesis of these compounds. Such a modulation is possible by using isoflavones, particularly genistein, which interfere with a signal transduction process necessary for stimulation of expression of certain genes. Results of in vitro experiments and studies on animal models indicated a
high efficiency of GET IT, including correction of behavior of affected mice. However, clinical trials, performed with soy isoflavone extracts, revealed only limited efficacy. This caused a controversy about GET IT as a potential, effective treatment of patients suffering from MPS, especially neuronopathic forms of these diseases. It this critical review, I present possible molecular mechanisms of therapeutic action of isoflavones (particularly genistein) and suggest that efficacy of GET IT might be sufficiently high when using relatively high doses of synthetic genistein (which was employed in experiments on cell cultures and mouse models) rather than low doses of soy isoflavone extracts (which were used in clinical trials). This proposal can be tested in double-blinded, placebo-controlled clinical trials