Amino acid substitutions [K16A] and [K28A] distinctly affect amyloid <Emphasis Type="Italic">β</Emphasis>-protein oligomerization

Amyloid β-protein (A β) assembles into oligomers that play a seminal role in Alzheimer’s disease (AD), a leading cause of dementia among the elderly. Despite undisputed importance of A β oligomers, their structure and the basis of their toxicity remain elusive. Previous experimental studies revealed that the [K16A] substitution strongly inhibits toxicity of the two predominant A β alloforms in the brain, A β ₄₀ and A β ₄₂, whereas the [K28A] substitution exerts only a moderate effect. Here, folding and oligomerization of [A16]A β ₄₀, [A28]A β ₄₀, [A16]A β ₄₂, and [A28]A β ₄₂ are examined by discrete molecular dynamics (DMD) combined with a four-bead implicit solvent force field, DMD4B-HYDRA, and compared to A β ₄₀ and A β ₄₂ oligomer formation. Our results show that both substitutions promote A β ₄₀ and A β ₄₂ oligomerization and that structural changes to oligomers are substitution- and alloform-specific. The [K28A] substitution increases solvent-accessible surface area of hydrophobic residues and the intrapeptide N-to-C terminal distance within oligomers more than the [K16A] substitution. The [K16A] substitution decreases the overall β-strand content, whereas the [K28A] substitution exerts only a modest change. Substitution-specific tertiary and quaternary structure changes indicate that the [K16A] substitution induces formation of more compact oligomers than the [K28A] substitution. If the structure-function paradigm applies to A β oligomers, then the observed substitution-specific structural changes in A β ₄₀ and A β ₄₂ oligomers are critical for understanding the structural basis of A β oligomer toxicity and correct identification of therapeutic targets against AD.     

The stacking interactions of bipyridine complexes: the influence of the metal ion type on the strength of interactions

The strength of the stacking interactions in the bipy complexes of nickel, palladium, and platinum, [M(CN)₂ bipy]₂ (M?=?Ni, Pd, Pt), was calculated using the ωB97xD/def2-TZVP method. The results show that for all considered geometries, interactions are the strongest for platinum, and weakest for nickel complexes, as a result of higher dispersion contributions of platinum over the palladium and nickel complexes. It was also shown that strength of interactions considerably rises with an increase of the stacking overlap area. As a consequence of the favorable electrostatic term, the strength of interactions also rises when metal atom and cyano ligands are involved in the overlap with bipy ligand. The strongest interaction was calculated in the platinum complex, for the geometry that has overlap of metal and cyano ligands with bipy ligand with an energy of -39.80 kcal mol^-1. The energies for similar geometries of palladium and nickel complexes are -34.60 and -32.45 kcal mol^-1. These energies, remarkably, exceed the strength of the stacking interactions between organic aromatic molecules. These results can be of importance in all systems with stacking interactions, from materials to biomolecules.     

Quantum-chemical study of interactions of <Emphasis Type="Italic">trans-</Emphasis>resveratrol with guanine-thymine dinucleotide and DNA-nucleobases

Trans-resveratrol, a natural phytoalexin present in red wine and grapes, has gained considerable attention because of its antiproliferative, chemopreventive and proapoptotic activity against human cancer cells. The accurate quantum-chemical computations based on the density functional theory (DFT) and ab initio second-order Møller-Plesset perturbation method (MP2) have been performed for the first time to study interactions of trans-resveratrol with guanine-thymine dinucleotide and DNA-derived nitrogenous bases: adenine, guanine, cytosine and thymine in vacuum and water medium. This compound is found to show high affinity to nitrogenous bases and guanine-thymine dinucleotide. The electrostatic interactions from intermolecular hydrogen bonding increase the stability of complexes studied. In particular, significantly strong hydrogen bonds between 4′-H atom of trans-resveratrol and imidazole nitrogen as well as carbonyl oxygen atoms of nucleobases studied stabilize these systems. The stabilization energies computed reveal that the negatively charged trans-resveratrol-dinucleotide complex is more energetically stable in water medium than in vacuum. MP2 method gives more reliable and significantly high values of stabilization energy of trans-resveratrol-dinucleotide, trans-resveratrol-guanine and trans-resveratrol-thymine complexes than B3LYP exchange-correlation functional because it takes into account London dispersion energy. According to the results, in the presence of trans-resveratrol the 3′-5′ phosphodiester bond in dinucleotide can be cleaved and the proton from 4′-OH group of trans-resveratrol migrates to the 3′-O atom of dinucleotide. It is concluded that trans-resveratrol is able to break the DNA strand. Hence, the findings obtained help understand antiproliferative and anticancer properties of this polyphenol.     

Molecular biological taxonomy of some free-living nitrogen-fixing bacteria

A novel approach to the taxonomy of several free-living nitrogen-fixing bacteria is proposed on the basis of two DNA parameters. 1) DNA base composition, expressed as average molar (guanine + cytosine) content, determined by thermal denaturation and 2) DNA homology, determined by DNA hybridization with bothPseudomonas fluorescens andPseudomonas putida. The following taxonomic conclusions emerged:

1. 1.
   The existence ofBeijerinckia andDerxia as individual genera seems justified.     
   
   

The mathematical model of concentration polarization coefficient in membrane transport and volume flows

In this paper, the authors investigate the membrane transport of aqueous non-electrolyte solutions in a single-membrane system with the membrane mounted horizontally. The purpose of the research is to analyze the influence of volume flows on the process of forming concentration boundary layers (CBLs). A mathematical model is provided to calculate dependences of a concentration polarization coefficient (ζ _s ) on a volume flux (J _vm ), an osmotic force (Δπ) and a hydrostatic force (ΔP) of different values. Property ζ _s ?=?f(J _vm ) for J _vm ?>?0 and for J _vm ?≈?0 and property ζ _s ?=?f(ΔC ₁) are calculated. Moreover, results of a simultaneous influence of ΔP and Δπ on a value of coefficient ζ _s when J _vm ?=?0 and J _vm ?≠?0 are investigated and a graphical representation of the dependences obtained in the research is provided. Also, mathematical relationships between the coefficient ζ _s and a concentration Rayleigh number (R _C ) were studied providing a relevant graphical representation. In an experimental test, aqueous solutions of glucose and ethanol were used.     

A novel NADPH-dependent reductase of <Emphasis Type="Italic">Sulfobacillus acidophilus</Emphasis> TPY phenol hydroxylase: expression,characterization, and functional analysis

The reductase component (MhpP) of the Sulfobacillus acidophilus TPY multicomponent phenol hydroxylase exhibits only 40 % similarity to Pseudomonas sp. strain CF600 phenol hydroxylase reductase. Amino acid sequence alignment analysis revealed that four cysteine residues (Cys-X ₄ -Cys-X ₂ -Cys-X _29-35 -Cys) are conserved in the N terminus of MhpP for [2Fe-2S] cluster binding, and two other motifs (RXYS and GXXS/T) are conserved in the C terminus for binding the isoalloxazine and phosphate groups of flavin adenine dinucleotide (FAD). Two motifs (S/T-R and yXCGp) responsible for binding to reduce nicotinamide adenine dinucleotide phosphate (NADPH) are also conserved in MhpP, although some residues differ. To confirm the function of this reductase, MhpP was heterologously expressed in Escherichia coli BL21(DE3) and purified. UV-visible spectroscopy and electron paramagnetic resonance spectroscopy revealed that MhpP contains a [2Fe-2S] cluster. MhpP mutants in which the four cysteine residues were substituted via site-directed mutagenesis lost the ability to bind the [2Fe-2S] cluster, resulting in a decrease in enzyme-specific oxidation of NADPH. Thin-layer chromatography revealed that MhpP contains FAD. Substrate specificity analyses confirmed that MhpP uses NADPH rather than NADH as an electron donor. MhpP oxidizes NADPH using cytochrome c, potassium ferricyanide, or nitro blue tetrazolium as an electron acceptor, with a specific activity of 1.7 ± 0.36, 0.78 ± 0.13, and 0.16 ± 0.06 U/mg, respectively. Thus, S. acidophilus TPY MhpP is a novel NADPH-dependent reductase component of phenol hydroxylase that utilizes FAD and a [2Fe-2S] cluster as cofactors.     

Altered somatic mutation level and DNA repair gene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> exposed to ultraviolet C,salt, and cadmium stresses

It is known that somatic mutations arising during animal growth and ageing contribute to the development of neurodegenerative and other animal diseases. For plants, several studies showed that small-scale somatic DNA mutations accumulated during Arabidopsis life cycle. However, there is a lack of data on the influence of environmental stresses on somatic DNA mutagenesis in plants. In this study, we analyzed the effects of ultraviolet C (UV-C) irradiation, high soil salinity, and cadmium (CdI₃) stresses on the level of small-scale somatic DNA mutations in Arabidopsis thaliana. The number of DNA mutations was examined in the Actin2 3′UTR (Actin-U1), ITS1-5.8rRNA-ITS2 (ITS), and ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) DNA regions. We found that somatic mutation levels considerably increased in CdI₃-treated Arabidopsis plants, while the mutation levels declined in the UV-C- and NaCl-treated A. thaliana. Cadmium is a mutagen that is known to inhibit DNA repair processes. The detected stress-induced alterations in somatic DNA mutation levels were accompanied by markedly increased expression of base excision repair genes (AtARP, AtDME, AtDML2, AtDML3, AtMBD4, AtROS, AtUNG, and AtZDP), nucleotide excision repair genes (AtDDB1a, AtRad4, and AtRad23a), mismatch repair genes (AtMSH2, AtMSH3, and AtMSH7), and photoreactivation genes (AtUVR2, AtUVR3). Thus, the results demonstrated that UV-C, high soil salinity, and cadmium stresses influence both the level of DNA mutations and expression of DNA repair genes. Salt- and UV-induced activation of DNA repair genes could contribute to the stress-induced decrease in somatic mutation level.     

Natively oxidized amino acid residues in the spinach cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex

The cytochrome b ₆ f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b ₆ f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc₁ complex. The types of ROS produced (O₂^{??, 1}O₂, and, possibly, H₂O₂) and the site(s) of ROS production within the b ₆ f complex have been the subject of some debate. Proposed sources of ROS have included the heme b _p , PQ _p ^?? (possible sources for O₂^??), the Rieske iron–sulfur cluster (possible source of O₂^?? and/or ¹O₂), Chl a (possible source of ¹O₂), and heme c _n (possible source of O₂^?? and/or H₂O₂). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b ₆ f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b ₆ f complex.     

The single-stranded DNA-binding protein of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Background

Deinococcus radiodurans R1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage without induced mutation. Single-stranded DNA-binding (SSB) protein is an essential protein in all organisms and is involved in DNA replication, recombination and repair. The published genomic sequence from Deinococcus radiodurans includes a putative single-stranded DNA-binding protein gene (ssb; DR0100) requiring a translational frameshift for synthesis of a complete SSB protein. The apparently tripartite gene has inspired considerable speculation in the literature about potentially novel frameshifting or RNA editing mechanisms. Immediately upstream of the ssb gene is another gene (DR0099) given an ssb-like annotation, but left unexplored.

Results

A segment of the Deinococcus radiodurans strain R1 genome encompassing the ssb gene has been re-sequenced, and two errors involving omitted guanine nucleotides have been documented. The corrected sequence incorporates both of the open reading frames designated DR0099 and DR0100 into one contiguous ssb open reading frame (ORF). The corrected gene requires no translational frameshifts and contains two predicted oligonucleotide/oligosaccharide-binding (OB) folds. The protein has been purified and its sequence is closely related to the Thermus thermophilus and Thermus aquaticus SSB proteins. Like the Thermus SSB proteins, the SSB_Dr functions as a homodimer. The Deinococcus radiodurans SSB homodimer stimulates Deinococcus radiodurans RecA protein and Escherichia coli RecA protein-promoted DNA three-strand exchange reactions with at least the same efficiency as the Escherichia coli SSB homotetramer.

Conclusions

The correct Deinococcus radiodurans ssb gene is a contiguous open reading frame that codes for the largest bacterial SSB monomer identified to date. The Deinococcus radiodurans SSB protein includes two OB folds per monomer and functions as a homodimer. The Deinococcus radiodurans SSB protein efficiently stimulates Deinococcus radiodurans RecA and also Escherichia coli RecA protein-promoted DNA strand exchange reactions. The identification and purification of Deinococcus radiodurans SSB protein not only allows for greater understanding of the SSB protein family but provides an essential yet previously missing player in the current efforts to understand the extraordinary DNA repair capacity of Deinococcus radiodurans.

     

Interaction of single-stranded DNA with the second DNA-binding site of the RecA nucleoprotein filament

RecA first forms a filament on single-stranded DNA (ssDNA), thereby forming the first site for ssDNA binding and, simultaneously, the second site for binding double-stranded DNA (dsDNA). Then, the nucleoprotein filament interacts with dsDNA, although it can bind ssDNA as well. The resulting complex searches for homology sites and performs strand exchange between homologous DNA molecules. The interaction of various ssDNAs with the second DNA-recognizing site of RecA was studied by gradually increasing the structural complexity of the DNA ligand. Recognizing ssDNA with the second site, the protein interacts with each nucleotide of the ligand, forming contacts with both internucleotide phosphate groups and nitrogen bases. Pyrimidine oligonucleotides d(pC) _n and d(pT) _n interacted with the second site of the RecA filament more efficiently than d(pA) _n did. This was due to a more efficient interaction of the RecA filament with the 5′-terminal nucleotide of pyrimidinic DNA and to the difference in specific conformational changes of the nucleoprotein filament in the presence of purinic and pyrimidinic DNAs. A comparison of thermodynamic characteristics of DNA recognition at the first and second DNA-binding sites of the filament showed that, at n > 10, d(pC) _n and d(pN) _n were bound at the second site less tightly than at the first site. At n > 20, the second site bound d(pA) _n more efficiently than the first site. The difference in d(pN) _n affinity for the first and second sites increased monotonically with increasing n. Possible mechanisms of a RecA-dependent search for homology and DNA strand exchange are discussed.     

Analysis of the expression of the <Emphasis Type="Italic">Rhodobacter sphaeroides lexA</Emphasis> gene

The regulation of the Rhodobacter sphaeroides lexA gene has been analyzed using both gel-mobility experiments and lacZ gene fusions. PCR-mediated mutagenesis demonstrated that the second GAAC motif in the sequence GAACN₇GAACN₇GAAC located upstream of the R. sphaeroides lexA gene is absolutely necessary for its DNA damage-mediated induction. Moreover, mutagenesis of either the first or the third GAAC motif in this sequence reduced, but did not abolish, the inducibility of the R. sphaeroides lexA gene. A R. sphaeroides lexA-defective (Def) mutant has also been constructed by replacing the active lexA gene with an inactivated gene copy constructed in vitro. Crude extracts of the R. sphaeroides lexA(Def) strain are unable to form any protein-DNA complex when added to the wild-type lexA promoter of R. sphaeroides. Likewise, the R. sphaeroides lexA(Def) cells constitutively express the recA and lexA genes. All these data clearly indicate that the lexA gene product is the negative regulator of the R. sphaeroides SOS response. Furthermore, the morphology, growth and viability of R. sphaeroides lexA(Def) cultures do not show any significant change relative to those of the wild-type strain. Hence, R. sphaeroides is so far the only bacterial species whose viability is known not to be affected by the presence of a lexA(Def) mutation.     

Purification and properties of recombinant DNA methyltransferase M2.<Emphasis Type="Italic">Bst</Emphasis>SE of the <Emphasis Type="Italic">Bst</Emphasis>SEI nickase-modification system

The operon for the Bacillus stearothermophilus SE-589 nickase-modification system (NM.BstSEI, recognition site 5′-GAGTC-3′) includes two DNA methyltransferase (M.) genes, bstSEIM1 and bstSEIM2. The gene encoding M2.BstSEI was cloned in pJW and expressed in Escherichia coli cells. M2.BstSEI was purified by chromatography and displayed maximal activity at 55° C and pH 7.5. The enzyme modified adenine in the nickase recognition site 5′-GAGTC-3′ and was specific for 5′-GASTC-3′ substrates. The kinetic parameters of the methylation reaction were determined. The catalytic constant was 2.2 min^?1, and the Michaelis constant was 9.8 nM on T7 DNA and 5.8 μM on SAM.     

Genetic differentiation among natural populations of the lizard complex <Emphasis Type="Italic">Darevskia raddei</Emphasis> as inferred from genome microsatellite marking

The article presents the genetic parameters of the populations of lizards of the Darevskia raddei complex (D. raddei nairensis and D. raddei raddei) and the populations of D. valentini calculated on the basis of the analysis of variability of 50 allelic variants of the three nuclear genome microsatellite-containing loci of 83 individuals. It was demonstrated that the F_st genetic distances between the populations of D. raddei nairensis and D. raddei raddei were not statistically significantly different from the F_st genetic distances between the populations of different species, D. raddei and D. valentini. At the same time, these distances were statistically significantly higher than the F_st distances between the populations belonging to one species within the genus Darevskia. These data suggest deep divergence between the populations of D. raddei raddei and D. raddei nairensis of the D. raddei complex and there arises the question on considering them as separate species.