Ribonuclease binase apoptotic signature in leukemic Kasumi-1 cells

Cytotoxic exogenous RNases triggering apoptotic response in malignant cells have potential as anticancer drugs; surprisingly, detailed characterization of the RNase-induced apoptosis has not been conducted so far. Here we show that a cytotoxic RNase from Bacillus intermedius (binase) induces extrinsic and intrinsic apoptotic pathways in leukemic Kasumi-1 cells. The experiments were performed using TaqMan Array Human Apoptosis 96-well Plate for gene expression analysis, and flow cytometry. Cytometric studies demonstrated dissipation of the mitochondrial membrane potential, opening of mitochondrial permeability transition pores, activation of caspases, increase of intracellular Ca²⁺ and decrease of reactive oxygen species levels. We found that expression of 62 apoptotic genes is up-regulated, including 16 genes that are highly up-regulated, and only one gene was found to be down-regulated. The highest, 16 fold increase of the expression level was observed for TNF gene. Highly up-regulated genes also include the non-canonical NF-κB signaling pathway and inflammatory caspases 1,4. The obtained results suggest that binase induces evolutionary acquired cellular response to a microbial agent and triggers unusual apoptosis pathway.     

Delivery and processing of exogenous double-stranded DNA in mouse CD34 + hematopoietic progenitor cells and their cell cycle changes upon combined treatment with cyclophosphamide and double-stranded DNA

We previously reported that fragments of exogenous double-stranded DNA can be internalized by mouse bone marrow cells without any transfection. Our present analysis shows that only 2% of bone marrow cells take up the fragments of extracellular exogenous DNA. Of these, ~ 45% of the cells correspond to CD34 + hematopoietic stem cells. Taking into account that CD34 + stem cells constituted 2.5% of the total cell population in the bone marrow samples analyzed, these data indicate that as much as 40% of CD34 + cells readily internalize fragments of extracellular exogenous DNA. This suggests that internalization of fragmented dsDNA is a general feature of poorly differentiated cells, in particular CD34 + bone marrow cells.     

Characterization of the Structure and Melting Behavior of the Loop I fragment of ColE1 RNA I

Abstract

We have synthesized two RNA fragments: a 42-mer corresponding to the full loop I sequence of the loop I region of ColE1 antisense RNA (RNA I), plus three additional Gs at the 5′-end, and a 31-mer which has 11 5′-end nucleotides (G(-2)-U9) deleted. The secondary structure of the 42-mer, deduced from one- and two-dimensional NMR spectra, consists of a stem of 11 base-pairs which contains a U-U base-pair and a bulged C base, a 7 nucleotide loop, and a single-stranded 5′ end of 12 nucleotides. The UV-melting study of the 42-mer further revealed a multi-step melting behavior with transition temperatures 32°C and 71°C clearly discernible. In conjunction with NMR melting study the major transition at 71°C is assigned to the overall melting of the stem region and the 32°C transition is assigned to the opening of the loop region. The deduced secondary structure agrees with that proposed for the intact RNA I and provides structural bases for understanding the specificity of RNase E.     

Comparison of Complex DNA Mixtures with Generic Oligonucleotide Microchips

Abstract

The reproducibility of melting curves for repeated hybridizations of target DNA with generic oligonucleotide microchips is shown experimentally to depend on the character of matching between fragments of target DNA and immobilized oligonucleotides. The reproducibility of melting curves is higher for the perfect match duplexes and decreases as the number of mismatched pairs within duplexes increases. This effect was applied to the comparative analysis of complex DNA mixtures. We developed a scheme in which we can identify and discriminate between the probe oligonucleotides responsible for the distinctions between target DNA mixtures. A scheme is illustrated by comparing DNA mixtures corresponding to VD-J genes connected with populations of mRNAs CDR3 TCR Vb (T-cell receptor beta complementarity determining region 3) from the thymus and pancreas of NOD mice. Our results demonstrate that generic microchips can be applied efficiently to the analysis of DNA mixtures.     

96 Study of the stacking of DNA homoassociates by the extended cluster approach

Metropolis Monte Carlo method based on the extended cluster approach (Danilov, Dailidonis, van Mourik, & Fruchtl, 2011a, 2011b; Dailidonis, Danilov, Früchtl, & van Mourik, 2011) is used to investigate adenine–adenine (AA), guanine–guanine (GG), thymine–thymine (TT), and cytosine–cytosine (CC) homoassociates in a cluster consisting of 400 water molecules. The starting structures taken were AA N(7) amino symmetric, TT N(3) – O(4) symmetric, GG N(1) – O(6) symmetric, and CC N(3) amino symmetric base pairs. A water spherical cluster with the density of water at room temperature and a radius sphere equal 13.9 Å was used, which corresponds to the most difficult conditions for the formation of stacks (see Abraham, 1982). In spite of such initial conditions, it is shown that during the simulation, each base pair is transformed into a more favorable stacked configuration. The results obtained allow to observe the whole process of convergence for the first time (for more information, visit the Website http://biophys.in.ua/).     

Structural analysis of the envelope gp120 V3 loop for some HIV-1 variants circulating in the countries of Eastern Europe

The V3 loop on gp120 from human immunodeficiency virus type 1 (HIV-1) is a focus of many research groups involved in anti-AIDS drug development because this region of the protein is a principal target for neutralizing antibodies and a major determinant for cell tropism and syncytium formation. In this study, the nucleotide sequences of the env gene region coding the V3 loop were determined by DNA sequencing methods for four novel HIV-1 strains that circulate in the countries of Eastern Europe, such as Russia, Belarus, Ukraine, etc. Based on the empirical data obtained, the 3D structures of the V3 loops associated with these viral modifications were generated by computer modeling and then compared to discover similarities in the spatial arrangement of this functionally important site of gp120. Despite the HIV-1 genetic variety, several regions of the V3 loop that contain residues critical for cell tropism were shown to be structurally invariant, which may explain its exceptional role in a co-receptor usage. These data together with those on the biological activity of the V3 individual residues clearly show that these conserved structural motifs of gp120 represent potential HIV-1 weak points most suitable for therapeutic intervention.     

Crystal structure of the CN-hydrolase SA0302 from the pathogenic bacterium Staphylococcus aureus belonging to the Nit and NitFhit Branch of the nitrilase superfamily

The nitrilases include a variety of enzymes with functional specificities of nitrilase, amidase, and hydrolase reactions. The crystal structure of the uncharacterized protein SA0302 from the pathogenic microorganism Staphylococcus aureus is solved at 1.7?Å resolution. The protein contains 261 amino acids and presents a four-layer αββα sandwich with a chain topology similar to that of a few known CN-hydrolase folds. In the crystal, the proteins are arranged as dimers whose monomers are related by a pseudo twofold rotation symmetry axis. Analysis of the sequences and structures of CN-hydrolases with known 3D structures shows that SA0302 definitely is a member of Branch 10 (Nit and NitFhit) of the nitrilase superfamily. Enzyme activities and substrate specificities of members of this branch are not yet characterized, in contrast to those of the members of Branches 1–9. Although the sequence identities between Branch 10 members are rather low, less than 30%, five conserved regions are common in this subfamily. Three of them contain functionally important catalytic residues, and the two other newly characterized ones are associated with crucial intramolecular and intermolecular interactions. Sequence homology of the area near the active site shows clearly that the catalytic triad of SA0302 is Glu41-Lys110-Cys146. We suggest also that the active site includes a fourth residue, the closely located Glu119. Despite an extensive similarity with other Nit-family structural folds, SA0302 displays an important difference. Protein loop 111–122, which follows the catalytic Lys110, is reduced to half the number of amino acids found in other Nit-family members. This leaves the active site fully accessible to solvent and substrates. We have identified conservative sequence motifs around the three core catalytic residues, which are inherent solely to Branch 10 of the nitrilase superfamily. On the basis of these new sequence fingerprints, 10 previously uncharacterized proteins also could be assigned to this hydrolase subfamily.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:19     

Pyrosequencing reveals how pulses influence rhizobacterial communities with feedback on wheat growth in the semiarid Prairie

Background and Aims

Evidence shows that plants modify their microbial environment leading to the “crop rotation effect”, but little is known about the changes in rhizobacterial community structure and functionality associated with beneficial rotation effects.

Methods

Polymerase chain reaction (PCR) and 454 GS FLX amplicon pyrosequencing were used to describe the composition of the rhizobacterial community evolving under the influence of pea, a growth promoting rotation crop, and the influence of three genotypes of chickpea, a plant known as an inferior rotation crop. The growth promoting properties of these rhizobacterial communities were tested on wheat in greenhouse assays.

Results

The rhizobacterial communities selected by pea and the chickpea CDC Luna in 2008, a wet year, promoted durum wheat growth, but those selected by CDC Vanguard or CDC Frontier had no growth-promoting effect. In 2009, a dry year, the influence of plants was mitigated, indicated that moisture availability is a major driver of soil bacterial community dynamics.

Conclusion

The effect of pulse crops on soil biological quality varies with the crop species and genotypes, and certain chickpea genotypes may induce positive rotation effects on wheat. The strength of a rotation effect on soil biological quality is modulated by the abundance of precipitation.     

New psychopsoid Neuroptera from the Early Cretaceous of Baissa,Transbaikalia

Two new genera and fi ve new species of the psychopsoid Neuroptera (one of which is unnamed) are described from the Early Cretaceous locality at Baissa, Transbaikalia, Russia: Epipsychopsis fusca n. gen., n. sp., E. variegata n. sp. (Psychopsidae), Pseudopsychopsis gradata n. gen., n. sp., P. baissensis n. sp. (Brongniartiellidae), and Sophogramma sp. (Kalligrammatidae).