Possibility of analytical application of the partition in aqueous biphasic polymeric systems technique

The partition behaviour of a number of ionic and nonionic surface-active substances in the dextran-polyethylene glycol system was examined. The strictly linear dependence of the logarithm of the partition coefficient on the alkyl chain length for a homologous series of nonionic surfactants provides a measure of the difference in the relative hydrophobicity between the two phases of the system, in terms of the free energy of transfer of a CH₂ group from the bottom phase to the top phase of the system. This difference is found to be altered in the presence of NaCl or KCl depending on the salt concentration. It is concluded that the influence of the salt composition of the system on the distributed solutes' behaviour may be due to the effect of the ions on the hydrophobicity difference between the phases.The partition of ionic amphiphiles is found to be dependent on the relative hydrophobicity of the compounds as well as on their charge. It is shown that at salt concentrations up to about 0.1 M NaCl the charged solute partition is determined by its charge as well as its relative hydrophobicity, in the presence of 0.1–0.2 M NaCl the substance distribution is highly dependent on its charge and slightly on its lipophility. At the salt concentrations above 0.2 M the solute partition is determined just by its hydrophobic character and seems to be totally independent of its charge. It is concluded that the partition technique can be used for analytical purposes. The method seems to be unique in providing quantitative information on the amphiphilic surface properties of the solutes being partitioned.     

Sequence-specific ultrasonic cleavage of DNA

We investigated the phenomenon of ultrasonic cleavage of DNA by analyzing a large set of cleavage patterns of DNA restriction fragments using polyacrylamide gel electrophoresis. The cleavage intensity of individual phosphodiester bonds was found to depend on the nucleotide sequence and the position of the bond with respect to the ends of the fragment. The relative intensities of cleavage of the central phosphodiester bond in 16 dinucleotides and 256 tetranucleotides were determined by multivariate statistical analysis. We observed a remarkable enhancement of the mean values of the relative intensities of cleavage (cleavage rates) in phosphodiester bonds following deoxycytidine, which diminished in the row of dinucleotides: d(CpG) > d(CpA) > d(CpT) >> d(CpC). The cleavage rates for all pairs of complementary dinucleotides were significantly different from each other. The effect of flanking nucleotides in tetranucleotides on cleavage rates of all 16 types of central dinucleotides was also statistically significant. The sequence-dependent ultrasonic cleavage rates of dinucleotides are consistent with reported data on the intensity of the conformational motion of their 5′-deoxyribose. As a measure of local conformational dynamics, cleavage rates may be useful for characterizing functional regions of the genome.     

Matrix metalloproteinases as target genes for gene regulatory networks driving molecular and cellular pathways related to a multistep pathogenesis of cerebrovascular disease

The present study investigated a joint contribution of matrix metalloproteinases (MMPs) genes to ischemic stroke (IS) development and analyzed interactions between MMP genes and genome-wide associated loci for IS. A total of 1288 unrelated Russians (600 IS patients and 688 healthy individuals) from Central Russia were recruited for the study. Genotyping of seven single nucleotide polymorphisms (SNPs) of MMP genes (rs1799750, rs243865, rs3025058, rs11225395, rs17576, rs486055, and rs2276109) and eight genome-wide associated loci for IS were done using Taq-Man–based assays and MALDI-TOF mass spectrometry iPLEX platform, respectively. Allele − 799T at rs11225395 of the MMP8 gene was significantly associated with a decreased risk of IS after adjustment for sex and age (OR = 0.82; 95%CI, 0.70-0.96; P = 0.016). The model-based multifactor dimensionality reduction method has revealed 21 two-order, 124 three-order, and 474 four-order gene-gene (G×G) interactions models meaningfully (P_perm < 0.05) associated with the IS risk. The bioinformatic analysis enabled establishing the studied MMP gene polymorphisms possess a clear regulatory potential and may be targeted by gene regulatory networks driving molecular and cellular pathways related to the pathogenesis of IS. In conclusion, the present study was the first to identify an association between polymorphism rs11225395 of the MMP8 gene and IS risk. The study findings also indicate that MMPs deserve special attention as a potential class of genes influencing the multistep mechanisms of cerebrovascular disease including atherosclerosis in cerebral arteries, acute cerebral artery occlusion as well as the ischemic injury of the brain and its recovery.     

Higher-order interhelical spatial interactions in membrane proteins

Higher-order interactions are important for protein folding and assembly. We introduce the concept of interhelical three-body interactions as derived from Delaunay triangulation and alpha shapes of protein structures. In addition to glycophorin A, where triplets are strongly correlated with protein stability, we found that tight interhelical triplet interactions exist extensively in other membrane proteins, where many types of triplets occur far more frequently than in soluble proteins. We developed a probabilistic model for estimating the value of membrane helical interaction triplet (MHIT) propensity. Because the number of known structures of membrane proteins is limited, we developed a bootstrap method for determining the 95% confidence intervals of estimated MHIT values. We identified triplets that have high propensity for interhelical interactions and are unique to membrane proteins, e.g. AGF, AGG, GLL, GFF and others. A significant fraction (32%) of triplet types contains triplets that may be involved in interhelical hydrogen bond interactions, suggesting the prevalent and important roles of H-bond in the assembly of TM helices. There are several well-defined spatial conformations for triplet interactions on helices with similar parallel or antiparallel orientations and with similar right-handed or left-handed crossing angles. Often, they contain small residues and correspond to the regions of the closest contact between helices. Sequence motifs such as GG4 and AG4 can be part of the three-body interactions that have similar conformations, which in turn can be part of a higher-order cooperative four residue spatial motif observed in helical pairs from different proteins. In many cases, spatial motifs such as serine zipper and polar clamp are part of triplet interactions. On the basis of the analysis of the archaeal rhodopsin family of proteins, tightly packed triplet interactions can be achieved with several different choices of amino acid residues.     

GIS and Remote Sensing for Detecting Yield Loss in Cranberry Culture

The primary goal of our research is to develop key elements of a precision agriculture program applicable to high-value woody perennial crops, such as cranberries. These crop systems exhibit tremendous variability in crop yields and quality as imposed by variations in soil properties (water availability and nutrient deficiency) that lead to crop stress (disease development and weed competition). Some of the variability present in the growing environment results in persistent yield losses as well as crop-quality reductions. We are using state-of-the-art methodologies (GIS, GPS, remote sensing) to identify and map spatial variations of the crop. Through image-processing methods (NDVI and unsupervised classification), approximately 65% of the variation in yield was described using 4-m multispectral satellite data as a base image.     

Oxadiazols: a new class of rationally designed anti-human immunodeficiency virus compounds targeting the nuclear localization signal of the viral matrix protein

Despite recent progress in anti-human immunodeficiency virus (HIV) therapy, drug toxicity and emergence of drug-resistant isolates during long-term treatment of HIV-infected patients necessitate the search for new targets that can be used to develop novel antiviral agents. One such target is the process of nuclear translocation of the HIV preintegration complex. Previously we described a class of arylene bis(methylketone) compounds that inhibit HIV-1 nuclear import by targeting the nuclear localization signal (NLS) in the matrix protein (MA). Here we report a different class of MA NLS-targeting compounds that was selected using computer-assisted drug design. The leading compound from this group, ITI-367, showed potent anti-HIV activity in cultures of T lymphocytes and macrophages and also inhibited HIV-1 replication in ex vivo cultured lymphoid tissue. The virus carrying inactivating mutations in MA NLS was resistant to ITI-367. Analysis by real-time PCR demonstrated that the compound specifically inhibited nuclear import of viral DNA, measured by two-long terminal repeat circle formation. Evidence of the existence of this mechanism was provided by immunofluorescent microscopy, using fluorescently labeled HIV-1, which demonstrated retention of the viral DNA in the cytoplasm of drug-treated macrophages. Compounds inhibiting HIV-1 nuclear import may be attractive candidates for further development.     

On the mechanism of formation and spectral properties of radical anions generated by the reduction of -[Re(CO)3(5-nitro-1,10-phenanthroline)] and -[Re(CO)3(3,4,7,8-tetramethyl-1,10-phenanthroline)] pendants in poly-4-vinylpyridine polymers

The electrochemical reduction in aprotic media of -[Re^I(CO)₃L]⁺ pendants in poly-4-vinylpyridine polymers is compared to that of [Re^I(CO)₃L]⁺ complexes (L = 5-nitro-1,10-phenanthroline and 3,4,7,8-tetramethyl-1,10-phenanthroline). The UV-Vis absorption spectra of the reduced radical anions of 5-nitro-1,10-phenanthroline (NO₂-phen) and 3,4,7,8-tetramethyl-1,10-phenanthroline (tmphen) were obtained by spectro-electrochemistry of [Re^I(CO)₃(NO₂-phen)(CH₃CN)]⁺ and [Re^I(CO)₃(tmphen)(CH₃CN)]⁺, respectively. Similar spectra were obtained for the radical anions -phen and tmphen⁻ after pulse radiolysis experiments with -[Re^I(CO)₃L]⁺-containing polymers. The analysis of the time-resolved difference spectra was performed using “multivariate curve resolution” (MCR) techniques. Unlike , CH₂OH radicals were unable to reduce tmphen ligands. The reaction of and/or CH₂OH with -[Re^I(CO)₃(NO₂-phen)]⁺-containing polymers generates -[Re^I(CO)₃(-phen)] pendants which after disproportionation give rise to products with λ_max = 380 nm. The kinetic behavior of -[Re^I(CO)₃(-phen)] pendants under different experimental conditions is discussed.     

A FAK/Src chimera with gain-of-function properties promotes formation of large peripheral adhesions associated with dynamic actin assembly

Formation of a complex between the tyrosine kinases FAK and Src is a key integrin-mediated signaling event implicated in cell motility, survival, and proliferation. Past studies indicate that FAK functions in the complex primarily as a "scaffold," acting to recruit and activate Src within cell/matrix adhesions. To study the cellular impact of FAK-associated Src signaling we developed a novel gain-of-function approach that involves expressing a chimeric protein with the FAK kinase domain replaced by the Src kinase domain. This FAK/Src chimera is subject to adhesion-dependent activation and promotes tyrosine phosphorylation of p130Cas and paxillin to higher steady-state levels than is achieved by wild-type FAK. When expressed in FAK -/- mouse embryo fibroblasts, the FAK/Src chimera resulted in a striking cellular phenotype characterized by unusual large peripheral adhesions, enhanced adhesive strength, and greatly reduced motility. Live cell imaging of the chimera-expressing FAK -/- cells provided evidence that the large peripheral adhesions are associated with a dynamic actin assembly process that is sensitive to a Src-selective inhibitor. These findings suggest that FAK-associated Src kinase activity has the capacity to promote adhesion integrity and actin assembly.     

Stable Isotope Ecology of Extant Tapirs from the Americas

Understanding the ecology of modern and ancient forests can help clarify the evolution of forest dwelling mammals. It is first necessary, however, to elucidate the source and extent of stable isotope variation in forest taxa. Tapirs are of particular interest because they are model organisms for identifying forest environments due to their highly conservative diet and habitat preferences. Here, stable carbon and oxygen isotopic compositions of extant tapirs are quantified to test hypotheses regarding ontogenetic diet shifts, stable isotope variation at the population level, and relationships between stable isotopes and climatic variables. A population of extant tapirs (Tapirus bairdii) demonstrates low δ¹³C variation (～2–3‰) and increased δ¹³C values in late erupting teeth, indicating that juveniles consume ¹³C deplete milk and/or browse in denser forests. Disparate δ¹⁸O values of late erupting teeth are instead reflecting seasonal variation. Extant tapir (T. bairdii, Tapirus pinchaque, Tapirus terrestris) δ¹⁸O values are constrained by climatic and geographic variables. Most notably, δ¹⁸O values of T. bairdii decrease with decreasing precipitation frequency. Tapirus terrestris is typically present in areas with greater precipitation than T. bairdii and δ¹⁸O values instead positively correlate with δ¹³C values. These data indicate that tapirs in wetter areas are getting a larger proportion of their water from leaves experiencing less evaporation in denser canopies, while T. bairdii is interpreted to increase its consumption of water via drinking when present in drier areas. An understanding of extant tapir stable isotope ecology improves ecological interpretations of these elusive mammals both today and in the past. Abstract in Spanish is available at http://www.blackwell‐synergy.com/loi/btp .     

Structural signatures of enzyme binding pockets from order-independent surface alignment: a study of metalloendopeptidase and NAD binding proteins

Detecting similarities between local binding surfaces can facilitate identification of enzyme binding sites and prediction of enzyme functions, and aid in our understanding of enzyme mechanisms. Constructing a template of local surface characteristics for a specific enzyme function or binding activity is a challenging task, as the size and shape of the binding surfaces of a biochemical function often vary. Here we introduce the concept of signature binding pockets, which captures information on preserved and varied atomic positions at multiresolution levels. For proteins with complex enzyme binding and activity, multiple signatures arise naturally in our model, forming a signature basis set that characterizes this class of proteins. Both signatures and signature basis sets can be automatically constructed by a method called SOLAR (Signature Of Local Active Regions). This method is based on a sequence-order-independent alignment of computed binding surface pockets. SOLAR also provides a structure-based multiple sequence fragment alignment to facilitate the interpretation of computed signatures. By studying a family of evolutionarily related proteins, we show that for metzincin metalloendopeptidase, which has a broad spectrum of substrate binding, signature and basis set pockets can be used to discriminate metzincins from other enzymes, to predict the subclass of metzincins functions, and to identify specific binding surfaces. Studying unrelated proteins that have evolved to bind to the same NAD cofactor, we constructed signatures of NAD binding pockets and used them to predict NAD binding proteins and to locate NAD binding pockets. By measuring preservation ratio and location variation, our method can identify residues and atoms that are important for binding affinity and specificity. In both cases, we show that signatures and signature basis set reveal significant biological insight.