Role of Kv7 channels in responses of the pulmonary circulation to hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is a beneficial mechanism that diverts blood from hypoxic alveoli to better ventilated areas of the lung, but breathing hypoxic air causes the pulmonary circulation to become hypertensive. Responses to airway hypoxia are associated with depolarization of smooth muscle cells in the pulmonary arteries and reduced activity of K(+) channels. As Kv7 channels have been proposed to play a key role in regulating the smooth muscle membrane potential, we investigated their involvement in the development of HPV and hypoxia-induced pulmonary hypertension. Vascular effects of the selective Kv7 blocker, linopirdine, and Kv7 activator, flupirtine, were investigated in isolated, saline-perfused lungs from rats maintained for 3-5 days in an isobaric hypoxic chamber (FiO2 = 0.1) or room air. Linopirdine increased vascular resistance in lungs from normoxic, but not hypoxic rats. This effect was associated with reduced mRNA expression of the Kv7.4 channel α-subunit in hypoxic arteries, whereas Kv7.1 and Kv7.5 were unaffected. Flupirtine had no effect in normoxic lungs but reduced vascular resistance in hypoxic lungs. Moreover, oral dosing with flupirtine (30 mg/kg/day) prevented short-term in vivo hypoxia from increasing pulmonary vascular resistance and sensitizing the arteries to acute hypoxia. These findings suggest a protective role for Kv7.4 channels in the pulmonary circulation, limiting its reactivity to pressor agents and preventing hypoxia-induced pulmonary hypertension. They also provide further support for the therapeutic potential of Kv7 activators in pulmonary vascular disease.

Cationic azacryptands as selective three-way DNA junction binding agents.

DNA damaging agents are among the most powerful anticancer drugs currently in clinical use. As an alternative to irreversible nucleobase damage and DNA strand breaks, the non-covalent stabilization of unusual, non-B DNA structures is currently emerging as a promising way to cause DNA damage with a high level of specificity. One of such non-B DNA structures is the three-way DNA junction: this Y-shaped multi-stranded architecture may act as an impediment to many DNA transactions, being therefore regarded as an invaluable target to create genomic defects that are improperly dealt with by cancer cells only. Herein, we report on a series of cationic azacryptands that make excellent candidates for assessing and harnessing the actual therapeutic potential of three-way DNA junction interacting compounds.

A twice-as-smart synthetic G-quartet: PyroTASQ is both a smart quadruplex ligand and a smart fluorescent probe.

Recent and unambiguous evidences of the formation of DNA and RNA G-quadruplexes in cells has provided solid support for these structures to be considered as valuable targets in oncology. Beyond this, they have lent further credence to the anticancer strategies relying on small molecules that selectively target these higher-order DNA/RNA architectures, referred to as G-quadruplex ligands. They have also shed bright light on the necessity of designing multitasking ligands, displaying not only enticing quadruplex interacting properties (affinity, structural selectivity) but also additional features that make them usable for detecting quadruplexes in living cells, notably for determining whether, when, and where these structures fold and unfold during the cell cycle and also for better assessing the consequences of their stabilization by external agents. Herein, we report a brand new design of such multitasking ligands, whose structure experiences a quadruplex-promoted conformational switch that triggers not only its quadruplex affinity (i.e., smart ligands, which display high affinity and selectivity for DNA/RNA quadruplexes) but also its fluorescence (i.e., smart probes, which behave as selective light-up fluorescent reporters on the basis of a fluorogenic electron redistribution). The first prototype of such multifunctional ligands, termed PyroTASQ, represents a brand new generation of quadruplex ligands that can be referred to as "twice-as-smart" quadruplex ligands.

Preparation and enantioselectivity binding studies of a new chiral cobalt(II)porphyrin-Tröger's base conjugate.

A new bis[cobalt(II)porphyrin]-Tröger's base conjugate was studied as a potential receptor for methyl esters of several amino acids. The conjugate was prepared as racemate, and then resolved via preparative high-performance liquid chromatography (HPLC) on a chiral column. The high affinity to lysine, histidine, and proline methyl esters was found by complexation studies followed by UV-Vis spectroscopy. The studies of pure enantiomers, followed by UV-Vis and electronic circular dichroism spectroscopy, revealed the highest enantioselectivity for lysine methyl ester.

Hyperdense cerebral artery computed tomography sign is associated with stroke severity rather than stroke subtype.

BACKGROUND: The hyperdense cerebral artery sign (HCAS) on unenhanced computed tomography (CT) in acute ischemic stroke is a valuable clinical marker, but it remains unclear if HCAS reflects clot composition or stroke etiology. Therefore, variables independently associated with HCAS were identified from a large international data set of patients treated with intravenous thrombolysis. METHODS: All stroke patients undergoing intravenous thrombolysis from the Safe Implementation of Treatments in Stroke-EAST (SITS-EAST) database between February 2003 and December 2011 were analyzed. A general estimating equation model accounting for within-center clustering was used to identify factors independently associated with HCAS. RESULTS: Of all 8878 consecutive patients, 8375 patients (94%) with available information about HCAS were included in our analysis. CT revealed HCAS in 19% of patients. Median baseline National Institutes of Health Stroke Scale (NIHSS) score was 12, mean age was 67 ± 12 years, and 3592 (43%) patients were females. HCAS was independently associated with baseline NIHSS (odds ratio [OR], 1.11; 95% confidence interval [CI], 1.10-1.12), vessel occlusion (OR, 5.02; 95% CI, 3.31-7.63), early ischemic CT changes (OR, 1.63; 95% CI, 1.31-2.04), year (OR, 1.07; 95% CI, 1.02-1.12), and age (10-year increments; OR, .90; 95% CI, .84-.96). Cardioembolic stroke was not associated with HCAS independently of baseline NIHSS. In different centers, HCAS was reported in 0%-50% of patients. CONCLUSIONS: This study illustrates significant variation in detection of HCAS among stroke centers in routine clinical practice. Accounting for within-center data clustering, stroke subtype was not independently associated with HCAS; HCAS was associated with the severity of neurologic deficit.

Determination of absolute configuration and conformation of a cyclic dipeptide by NMR and chiral spectroscopic methods.

Increasing precision of contemporary computational methods makes spectroscopies such as vibrational (VCD) and electronic (ECD) circular dichroism attractive for determination of absolute configurations (AC) of organic compounds. This is, however, difficult for polar, flexible molecules with multiple chiral centers. Typically, a combination of several methods provides the best picture of molecular behavior. As a test case, all possible stereoisomers with known AC (RS, SR, SS, and RR) of the cyclic dipeptide cyclo(Arg-Trp) (CAT) were synthesized, and the performances of the ECD, infrared (IR), VCD, Raman, Raman optical activity (ROA), and nuclear magnetic resonance (NMR) techniques for AC determination were investigated. The spectra were interpreted with the aid of density functional theory (DFT) calculations. Folded geometries stabilized by van der Waals and electrostatic interactions between the diketopiperazine (DKP) ring and the indole group are predicted to be preferred for CAT, with more pronounced folding due to Arg-Trp stacking in the case of SS/RR-CAT. The RS/SR isomers prefer a twist-boat puckering of the DKP ring, which is relatively independent of the orientation of the side chains. Calculated conformer-averaged VCD and ECD spectra explain most of the experimentally observed bands and allow for AC determination of the tryptophan side-chain, whereas the stereochemical configuration of the arginine side-chain is visible only in VCD. NMR studies provide characteristic long-range (2)J(C,H) and (3)J(C,H) coupling constants, and nuclear Overhauser effect (NOE) correlations, which in combination with either ECD or VCD also allow for complete AC determination of CAT.

Determining the absolute configuration of two marine compounds using vibrational chiroptical spectroscopy.

Chiroptical techniques are increasingly employed for assigning the absolute configuration of chiral molecules through comparison of experimental spectra with theoretical predictions. For assignment of natural products, electronic chiroptical spectroscopies such as electronic circular dichroism (ECD) are routinely applied. However, the sensitivity of electronic spectral parameters to experimental conditions and the theoretical methods employed can lead to incorrect assignments. Vibrational chiroptical methods (vibrational circular dichroism, VCD, and Raman optical activity, ROA) provide more reliable assignments, although they, in particular ROA, have been little explored for assignments of natural products. In this study, the ECD, VCD, and ROA chiroptical spectroscopies are evaluated for the assignment of the absolute configuration of a highly flexible natural compound with two stereocenters and an asymmetrically substituted double bond, the marine antibiotic Synoxazolidinone A (SynOxA), recently isolated from the sub-Arctic ascidian Synoicum pulmonaria. Conformationally averaged nuclear magnetic resonance (NMR), ECD, Raman, ROA, infrared (IR) and VCD spectral parameters are computed for the eight possible stereoisomers of SynOxA and compared to experimental results. In contrast to previously reported results, the stereochemical assignment of SynOxA based on ECD spectral bands is found to be unreliable. On the other hand, ROA spectra allow for a reliable determination of the configuration at the double bond and the ring stereocenter. However, ROA is not able to resolve the chlorine-substituted stereogenic center on the guanidinium side chain of SynOxA. Application of the third chiroptical method, VCD, indicates unique spectral features for all eight SynOxA isomers in the theoretical spectra. Although the experimental VCD is weak and restricted by the limited amount of sample, it allows for a tentative assignment of the elusive chlorine-substituted stereocenter. VCD chiroptical analysis of a SynOxA derivative with three stereocenters, SynOxC, results in the same absolute configuration as for SynOxA. Despite the experimental challenges, the results convincingly prove that the assignment of absolute configuration based on vibrational chiroptical methods is more reliable than for ECD.

Stacked and continuous helical self-assemblies of guanosine monophosphates detected by vibrational circular dichroism.

The aim of this study was to characterize self-assembled structures of guanosine derivatives in aqueous solutions by vibrational circular dichroism (VCD) and electronic circular dichroism (ECD). Three guanosine derivatives were studied [5'-guanosine monophosphate (GMP), diphosphate (GDP), and triphosphate (GTP)] using a broad range of concentrations and various metal/guanosine ratios. VCD was used for the first time in this field and showed itself to be a powerful method for obtaining specific structural information in solution. It can also help to determine the impact that the cations have, when added to the solution, on the versatile structures of guanine derivatives in terms of their association and disassociation. Based on the markedly different intensities and signs of the VCD signals observed for different concentrations of guanosine derivatives, we propose various structures based on guanine quartets for high guanosine concentrations and high K(+)/guanosine ratios (i.e., columnar helical organization of the quartets, which are rearranged into a continuous helix). We performed a degenerate coupled oscillator (DCO) calculation to interpret the VCD spectra obtained and how they vary during the assembly of guanosine derivatives. The calculations correctly predicted the VCD spectra and enabled us to identify the structures of the metal cation/guanosine monophosphate aggregates. ECD in the ultraviolet region was used as a diagnostic tool to characterize the studied systems and as a contact point between the previously defined structures of the guanine derivative assemblies and the molecular systems studied here. These studies revealed that the VCD technique is a powerful new method for determining the structures of optically active guanosine motifs.

Absolute configuration of a cyclic dipeptide reflected in vibrational optical activity: ab initio and experimental investigation.

The ability of Raman optical activity (ROA) and vibrational circular dichroism (VCD) experiments to determine the absolute configuration of chiral molecules with multiple stereogenic centers was explored for four diastereoisomers of a conformationally flexible cyclic dipeptide, cyclo(Arg-Tyr(OMe)). The reliability of the interpretation depended on the correct description of the molecular conformation, which was found to be strongly affected by intramolecular interactions. In particular, when dispersion corrections were included in the density functional theory calculations, the simulated spectra matched the experimental observations well. Experimental and theoretical ROA and VCD spectra were well correlated for all the absolute configurations (RS, SR, SS, and RR) of protonated cyclo(Arg-Tyr(OMe)). These spectroscopies thus appear useful not only for reliable determination of the absolute configuration and conformation but also in revealing the role of hydrogen bonds and C-H···π interactions in the structure stabilization, which can potentially be used when designing enzyme inhibitors and supramolecular architectures.

Supramolecular arrangement of guanosine/5-guanosine monophosphate binary mixtures studied by methods of circular dichroism.

Self-assembly of molecules is one of the fundamental processes in biology and in supramolecular chemistry. Guanosine (Guo) and its derivatives are among the widely studied molecules because of self-assembly abilities. Their tetrameric associates are the nature of telomeric DNA, and furthermore they are fundamental building blocks of supramolecular reversible gels, which may arise in certain physical and chemical conditions. Although poorly soluble in water, Guo forms interesting structures with guanosine 5'-monophosphate salt (GMP) in the TRIS buffer. We used electronic circular dichroism and vibrational circular dichroism to describe the thermal response of gels formed by the Guo/GMP binary mixture. Using these complementary techniques suitable to study conformational changes of chiral compounds, we obtained information about the involvement of functional groups and weak interactions in the guanosine quartet (G(4)) and stacked G(4) structures.

Comparison of the Influence of Two Types of Plasma Treatment of Short Carbon Fibers on Mechanical Properties of Epoxy Composites Filled with These Treated Fibers.

The interfacial interface between fibers and matrix plays a key role for epoxy matrix composites and short recycled randomly arranged fibers. This study used short recycled carbon fiber (RCF) as a filler. Plasma treatment was used for carbon fiber surface treatment. This treatment was performed using radio (RF) and microwave (MW) frequencies at the same pressure and atmosphere. Appropriate chemical modification of the fiber surfaces helps to improve the wettability of the carbon fibers and, at the same time, allows the necessary covalent bonds to form between fibers and the epoxy matrix. The effect of the plasma treatment was analyzed and confirmed by X-ray photoelectron spectroscopy, Raman microscopy, scanning electron microscopy, transmission electron microscopy and wettability measurements. Composite samples filled with recycled carbon fibers with low concentrations (1 wt%, 2.5 wt% and 5 wt%) and high concentrations (20 wt% and 30 wt%) were made from selected treated fibers. The mechanical properties (impact toughness, 3PB) were analyzed on these samples. It was found that the modulus of elasticity and bending stress increase with the increasing content of recycled carbon fibers. A more significant change in impact strength occurred in samples with low concentration.

On 3-Coloring of ( 2 P 4 , C 5 )-Free Graphs.

The 3-coloring of hereditary graph classes has been a deeply-researched problem in the last decade. A hereditary graph class is characterized by a (possibly infinite) list of minimal forbidden induced subgraphs H 1 , H 2 , … ; the graphs in the class are called ( H 1 , H 2 , … ) -free. The complexity of 3-coloring is far from being understood, even for classes defined by a few small forbidden induced subgraphs. For H-free graphs, the complexity is settled for any H on up to seven vertices. There are only two unsolved cases on eight vertices, namely 2 P 4 and P 8 . For P 8 -free graphs, some partial results are known, but to the best of our knowledge, 2 P 4 -free graphs have not been explored yet. In this paper, we show that the 3-coloring problem is polynomial-time solvable on ( 2 P 4 , C 5 ) -free graphs.

Vertex Deletion into Bipartite Permutation Graphs.

A permutation graph can be defined as an intersection graph of segments whose endpoints lie on two parallel lines ℓ 1 and ℓ 2 , one on each. A bipartite permutation graph is a permutation graph which is bipartite. In this paper we study the parameterized complexity of the bipartite permutation vertex deletion problem, which asks, for a given n-vertex graph, whether we can remove at most k vertices to obtain a bipartite permutation graph. This problem is NP -complete by the classical result of Lewis and Yannakakis [20]. We analyze the structure of the so-called almost bipartite permutation graphs which may contain holes (large induced cycles) in contrast to bipartite permutation graphs. We exploit the structural properties of the shortest hole in a such graph. We use it to obtain an algorithm for the bipartite permutation vertex deletion problem with running time O ( 9 k · n 9 ) , and also give a polynomial-time 9-approximation algorithm.

Computational Modeling of Polymer Matrix Based Textile Composites.

A simple approach to the multiscale analysis of a plain weave reinforced composite made of basalt fabrics bonded to a high performance epoxy resin L285 Havel is presented. This requires a thorough experimental program to be performed at the level of individual constituents as well as formulation of an efficient and reliable computational scheme. The rate-dependent behavior of the polymer matrix is examined first providing sufficient data needed in the calibration step of the generalized Leonov model, which in turn is adopted in numerical simulations. Missing elastic properties of basalt fibers are derived next using nanoindentation. A series of numerical tests is carried out at the level of yarns to promote the ability of a suitably modified Mori-Tanaka micromechanical model to accurately describe the nonlinear viscoelastic response of unidirectional fibrous composites. The efficiency of the Mori-Tanaka method is then exploited in the formulation of a coupled two scale computational scheme, while at the level of textile ply the finite element computational homogenization is assumed, the two-point averaging format of the Mori-Tanaka method is applied at the level of yarn to serve as a stress updater in place of another finite element model representing the yarn microstructure as typical of FE2 based multiscale approach. Several numerical simulations are presented to support the proposed modeling methodology.

Karyotype analysis of the Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae) reveals a large X chromosome with rRNA and histone gene families.

The Russsian wheat aphid (RWA), Diuraphis noxia (Kurdjumov), is a worldwide pest of cereals. Despite its economic importance, little is known about its genome. Here we investigated physical genomic features in RWA by karyotype analysis using differential staining with AgNO(3), CMA(3), and DAPI, by chromosomal localization of ribosomal DNA (rDNA), H3 and H4 histone genes, and the "arthropod" telomeric sequence (TTAGG)(n) using fluorescence in situ hybridization (FISH), and by measuring the RWA genome size using flow cytometry. The female karyotype, 2n = 10, is composed of four autosome pairs and a pair of X chromosomes, whereas the male karyotype, 2n = 9, has a single X. The X chromosome is the largest element in the karyotype. All three molecular markers used, i.e., 18S rRNA and both H3 and H4 probes are co-localized at one end of the X chromosome. The FISH probes revealed that the AgNO(3)-positive bridge between two prometaphase X chromosomes of females, which is believed to be responsible for the elimination of one X chromosome in aphid oocytes determined to undergo male development, contains clusters of both histone genes, in addition to an rDNA cluster. Interestingly, RWA lacks the (TTAGG)(n) telomeric sequence in its genome, in contrast to several previously investigated aphid species. Additionally, we compared female and male genome sizes. The female genome size is 2C = 0.86 pg, whereas the male genome size is 2C = 0.70 pg. The difference between the DNA content in the two genders suggests that the RWA X chromosome occupies about 35% of the female haploid genome (1C = 0.43 pg), which makes it one of the largest sex chromosomes in the animal kingdom.

Subexponential-Time Algorithms for Finding Large Induced Sparse Subgraphs.

Let C and D be hereditary graph classes. Consider the following problem: given a graph G ∈ D , find a largest, in terms of the number of vertices, induced subgraph of G that belongs to C . We prove that it can be solved in 2 o ( n ) time, where n is the number of vertices of G, if the following conditions are satisfied:the graphs in C are sparse, i.e., they have linearly many edges in terms of the number of vertices;the graphs in D admit balanced separators of size governed by their density, e.g., O ( Δ ) or O ( m ) , where Δ and m denote the maximum degree and the number of edges, respectively; andthe considered problem admits a single-exponential fixed-parameter algorithm when parameterized by the treewidth of the input graph. This leads, for example, to the following corollaries for specific classes C and D :a largest induced forest in a P t -free graph can be found in 2 O ~ ( n 2 / 3 ) time, for every fixed t; anda largest induced planar graph in a string graph can be found in 2 O ~ ( n 2 / 3 ) time.

Growth of vascular smooth muscle cells on collagen I exposed to RBL-2H3 mastocytoma cells.

Remodeling of the peripheral pulmonary vasculature during chronic hypoxia is characterized by accelerated collagenolysis and thickening of the vascular wall. Low molecular weight peptides, products of cleavage by interstitial collagenase and muscular layer in the peripheral pulmonary vessels, are typically present. The aim of this "in vitro" study was to verify that mast cells (RBL-2H3) as a potent source of a variety of biomolecules which can affect vessel wall remodeling are capable of splitting collagen and then facilitating the growth of vascular smooth muscle cells (VSMC). Collagen I was exposed to RBL-2H3 cells cultured 48 hours under normoxic or hypoxic (3% O(2)) conditions and then seeded with VSMC. The VSMC proliferated with the shortest doubling time and reached the highest cell population density on the collagen pre-modified with hypoxic RBL-2H3 cells. This increased growth activity of VSMC was probably due to the fragmentation of collagen by proteases released from RBL-2H3 cells. Absolute amount of collagen fragments was similar in samples exposed to normoxic and hypoxic RBL-2H3 cells, but the concentration of at least one collagen fragment was significantly higher under hypoxic conditions. Mast cells exposed to hypoxia are more capable to split collagen and facilitate the growth of VSMC.

Prevention of mast cell degranulation by disodium cromoglycate delayed the regression of hypoxic pulmonary hypertension in rats.

BACKGROUND: Pulmonary vascular remodeling induced by chronic hypoxia regresses after return to normoxia. This regression is associated with an increased amount of collagenase in pulmonary mast cells and increased collagenolytic and elastolytic activity in the lung tissue. OBJECTIVE: The role of lung mast cells during recovery from chronic hypoxia was tested by the inhibition of their degranulation by disodium cromoglycate (DSCG). METHODS: Male Wistar rats (n = 46) were exposed to isobaric hypoxia (3 weeks, F(i)O(2) 0.1). Thirteen of them were tested immediately at the end of exposure, 17 were treated with DSCG during the first 4 days of recovery and tested on the 5th or 14th day of recovery, 16 untreated animals were measured at the same time intervals. These groups were compared with 12 animals kept in normoxia. The rats were anesthetized (Thiopental) and their pulmonary arterial blood pressure (PAP), cardiac output and heart weight were tested, as well as the collagen composition of the walls of the peripheral pulmonary arteries. RESULTS: DSCG applied during the first 4 days of recovery from chronic hypoxia blocked the decrease in PAP during the early phase of recovery and had no influence on PAP at a later phase. DSCG administration prevents collagen splitting in peripheral pulmonary vessels at the early phase of recovery. PAP and right ventricle hypertrophy were normalized after 14 days of return to normoxia. CONCLUSIONS: Mast cell degranulation plays a role in the regression of pulmonary hypertension during the early phase of recovery from chronic hypoxia.

Prevention of mast cell degranulation by disodium cromoglycate attenuates the development of hypoxic pulmonary hypertension in rats exposed to chronic hypoxia.

BACKGROUND: Chronic hypoxia induces lung vascular remodeling, which results in pulmonary hypertension. Vascular remodeling is associated with collagenolysis and activation of matrix metalloproteinases (MMPs). One of the possible sources of MMPs in hypoxic lung are mast cells. OBJECTIVE: The role of lung mast cell collagenolytic activity in hypoxic pulmonary hypertension was tested by the inhibitor of mast cell degranulation disodium cromoglycate (DSCG). METHODS: Rats were treated with DSCG in an early or later phase of isobaric hypoxia. Control groups were exposed to hypoxia only or to normoxia. Lung hemodynamics, muscularization and collagen metabolism in the walls of peripheral pulmonary vessels in the lungs were measured. RESULTS: DSCG applied at an early phase of exposure to hypoxia reduced the development of pulmonary hypertension, inhibited muscularization in peripheral pulmonary arteries and decreased the amount of collagen cleavage fragments in prealveolar vessels. CONCLUSIONS: Mast cell degranulation plays a role in the initiation of hypoxic pulmonary vascular remodeling.

Characterization of a eukaryotic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and identification of kinase substrates.

Searching the genome sequence of Streptococcus pneumoniae revealed the presence of a single Ser/Thr protein kinase gene stkP linked to protein phosphatase phpP. Biochemical studies performed with recombinant StkP suggest that this protein is a functional eukaryotic-type Ser/Thr protein kinase. In vitro kinase assays and Western blots of S. pneumoniae subcellular fractions revealed that StkP is a membrane protein. PhpP is a soluble protein with manganese-dependent phosphatase activity in vitro against a synthetic substrate RRA(pT)VA. Mutations in the invariant aspartate residues implicated in the metal binding completely abolished PhpP activity. Autophosphorylated form of StkP was shown to be a substrate for PhpP. These results suggest that StkP and PhpP could operate as a functional pair in vivo. Analysis of phosphoproteome maps of both wild-type and stkP null mutant strains labeled in vivo and subsequent phosphoprotein identification by peptide mass fingerprinting revealed two possible substrates for StkP. The evidence is presented that StkP can phosphorylate in vitro phosphoglucosamine mutase GlmM which catalyzes the first step in the biosynthetic pathway leading to the formation of UDP-N-acetylglucosamine, an essential common precursor to cell envelope components.