Mediator mechanisms involved in TRPV1 and P2X receptor-mediated, ROS-evoked bradypneic reflex in anesthetized rats.

Inhalation of H2O2 is known to evoke bradypnea followed by tachypnea, which are reflexes resulting from stimulation by reactive oxygen species of vagal lung capsaicin-sensitive and myelinated afferents, respectively. This study investigated the pharmacological receptors and chemical mediators involved in triggering these responses. The ventilatory responses to 0.2% aerosolized H2O2 were studied before and after various pharmacological pretreatments in anesthetized rats. The initial bradypneic response was reduced by a transient receptor potential vanilloid 1 (TRPV1) receptor antagonist [capsazepine; change (Delta) = -53%] or a P2X purinoceptor antagonist [iso-pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (PPADS); Delta = -47%] and was further reduced by capsazepine and iso-PPADS in combination (Delta = -78%). The initial bradypneic response was reduced by a cyclooxygenase inhibitor (indomethacin; Delta = -48%), ATP scavengers (apyrase and adenosine deaminase in combination; Delta = -50%), or capsazepine and indomethacin in combination (Delta = -47%), was further reduced by iso-PPADS and indomethacin in combination (Delta = -75%) or capsazepine and ATP scavengers in combination (Delta = -83%), but was not affected by a lipoxygenase inhibitor (nordihydroguaiaretic acid) or by any of the various vehicles. No pretreatment influenced delayed tachypnea. We concluded that 1) the initial bradypneic response to H2O2 results from activation of both TRPV1 and P2X receptors, possibly located at terminals of vagal lung capsaicin-sensitive afferent fibers; 2) the functioning of the TRPV1 and P2X receptors in triggering the initial bradypnea is, in part, mediated through the actions of cyclooxygenase metabolites and ATP, respectively; and 3) these mechanisms do not contribute to the H2O2-evoked delayed tachypnea.     

Time-resolved fluorescence biosensor for adenosine detection based on home-made europium complexes

In this protocol, the authors report a time-resolved fluorescence biosensor based on home-made europium complexes for highly sensitive detection of small molecules using adenosine as a model analyte. The fluorophore that used is europium complexes. Its signal can be measured in a time-resolved manner that eliminates most of the unspecific fluorescent background. The amino modified aptamer probe, which is designed to specifically recognize adenosine, is combined to the aldehyde-group modified glass slide by covalent bond. Europium complex-labeled a short ssDNA, designed to segment hybridize with aptamer probe is immobilized on the glass slide by hybridization reaction. In the presence of adenosine, the aptamer part is more inclined to bounds with adenosine and triggers structure-switching of the aptamer from aptamer/ssDNA duplex to aptamer/target complex. As a result, europium complexes-labeled ssDNA is forced to dissociate from the sensor interface, resulting in time-resolved fluorescence intensity decrease. The decrement intensity is proportional to the amount of adenosine. Under optimized assay conditions, a linear range (1.0×10(-8)M to 1.0×10(-7)M) is got with low detection limit of 5.61nM. The biosensor exhibits excellent selectivity and can provide a promising potential for aptamer-based adenosine detection.     

Positive Charges on Lysine Residues of the Extrinsic 18 kDa Protein Are Important to Its Electrostatic Interaction with Spinach Photosystem Ⅱ Membranes

To determine the contribution of charged amino acids to binding with the photosystem II complex （PSII）, the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N- succinimidyl propionate （NSP） or glycine methyl ester （GME） in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4-10 and 10-14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes.     

The first complete mitochondrial genome sequence of Nanorana parkeri and Nanorana ventripunctata (Amphibia: Anura: Dicroglossidae), with related phylogenetic analyses

Members of the Nanorana genus (family Dicroglossidae) are often referred to as excellent model species with which to study amphibian adaptations to extreme environments and also as excellent keystone taxa for providing insights into the evolution of the Dicroglossidae. However, a complete mitochondrial genome is currently only available for Nanorana pleskei. Thus, we analyzed the complete mitochondrial genomes of Nanorana parkeri and Nanorana ventripunctata to investigate their evolutionary relationships within Nanorana and their phylogenetic position in the family Dicroglossidae. Our results showed that the genomes of N. parkeri (17,837 bp) and N. ventripunctata (18,373 bp) encode 13 protein‐coding genes (PCGs), two ribosomal RNA genes, 23 transfer RNA (tRNA) genes, and a noncoding control region. Overall sequences and genome structure of the two species showed high degree of similarity with N. pleskei, although the motif structures and repeat sequences of the putative control region showed clear differences among these three Nanorana species. In addition, a tandem repeat of the tRNA‐Met gene was found located between the tRNA‐Gln and ND2 genes. On both the 5′ and 3′‐sides, the control region possessed distinct repeat regions; however, the CSB‐2 motif was not found in N. pleskei. Based on the nucleotide sequences of 13 PCGs, our phylogenetic analyses, using Bayesian inference and maximum‐likelihood methods, illustrate the taxonomic status of Nanorana with robust support showing that N. ventripunctata and N. pleskei are more closely related than they are to N. parkeri. In conclusion, our analyses provide a more robust and reliable perspective on the evolutionary history of Dicroglossidae than earlier analyses, which used only a single species (N. pleskei).     

Role of two-component regulatory systems in intracellular survival of Mycobacterium tuberculosis

The typical two-component regulatory systems (TCSs), consisting of response regulator and histidine kinase, play a central role in survival of pathogenic bacteria under stress conditions such as nutrient starvation, hypoxia, and nitrosative stress. A total of 11 complete paired two-component regulatory systems have been found in Mycobacterium tuberculosis, including a few isolated kinase and regulatory genes. Increasing evidence has shown that TCSs are closely associated with multiple physiological process like intracellular persistence, pathogenicity, and metabolism. This review gives the two-component signal transduction systems in M. tuberculosis and their signal transduction roles in adaption to the environment.     

Identification and Severity Determination of Wheat Stripe Rust and Wheat Leaf Rust Based on Hyperspectral Data Acquired Using a Black-Paper-Based Measuring Method

It is important to implement detection and assessment of plant diseases based on remotely sensed data for disease monitoring and control. Hyperspectral data of healthy leaves, leaves in incubation period and leaves in diseased period of wheat stripe rust and wheat leaf rust were collected under in-field conditions using a black-paper-based measuring method developed in this study. After data preprocessing, the models to identify the diseases were built using distinguished partial least squares (DPLS) and support vector machine (SVM), and the disease severity inversion models of stripe rust and the disease severity inversion models of leaf rust were built using quantitative partial least squares (QPLS) and support vector regression (SVR). All the models were validated by using leave-one-out cross validation and external validation. The diseases could be discriminated using both distinguished partial least squares and support vector machine with the accuracies of more than 99%. For each wheat rust, disease severity levels were accurately retrieved using both the optimal QPLS models and the optimal SVR models with the coefficients of determination (R²) of more than 0.90 and the root mean square errors (RMSE) of less than 0.15. The results demonstrated that identification and severity evaluation of stripe rust and leaf rust at the leaf level could be implemented based on the hyperspectral data acquired using the developed method. A scientific basis was provided for implementing disease monitoring by using aerial and space remote sensing technologies.     

Identification of highly potent and selective PI3Kδ inhibitors

Selective PI3Kδ inhibitors have recently been hypothesized to be appropriate immunosuppressive agents for the treatment of immunological disorders such as rheumatoid arthritis. However, few reports have highlighted molecules that are highly selective for PI3Kδ over the other PI3K isoforms. In this letter, isoform and kinome selective PI3Kδ inhibitors are presented. The Structural Activity Relationship leading to such molecules is outlined.     

Arylsulfonamidopiperidone derivatives as a novel class of factor Xa inhibitors

The design, synthesis and SAR of a novel class of valerolactam-based arylsulfonamides as potent and selective FXa inhibitors is reported. The arylsulfonamide–valerolactam scaffold was derived based on the proposed bioisosterism to the arylcyanoguanidine-caprolactam core in known FXa inhibitors. The SAR study led to compound 46 as the most potent FXa inhibitor in this series, with an IC₅₀ of 7 nM and EC_2×PT of 1.7 μM. The X-ray structure of compound 40 bound to FXa shows that the sulfonamide–valerolactam scaffold anchors the aryl group in the S1 and the novel acylcytisine pharmacophore in the S4 pockets.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.