ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are key signalling molecules produced in response to various stimuli and involved in a diverse range of plant signal transduction processes. Nitric oxide and H(2)O(2) have been identified as essential components of the complex signalling network inducing stomatal closure in response to the phytohormone abscisic acid (ABA). A close inter-relationship exists between ABA and the spatial and temporal production and action of both NO and H(2)O(2) in guard cells. This study shows that, in Arabidopsis thaliana guard cells, ABA-mediated NO generation is in fact dependent on ABA-induced H(2)O(2) production. Stomatal closure induced by H(2)O(2) is inhibited by the removal of NO with NO scavenger, and both ABA and H(2)O(2) stimulate guard cell NO synthesis. Conversely, NO-induced stomatal closure does not require H(2)O(2) synthesis nor does NO treatment induce H(2)O(2) production in guard cells. Tungstate inhibition of the NO-generating enzyme nitrate reductase (NR) attenuates NO production in response to nitrite in vitro and in response to H(2)O(2) and ABA in vivo. Genetic data demonstrate that NR is the major source of NO in guard cells in response to ABA-mediated H(2)O(2) synthesis. In the NR double mutant nia1, nia2 both ABA and H(2)O(2) fail to induce NO production or stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant, responses to H(2)O(2) are not impaired. Importantly, we show that in the NADPH oxidase deficient double mutant atrbohD/F, NO synthesis and stomatal closure to ABA are severely reduced, indicating that endogenous H(2)O(2) production induced by ABA is required for NO synthesis. In summary, our physiological and genetic data demonstrate a strong inter-relationship between ABA, endogenous H(2)O(2) and NO-induced stomatal closure.     

Metagenomic analysis exploring taxonomic diversity of rumen microbial communities in Vechur and crossbred cattle of Kerala state,India

Rumen, one of the most productive diverse microbial habitats plays a vital role in the breakdown of feed to produce energy for maintenance and milk production in cattle. Culture-based procedures...     

Structural insights into the anti-cancer activity of quercetin on G-tetrad,mixed G-tetrad,and G-quadruplex DNA using quantum chemical and molecular dynamics simulations

Abstract

Human telomerase referred as ‘terminal transferase’ is a nucleoprotein enzyme which inhibits the disintegration of telomere length and act as a drug target for the anticancer therapy. The tandem repeating structure of telomere sequence forms the guanine-rich quadruplex structures that stabilize stacked tetrads. In our present work, we have investigated the interaction of quercetin with DNA tetrads using DFT. Geometrical analysis revealed that the influence of quercetin drug induces the structural changes into the DNA tetrads. Among DNA tetrads, the quercetin stacked with GCGC tetrad has the highest interaction energy of ?88.08?kcal/mol. The binding mode and the structural stability are verified by the absorption spectroscopy method. The longer wavelength was found at 380?nm and it exhibits bathochromic shift. The findings help us to understand the binding nature of quercetin drug with DNA tetrads and it also inhibits the telomerase activity. Further, the quercetin drug interacted with G-quadruplex DNA by using molecular dynamics (MD) simulation studies for 100?ns simulation at different temperatures and different pH levels (T?=?298 K, 320?K and pH = 7.4, 5.4). The structural stability of the quercetin with G-quadruplex structure is confirmed by RMSD. For the acidic condition (pH = 5.4), the binding affinity is higher toward G-quadruplex DNA, this result resembles that the quercetin drug is well interacted with G-quadruplex DNA at acidic condition (pH = 7.4) than the neutral condition. The obtained results show that quercetin drug stabilizes the G-quadruplex DNA, which regulates telomerase enzyme and it potentially acts as a novel anti-cancer agent.

Communicated by Ramaswamy H. Sarma     

The effects of hitchhiker antigens co-eluting with affinity-purified research antibodies

The popularity of Protein G for the purification of antibodies has given rise to an entire industry that supplies scientists with research grade immunoreagents; however, many times the supplied product is contaminated with antigens bound to the antibody's complementarity-determining regions (CDRs). These "hitchhikers" are a category of host cell proteins that are elusive to detect due to their interaction with the antibody in the final product and yet their impact on an experiment or an entire field of study can be far reaching. In an earlier work, the role of hitchhikers on a human anti-histone antibody destined for clinical usage was explored and a stringent purification scheme developed. Here we use a murine monoclonal, which reflects the type of commercial antibody usually purchased for research. We evaluate three purification schemes: a traditional approach using a one-step, low pH elution buffer (pH 2.5); a gentler approach using a pH gradient elution scheme (pH 7 down to pH 2.5); and finally, a more stringent purification patterned on our earlier published method that uses a quaternary amine guard column and a high salt wash during antibody immobilization on the Protein G. We stress that the stringent purification incorporates the pH gradient scheme and is gentler than the low-pH approach. The resulting product from all three purifications is directly compared for binding potency, histone content (using an ELISA based assay) and residual DNA (using quantitative PCR). The results demonstrate that the first two methods are inadequate for hitchhiker removal. The traditional one-step, low pH approach produces a single elution peak containing histone contaminated antibody with picogram quantities of residual DNA, however, the trailing end of the same peak is loaded with antibody complexed to nanogram amounts of DNA, in some cases, over 100 ng. The pH gradient approach provided antibodies accompanied by only picograms of residual DNA and, on average, 1 out of every 10-20 CDRs occupied by a histone antigen. The more stringent approach, using the salt wash prior to elution with the pH gradient, has an average of 1 out of every 75 CDRs contaminated with a histone while the majority of the residual DNA is captured by the quaternary amine column placed in front of the Protein G. The consequences of these contaminants is illustrated by showing how they manifest themselves in unusual antibody potency values ranging from 558% for antibody bound to histone hitchhikers down to 15% for antibody contaminated with DNA hitchhikers. Those samples purified by the recommended stringent approach show potency values between 90 and 101%. Most importantly, we repeatedly demonstrate in a simulated chromatin immunoprecipitation (ChIP) assay the ability to precipitate clean plasmid DNA with histone contaminated antibody that had been purified using the traditional one-step, low pH elution approach. Expectedly, those antibodies stringently purified and showing 100% binding potency were unable to precipitate DNA in the absence of histone hitchhikers.     

Pharmacological blockade of TRPM8 ion channels alters cold and cold pain responses in mice

TRPM8 (Transient Receptor Potential Melastatin-8) is a cold- and menthol-gated ion channel necessary for the detection of cold temperatures in the mammalian peripheral nervous system. Functioning TRPM8 channels are required for behavioral responses to innocuous cool, noxious cold, injury-evoked cold hypersensitivity, cooling-mediated analgesia, and thermoregulation. Because of these various roles, the ability to pharmacologically manipulate TRPM8 function to alter the excitability of cold-sensing neurons may have broad impact clinically. Here we examined a novel compound, PBMC (1-phenylethyl-4-(benzyloxy)-3-methoxybenzyl(2-aminoethyl)carbamate) which robustly and selectively inhibited TRPM8 channels in vitro with sub-nanomolar affinity, as determined by calcium microfluorimetry and electrophysiology. The actions of PBMC were selective for TRPM8, with no functional effects observed for the sensory ion channels TRPV1 and TRPA1. PBMC altered TRPM8 gating by shifting the voltage-dependence of menthol-evoked currents towards positive membrane potentials. When administered systemically to mice, PBMC treatment produced a dose-dependent hypothermia in wildtype animals while TRPM8-knockout mice remained unaffected. This hypothermic response was reduced at lower doses, whereas responses to evaporative cooling were still significantly attenuated. Lastly, systemic PBMC also diminished cold hypersensitivity in inflammatory and nerve-injury pain models, but was ineffective against oxaliplatin-induced neuropathic cold hypersensitivity, despite our findings that TRPM8 is required for the cold-related symptoms of this pathology. Thus PBMC is an attractive compound that serves as a template for the formulation of highly specific and potent TRPM8 antagonists that will have utility both in vitro and in vivo.     

Expression of receptors for VEGFs on normal human thyroid follicular cells and their role in follicle formation

Human thyroid follicular cells in culture expressed the mRNAs for the receptors for vascular endothelial growth factors (VEGFRs). The relative expression was neuropilin1 = neuropilin2 = VEGFR2 > VEGFR1 > VEGFR3. Western blotting for VEGFR2 showed labeling of proteins ~200-230 kDa. Clonal follicular thyroid cell lines (FRTL5 and FTC133) also expressed mRNAs for the VEGFR1 and 2 obviating concerns of endothelial cell contamination. In the primary cultures, TSH, which is essential for expression of differentiated function, reduced VEGFR2 mRNA levels by 60%. Immunostaining for VEGFRs and neuropilin2 (NRP2), showed expression on the plasma membrane but with the exception of neuropilin1 (NRP1), all VEGFRs were also found in the cytoplasm and nucleus. Antibody specific for phosphotyrosine 1214 in VEGFR2 showed that the receptor was phosphorylated in the primary cultures and the cell lines. When VEGFR signaling was blocked with a specific inhibitor, follicle formation in the primary cultures was enhanced suggesting that VEGFR activation was detrimental to follicle formation. Immunostaining of sections of normal thyroids and various pathologies showed staining for VEGFR2 and pVEGFR2. We conclude that normal thyroid follicular cell express VEGFRs. For VEGFR2 its subcellular localization suggests functions additional to that of a cell surface receptor and a role in follicular integrity.     

Cryo‐EM structures of pentameric autoinducer‐2 exporter from Escherichia coli reveal its transport mechanism

Bacteria utilize small extracellular molecules to communicate in order to collectively coordinate their behaviors in response to the population density. Autoinducer‐2 (AI‐2), a universal molecule for both intra‐ and inter‐species communication, is involved in the regulation of biofilm formation, virulence, motility, chemotaxis, and antibiotic resistance. While many studies have been devoted to understanding the biosynthesis and sensing of AI‐2, very little information is available on its export. The protein TqsA from Escherichia coli, which belongs to the AI‐2 exporter superfamily, has been shown to export AI‐2. Here, we report the cryogenic electron microscopic structures of two AI‐2 exporters (TqsA and YdiK) from E. coli at 3.35 Å and 2.80 Å resolutions, respectively. Our structures suggest that the AI‐2 exporter exists as a homo‐pentameric complex. In silico molecular docking and native mass spectrometry experiments were employed to demonstrate the interaction between AI‐2 and TqsA, and the results highlight the functional importance of two helical hairpins in substrate binding. We propose that each monomer works as an independent functional unit utilizing an elevator‐type transport mechanism.