Whole miRNome-wide Differential Co-expression of MicroRNAs

Co-regulation of genes has been extensively analyzed, however, rather limited knowledge is available on co-regulations within the miRNome. We investigated differential co-expression of microRNAs (miRNAs) based on miRNome profiles of whole blood from 540 individuals. These include patients suffering from different cancer and non-cancer diseases, and unaffected controls. Using hierarchi-cal clustering, we found 9 significant clusters of co-expressed miRNAs containing 2-36 individual miRNAs. Through analyzing multiple sequencing alignments in the clusters, we found that co-expression of miRNAs is associated with both sequence similarity and genomic co-localization. We calculated correlations for all 371,953 pairs of miRNAs for all 540 individuals and identified 184 pairs of miRNAs with high correlation values. Out of these 184 pairs of miRNAs, 16 pairs (8.7%) were differentially co-expressed in unaffected controls, cancer patients and patients with non-cancer diseases. By computing correlated and anti-correlated miRNA pairs, we constructed a network with 184 putative co-regulations as edges and 100 miRNAs as nodes. Thereby, we detected specific clusters of miRNAs with high and low correlation values. Our approach represents the most comprehensive co-regulation analysis based on whole miRNome-wide expression profiling. Our findings further decrypt the interactions of miRNAs in normal and human pathological processes.     

Mechanism of Action of Secreted Newt Anterior Gradient Protein

Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family.     

Seed biopriming with drought tolerant isolates of Trichoderma harzianum promote growth and drought tolerance in Triticum aestivum

Green house study was aimed to investigate the effect of seed biopriming with drought tolerant isolates of Trichoderma harzianum, viz. Th 56, 69, 75, 82 and 89 on growth of wheat under drought stress and to explore the mechanism underlying plant water stress resilience in response to Trichoderma inoculation. Measurements of relative water content, osmotic potential, osmotic adjustment, leaf gas exchange, chlorophyll fluorescence and membrane stability index were performed. In addition, analysis of the phenolics, proline, lipid peroxidation and measurements of phenylalanine ammonia‐lyase activity were carried out. Seed biopriming enhanced drought tolerance of wheat as drought induced changes like stomatal conductance, net photosynthesis and chlorophyll fluorescence were delayed. Drought stress from 4 to 13 days of withholding water induced an increase in the concentration of stress induced metabolites in leaves, while Trichoderma colonisation caused decrease in proline, malondialdehyde (MDA) and hydrogen peroxide (H₂O₂), and an increase in total phenolics. A common factor that negatively affects plants under drought stress conditions is accumulation of toxic reactive oxygen species (ROS), and we tested the hypothesis that seed biopriming reduced damages resulting from accumulation of ROS in stressed plants. The enhanced redox state of colonised plants could be explained by higher l ‐phenylalanine ammonia‐lyase (PAL) activity in leaves after 13 days of drought stress in Trichoderma treated plants. Similar activity was induced in untreated plants in response to drought stress but to a lower extent in comparison to treated plants. Our results support the hypothesis that seed biopriming in wheat with drought tolerant T. harzianum strains increased root vigour besides performing the process of osmoregulation. It ameliorates drought stress by inducing physiological protection in plants against oxidative damage, due to enhanced capacity to scavenge ROS and increased level of PAL, a mechanism that is expected to augment tolerance to abiotic stresses.     

Aza-annulation on the 16-dehydropregnenolone, via tandem intermolecular aldol process and intramolecular Michael addition

16-Dehydropregnenolone undergoes a smooth annulation with propan-1-amine and aromatic aldehydes. Several amine derivatives of 16- dehydropregnenolone were synthesized and evaluated as inhibitors of DPP-IV. The structures of compounds were confirmed by ¹H, ¹³C, NMR and mass spectral analysis. Among 17 compounds evaluated only five compounds 1, 9, 13, 15 and 16 demonstrated significant inhibition of DPP. This study suggest that introduction of appropriate substituents in the 16-dehydropregnenolone plays an important role in DPP-IV inhibitory activity.     

Mechanism of glucose-6-phosphate dehydrogenase-mediated regulation of coronary artery contractility

We previously identified glucose-6-phosphate dehydrogenase (G6PD) as a regulator of vascular smooth muscle contraction. In this study, we tested our hypothesis that G6PD activated by KCl via a phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-protein kinase C (PKC) pathway increases vascular smooth muscle contraction and that inhibition of G6PD relaxes smooth muscle by decreasing intracellular Ca(2+) ([Ca(2+)](i)) and Ca(2+) sensitivity to the myofilament. Here we show that G6PD is activated by membrane depolarization via PKC and PTEN pathway and that G6PD inhibition decreases intracellular free calcium ([Ca(2+)](i)) in vascular smooth muscle cells and thus arterial contractility. In bovine coronary artery (CA), KCl (30 mmol/l) increased PKC activity and doubled G6PD V(max) without affecting K(m). KCl-induced PKC and G6PD activation was inhibited by bisperoxo(pyridine-2-carboxyl)oxovanadate (Bpv; 10 μmol/l), a PTEN inhibitor, which also inhibited (P < 0.05) KCl-induced CA contraction. The G6PD blockers 6-aminonicotinamide (6AN; 1 mmol/l) and epiandrosterone (EPI; 100 μmol/l) inhibited KCl-induced increases in G6PD activity, [Ca(2+)](i), Ca(2+)-dependent myosin light chain (MLC) phosphorylation, and contraction. Relaxation of precontracted CA by 6AN and EPI was not blocked by calnoxin (10 μmol/l), a plasma membrane Ca(2+) ATPase inhibitor or by lowering extracellular Na(+), which inhibits the Na(+)/Ca(2+) exchanger (NCX), but cyclopiazonic acid (200 μmol/l), a sarcoplasmic reticulum Ca(2+) ATPase inhibitor, reduced (P < 0.05) 6AN- and EPI-induced relaxation. 6AN also attenuated phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Ser855, a site phosphorylated by Rho kinase, inhibition of which reduced (P < 0.05) KCl-induced CA contraction and 6AN-induced relaxation. By contrast, 6AN increased (P < 0.05) vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser239, indicating that inhibition of G6PD increases PKA or PKG activity. Inhibition of PKG by RT-8-Br-PET-cGMPs (100 nmol/l) diminished 6AN-evoked VASP phosphorylation (P < 0.05), but RT-8-Br-PET-cGMPs increased 6AN-induced relaxation. These findings suggest G6PD inhibition relaxes CA by decreasing Ca(2+) influx, increasing Ca(2+) sequestration, and inhibiting Rho kinase but not by increasing Ca(2+) extrusion or activating PKG.     

New national and regional bryophyte records, 46

Abstract

Bryophyte biomass and diversity vary strongly with altitude in the tropics. Low abundance and low species numbers in lowland rain forests are most likely due to reduced diurnal activity times combined with high nocturnal respiration rates at high temperatures. This may exclude many montane species from the warm lowlands. However, an alternative hypothesis explains the observed pattern, namely a limited desiccation tolerance of montane species, precipitation being more concentrated but less frequent in most lowland forests compared to montane cloud forests. To test this hypothesis, we studied the desiccation tolerance of four montane and four lowland bryophyte species. The effects of prolonged drought were quantified with chlorophyll fluorescence (F_v/F_m) and the extent of electrolyte leakage. Both montane and lowland species survived dry periods of ≧80 days, which far exceeds the duration of dry periods in the wet lowland tropics. We can thus exclude intolerance to long dry spells as an explaination for the absence of the tested montane species in the lowlands. We should continue to focus on other mechanisms to explain the altitudinal gradient of bryophyte abundance and diversity in the tropics, in order to understand this pattern, as well as to predict future trends under climatic warming.     

Preparation of cultured myofibers from larval salamander limbs for cellular plasticity studies

Identification of the mechanisms underlying cellular plasticity in salamander cells is important because these may give pointers to the restricted regenerative ability of mammals. The myofibers from salamanders are remarkable for their ability to undergo cellularization and cell-cycle re-entry during regeneration. Here, we describe a detailed method for the isolation and culture of larval salamander myofibers in numbers suitable for cellular plasticity studies. The basic protocol for isolation and purification of cells can be completed in 4 h.