Inhibition of the cardiac Na+ channel Nav1.5 by carbon monoxide

Sub-lethal carbon monoxide (CO) exposure is frequently associated with myocardial arrhythmias and our recent studies have demonstrated that these may be attributable to modulation of cardiac Na+ channels, causing an increase in the late current and an inhibition of the peak current. Using a recombinant expression system, we demonstrate that CO inhibits peak human Nav1.5 current amplitude without activation of the late Na+ current observed in native tissue. Inhibition was associated with a hyperpolarizing shift in the steady-state inactivation properties of the channels and was unaffected by modification of channel gating induced by anemone toxin (rATX-II). Systematic pharmacological assessment indicated that no recognised CO-sensitive intracellular signalling pathways appeared to mediate CO inhibition of Nav1.5. Inhibition was, however, markedly suppressed by inhibition of nitric oxide (NO) formation, but NO donors did not mimic or occlude channel inhibition by CO, indicating that NO alone did not account for the actions of CO. Exposure of cells to dithiothreitol immediately before CO exposure also dramatically reduced the magnitude of current inhibition. Similarly, L-cysteine and N-ethylmaleimide significantly attenuated the inhibition caused by CO. In the presence of DTT and the NO inhibitor L-NAME, the ability of CO to inhibit Nav1.5 was almost fully prevented. Our data indicate that inhibition of peak Na+ current (which can lead to Brugada-syndrome like arrhythmias) occurs via a mechanism distinct from induction of the late current, requires NO formation and is dependent on channel redox state.

Heme oxygenase-1 regulates cell proliferation via carbon monoxide-mediated inhibition of T-type Ca2+ channels

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically.

Claviceps purpurea expressing polygalacturonases escaping PGIP inhibition fully infects PvPGIP2 wheat transgenic plants but its infection is delayed in wheat transgenic plants with increased level of pectin methyl esterification

Claviceps purpurea is a biotrophic fungal pathogen of grasses causing the ergot disease. The infection process of C. purpurea on rye flowers is accompanied by pectin degradation and polygalacturonase (PG) activity represents a pathogenicity factor. Wheat is also infected by C. purpurea and we tested whether the presence of polygalacturonase inhibiting protein (PGIP) can affect pathogen infection and ergot disease development. Wheat transgenic plants expressing the bean PvPGIP2 did not show a clear reduction of disease symptoms when infected with C. purpurea. To ascertain the possible cause underlying this lack of improved resistance of PvPGIP2 plants, we expressed both polygalacturonases present in the C. purpurea genome, cppg1 and cppg2 in Pichia pastoris. In vitro assays using the heterologous expressed PGs and PvPGIP2 showed that neither PG is inhibited by this inhibitor. To further investigate the role of PG in the C. purpurea/wheat system, we demonstrated that the activity of both PGs of C. purpurea is reduced on highly methyl esterified pectin. Finally, we showed that this reduction in PG activity is relevant in planta, by inoculating with C. purpurea transgenic wheat plants overexpressing a pectin methyl esterase inhibitor (PMEI) and showing a high degree of pectin methyl esterification. We observed reduced disease symptoms in the transgenic line compared with null controls. Together, these results highlight the importance of pectin degradation for ergot disease development in wheat and sustain the notion that inhibition of pectin degradation may represent a possible route to control of ergot in cereals.

Propeptide-mediated inhibition of myostatin increases muscle mass through inhibiting proteolytic pathways in aged mice

Mammalian aging is accompanied by a progressive loss of skeletal muscle, a process called sarcopenia. Myostatin, a secreted member of the transforming growth factor-β family of signaling molecules, has been shown to be a potent inhibitor of muscle growth. Here, we examined whether muscle growth could be promoted in aged animals by antagonizing the activity of myostatin through the neutralizing activity of the myostatin propeptide. We show that a single injection of an AAV8 virus expressing the myostatin propeptide induced an increase in whole body weights and all muscles examined within 7 weeks of treatment. Our cellular studies demonstrate that muscle enlargement was due to selective fiber type hypertrophy, which was accompanied by a shift toward a glycolytic phenotype. Our molecular investigations elucidate the mechanism underpinning muscle hypertrophy by showing a decrease in the expression of key genes that control ubiquitin-mediated protein breakdown. Most importantly, we show that the hypertrophic muscle that develops as a consequence of myostatin propeptide in aged mice has normal contractile properties. We suggest that attenuating myostatin signaling could be a very attractive strategy to halt and possibly reverse age-related muscle loss.

Mastery-approach goals eliminate retrieval-induced forgetting: the role of achievement goals in memory inhibition

The present study examined how achievement goals affect retrieval-induced forgetting. Researchers have suggested that mastery-approach goals (i.e., developing one’s own competence) promote a relational encoding, whereas performance-approach goals (i.e., demonstrating one’s ability in comparison to others) promote item-specific encoding. These different encoding processes may affect the degree to which participants integrate the exemplars within a category and, as a result, we expected that retrieval-induced forgetting may be reduced or eliminated under mastery-approach goals. Three experiments were conducted using a retrieval-practice paradigm with different stimuli, where participants’ achievement goals were manipulated through brief written instructions. A meta-analysis that synthesized the results of the three experiments showed that retrieval-induced forgetting was not statistically significant in the mastery-approach goal condition, whereas it was statistically significant in the performance-approach goal condition. These results suggest that mastery-approach goals eliminate retrieval-induced forgetting, but performance-approach goals do not, demonstrating that motivation factors can influence inhibition and forgetting.

Diallyl disulfide-induced apoptosis in a breast-cancer cell line (MCF-7) may be caused by inhibition of histone de-acetylation

The health benefits of garlic have been proven by epidemiological and experimental studies. Diallyl disulphide (DADS), the major organosulfur compound found in garlic oil, is known to lower the incidence of breast cancer both in vitro and in vivo. The studies reported here demonstrate that DADS induces apoptosis in the MCF-7 breast-cancer cell line through interfering with cell-cycle growth phases in a way that increases the sub-G0 population and substantially halts DNA synthesis. DADS also induces phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane and activates caspase-3. Further studies revealed that DADS modulates the cellular levels of Bax, Bcl-2, Bcl-xL and Bcl-w in a dose-dependent manner, suggesting the involvement of Bcl-2 family proteins in DADS induced apoptosis. Histone deacetylation inhibitors (HDACi) are known to suppress cancer growth and induce apoptosis in cancer cells. Here it is shown that DADS has HDACi properties in MCF-7 cells as it lowers the removal of an acetyl group from an acetylated substrate and induces histone-4 (H4) hyper-acetylation. The data thus indicate that the HDACi properties of DADS may be responsible for the induction of apoptosis in breast cancer cells.

GRID and docking analyses reveal a molecular basis for flavonoid inhibition of src-family kinase activity

Flavonoids reduce cardiovascular disease risk through anti-inflammatory, anti-coagulant and anti-platelet actions. One key flavonoid inhibitory mechanism is blocking kinase activity that drives these processes. Flavonoids attenuate activities of kinases including phosphoinositide-3-kinase (PI3K), Fyn, Lyn, Src, Syk, PKC, PIM1/2, ERK, JNK, and PKA. X-ray crystallographic analyses of kinase-flavonoid complexes show that flavonoid ring systems and their hydroxyl substitutions are important structural features for their binding to kinases. A clearer understanding of structural interactions of flavonoids with kinases is necessary to allow construction of more potent and selective counterparts. We examined flavonoid (quercetin, apigenin and catechin) interactions with Src-family kinases (Lyn, Fyn and Hck) applying the Sybyl docking algorithm and GRID. A homology model (Lyn) was used in our analyses to demonstrate that high quality predicted kinase structures are suitable for flavonoid computational studies. Our docking results revealed potential hydrogen bond contacts between flavonoid hydroxyls and kinase catalytic site residues. Identification of plausible contacts indicated that quercetin formed the most energetically stable interactions, apigenin lacked hydroxyl groups necessary for important contacts, and the non-planar structure of catechin could not support predicted hydrogen bonding patterns. GRID analysis using a hydroxyl functional group supported docking results. Based on these findings, we predicted that quercetin would inhibit activities of Src-family kinases with greater potency than apigenin and catechin. We validated this prediction using in vitro kinase assays. We conclude that our study can be used as a basis to construct virtual flavonoid interaction libraries to guide drug discovery using these compounds as molecular templates.

Synergistic inhibition of Haemonchus contortus exsheathment by flavonoid monomers and condensed tannins

This study investigated the separate and combined anthelmintic (AH) effects of different phenolic compounds, including condensed tannins and flavonoids, all of which are known to occur in willow leaves, a potentially valuable dry season feed. A range of contrasting model tannins, which span the whole range of willow tannins, were isolated from tilia flowers, goat willow leaves, black currant leaves and red currant leaves. All together, the tested compounds represented the major tannin types (procyanidins and prodelphinidins) and flavonoid types (flavonols, flavones and flavanones). The larval exsheathment inhibition assay (LEIA) was used to assess their in vitro effects on Haemonchus contortus third stage larvae. Arbutin, vanillic acid, and taxifolin proved to be ineffective whereas naringenin, quercetin and luteolin were highly effective at 250 μM concentrations. Procyanidin (PC) tannins tended to be less active than prodelphinidin tannins (PD). Experiments with combinations of tannins and quercetin or luteolin revealed for the first time the existence of synergistic AH effects between tannins and flavonoid monomers. They also provided evidence that synergistic effects appear to occur at slightly lower concentrations of PC than PD. This suggests that the AH activity of condensed tannins can be significantly enhanced by the addition of quercetin or luteolin. This information may prove useful for plant breeding or selection and for designing optimal feed mixtures.

Methane cross-validation between three Fourier Transform Spectrometers: SCISAT ACE-FTS, GOSAT TANSO-FTS, and ground-based FTS measurements in the Canadian high Arctic

We present cross-validation of remote sensing measurements of methane profiles in the Canadian high Arctic. Accurate and precise measurements of methane are essential to understand quantitatively its role in the climate system and in global change. Here, we show a cross-validation between three datasets: two from spaceborne instruments and one from a ground-based instrument. All are Fourier Transform Spectrometers (FTSs). We consider the Canadian SCISAT Atmospheric Chemistry Experiment (ACE)-FTS, a solar occultation infrared spectrometer operating since 2004, and the thermal infrared band of the Japanese Greenhouse Gases Observing Satellite (GOSAT) Thermal And Near infrared Sensor for carbon Observation (TANSO)-FTS, a nadir/off-nadir scanning FTS instrument operating at solar and terrestrial infrared wavelengths, since 2009. The ground-based instrument is a Bruker 125HR Fourier Transform Infrared (FTIR) spectrometer, measuring mid-infrared solar absorption spectra at the Polar Environment Atmospheric Research Laboratory (PEARL) Ridge Lab at Eureka, Nunavut (80° N, 86° W) since 2006. For each pair of instruments, measurements are collocated within 500 km and 24 h. An additional criterion based on potential vorticity values was found not to significantly affect differences between measurements. Profiles are regridded to a common vertical grid for each comparison set. To account for differing vertical resolutions, ACE-FTS measurements are smoothed to the resolution of either PEARL-FTS or TANSO-FTS, and PEARL-FTS measurements are smoothed to the TANSO-FTS resolution. Differences for each pair are examined in terms of profile and partial columns. During the period considered, the number of collocations for each pair is large enough to obtain a good sample size (from several hundred to tens of thousands depending on pair and configuration). Considering full profiles, the degrees of freedom for signal (DOFS) are between 0.2 and 0.7 for TANSO-FTS and between 1.5 and 3 for PEARL-FTS, while ACE-FTS has considerably more information (roughly 1° of freedom per altitude level). We take partial columns between roughly 5 and 30 km for the ACE-FTS–PEARL-FTS comparison, and between 5 and 10 km for the other pairs. The DOFS for the partial columns are between 1.2 and 2 for PEARL-FTS collocated with ACE-FTS, between 0.1 and 0.5 for PEARL-FTS collocated with TANSO-FTS or for TANSO-FTS collocated with either other instrument, while ACE-FTS has much higher information content. For all pairs, the partial column differences are within ± 3 × 1022 molecules cm−2. Expressed as median ± median absolute deviation (expressed in absolute or relative terms), these differences are 0.11 ± 9.60 × 10^20 molecules cm−2 (0.012 ± 1.018 %) for TANSO-FTS–PEARL-FTS, −2.6 ± 2.6 × 10^21 molecules cm−2 (−1.6 ± 1.6 %) for ACE-FTS–PEARL-FTS, and 7.4 ± 6.0 × 10^20 molecules cm−2 (0.78 ± 0.64 %) for TANSO-FTS–ACE-FTS. The differences for ACE-FTS–PEARL-FTS and TANSO-FTS–PEARL-FTS partial columns decrease significantly as a function of PEARL partial columns, whereas the range of partial column values for TANSO-FTS–ACE-FTS collocations is too small to draw any conclusion on its dependence on ACE-FTS partial columns.

Climatology and variability of trace gases in extratropical double-tropopause regions from MLS, HIRDLS, and ACE-FTS measurements

Upper tropospheric and lower stratospheric measurements from the Aura Microwave Limb Sounder (MLS), the Aura High Resolution Dynamics Limb Sounder (HIRDLS), and the Atmospheric Chemistry Experiment-Fourier transform spectrometer (ACE-FTS) are used to present the first global climatological comparison of extratropical, nonpolar trace gas distributions in double-tropopause (DT) and single-tropopause (ST) regions. Stratospheric tracers, O3, HNO3, and HCl, have lower mixing ratios ∼2–8 km above the primary (lowermost) tropopause in DT than in ST regions in all seasons, with maximum Northern Hemisphere (NH) differences near 50% in winter and 30% in summer. Southern Hemisphere winter differences are somewhat smaller, but summer differences are similar in the two hemispheres. H2O in DT regions of both hemispheres shows strong negative anomalies in November through February and positive anomalies in July through October, reflecting the strong seasonal cycle in H2O near the tropical tropopause. CO and other tropospheric tracers examined have higher DT than ST values 2–7 km above the primary tropopause, with the largest differences in winter. Large DT-ST differences extend to high NH latitudes in fall and winter, with longitudinal maxima in regions associated with enhanced wave activity and subtropical jet variations. Results for O3 and HNO3 agree closely between MLS and HIRDLS, and differences from ACE-FTS are consistent with its sparse and irregular midlatitude sampling. Consistent signatures in climatological trace gas fields provide strong evidence that transport from the tropical upper troposphere into the layer between double tropopauses is an important pathway for stratosphere-troposphere exchange.

A tillering inhibition gene influences root-shoot carbon partitioning and pattern of water use to improve wheat productivity in rainfed environments

Genetic modification of shoot and root morphology has potential to improve water and nutrient 19 uptake of wheat crops in rainfed environments. Near-isogenic lines (NILs) varying for a tillering 20 inhibition (tin) gene and representing multiple genetic backgrounds were investigated in contrasting 21 controlled environments for shoot and root growth. Leaf area, shoot and root biomass were similar 22 until tillering whereupon reduced tillering in tin-containing NILs produced reductions of up to 60% in 23 total leaf area and biomass, and increases in total root length of up to 120% and root biomass to 24 145%. Together, root-to-shoot ratio increased two-fold with the tin gene. The influence of tin on shoot 25 and root growth was greatest in the cv. Banks genetic background, particularly in the biculm-selected 26 NIL, and was typically strongest in cooler environments. A separate de-tillering study confirmed 27 greater root-to-shoot ratios with regular tiller removal in non-tin containing genotypes. In validating 28 these observations in a rainfed field study, the tin allele had a negligible effect on seedling growth but 29 was associated with significantly (P<0.05) reduced tiller number (-37%), leaf area index (-26%) and 30 spike number (-35%) to reduce plant biomass (-19%) at anthesis. Root biomass, root-to-shoot ratio at 31 early stem elongation and root depth at maturity were increased in tin-containing NILs. Soil water use 32 was slowed in tin-containing NILs resulting in greater water availability, greater stomatal 33 conductance, cooler canopy temperatures and maintenance of green leaf area during grain-filling. 34 Together these effects contributed to increases in harvest index and grain yield. In both the controlled 35 and field environments, the tin gene was commonly associated with increased root length and biomass 36 but the significant influence of genetic background and environment suggests careful assessment of 37 tin-containing progeny in selection for genotypic increases in root growth.