Phosphatidic acid activates a wound-activated MAPK in Glycine max

Many plant species demonstrate a systemic increase in phosphatidic acid (PA) levels after being wounded (Lee et al., 1997). To understand the role of PA in wound signal transduction, we investigated if PA can activate protein kinases in soybean (Glycine max L.). We found that a MAPK is activated in soybean seedlings in both wounded and neighboring unwounded leaves. The wound-activated soybean kinase is specifically recognized by an antibody against the alfalfa MAPK, SIMK. When PA production is inhibited with n-butanol, an inhibitor of phospholipase D, the wound-induced activation of the MAPK is suppressed, suggesting that an elevation in PA levels is essential for its activation. Supporting this is the observation that exogenous PA activates the MAPK in suspension-cultured soybean cells. Activation of the 49 kDa MAPK occurs almost exclusively by PA, as other lipids are unable to or can only weakly activate the kinase. PA-induced activation of the MAPK is not a direct effect on the kinase but is mediated by upstream kinases. Our results suggest that PA acts as a second messenger in wound-induced MAPK signaling in plants.     

JH-4 reduces HMGB1-mediated septic responses and improves survival rate in septic mice

Inhibition of high mobility group box 1 (HMGB1) and restoration of endothelial integrity are emerging as attractive therapeutic strategies for the management of severe vascular inflammatory diseases. Recently, we found that JH-4, a synthesized decursin derivative, exhibited a strong anti-Hutchinson-Gilford progeria syndrome by efficiently blocking progerin-lamin A/C binding. In this study, we examined the effects of JH-4 on HMGB1-mediated septic responses and the survival rate in a mouse sepsis model. The anti-inflammatory activities of JH-4 were monitored based on its effects on lipopolysaccharide- or cecal ligation and puncture (CLP)-mediated release of HMGB1. The antiseptic activities of JH-4 were determined by measuring permeability, leukocyte adhesion, migration, and the activation of proinflammatory proteins in HMGB1-activated human umbilical vein endothelial cells and mice. JH-4 inhibited the release of HMGB1 and downregulated HMGB1-dependent inflammatory responses in human endothelial cells. JH-4 also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with JH-4 reduced CLP-induced release of HMGB1, sepsis-related mortality, and pulmonary injury in vivo. Our results indicate that JH-4 is a possible therapeutic agent to treat various severe vascular inflammatory diseases via the inhibition of the HMGB1 signaling pathway.     

Docosahexaenoic acid affects endothelial nitric oxide synthase in caveolae

n-3 Polyunsaturated fatty acids are assumed to play an important role in the prevention and treatment of atherosclerosis. Endothelial nitric-oxide synthase (eNOS) is responsible for cardiovascular homeostasis involving in regulation of vascular function, and the subcellular localization is critical for its activation. Here we determined the effect of docosahexaenoic acid (DHA, 22:6 n-3) on distribution of eNOS and its activity. DHA treatment markedly altered lipid environment of caveolae microdomains, which was coincided with selective displacement of caveolin-1 and eNOS from caveolae. Akt was not detected in caveolae fractions and CaM was distributed in both of caveolin-1-enriched membranes and non-caveolar fractions, whose distribution was unaffected by DHA. These data demonstrated for the first time that DHA altered caveolae microenvironment not only by modifying membrane lipid composition, but also by changing distribution of major structural proteins. DHA-induced alterations in caveolae lipid/protein environment may be an important mechanism in the development of pathogenesis of atherosclerosis.     

Eicosapentaenoic acid modifies lipid composition in caveolae and induces translocation of endothelial nitric oxide synthase

Endothelial nitric oxide synthase (eNOS) plays a crucial role in the regulation of a variety of cardiovascular functions. Many studies have shown that dietary n-3 polyunsaturated fatty acids (PUFAs) have beneficial effects on coronary atherosclerosis. However, the mechanisms of n-3 PUFAs regulation in eNOS activation remain unknown. In the present study we investigated the effects of eicosapentaenoic acid (EPA, 20:5 n-3) on subcellular distribution of eNOS and lipid composition of caveolae. We demonstrated for the first time that EPA treatment profoundly altered lipid composition and fatty acyl substitutions of phospholipids in caveolae. We found that caveolin-1 was solely located in caveolae fractions in control cells, and EPA treatment displaced caveolin-1 from caveolae. eNOS was detected in the caveolin-enriched fractions and noncaveolae fractions in control cells. EPA treatment induced the translocation of eNOS from caveolae fractions to soluble fractions. P-eNOS was also distributed in both fractions. After EPA treatment, the level of p-eNOS in each fraction was increased but the distribution of which was unaffected. Moreover, the results of immunofluorescence confirmed that EPA could redistribute caveolin-1 and eNOS in plasma membrane. eNOS activity in HUVEC cells was increased after EPA treatment, which was in a dose dependent manner. And incubation with 50 microM EPA had the maximum effect on eNOS activity. Our results suggested that eNOS translocation was paralleled by a stimulated capacity for NO production in the cells. We found that total Akt and p-Akt were primarily presented in heavy membranes in control cells, and the relative level of p-Akt increased but the distribution did not change after EPA treatment. The distribution of CaM was slightly changed after EPA treatment. Our results indicated that n-3 PUFAs profoundly altered caveolae microenvironment, thereby modifying location and function of proteins in caveolae. EPA-induced alterations of lipid and proteins in caveolae may be an important mechanism in the pathophysiologic process of atherosclerosis.     

Heat stress in grapevine: the pros and cons of acclimation

Heat stress is a major limiting factor of grapevine production and quality. Acclimation and recovery are essential to ensure plant survival, and the recovery mechanisms can be independent of the heat response mechanisms. An experimental set up with and without acclimation to heat followed by recovery [stepwise acclimation and recovery (SAR) and stepwise recovery (SR), respectively] was applied to two grapevine varieties, Touriga Nacional (TN), and Trincadeira (TR), with different tolerance to abiotic stress. Major differences were found between leaves of SAR and SR, especially after recovery; in SAR, almost all parameters returned to basal levels while in SR they remained altered. Acclimation led to a swifter and short‐term antioxidative response, affecting the plant to a lesser extent than SR. Significant differences were found among varieties: upon stress, TN significantly increased ascorbate and glutathione reduction levels, boosting the cell's redox‐buffering capacity, while TR needed to synthesize both metabolites, its response being insufficient to keep the redox state at working levels. TR was affected by stress for a longer period and the up‐regulation pattern of antioxidative stress genes was more obvious. In TN, heat shock proteins were significantly induced, but the canonical heat‐stress gene signature was not evident probably because no shutdown of the housekeeping metabolism was needed.     

Comparative kinetics of cofactor association and dissociation for the human and trypanosomal S-adenosylhomocysteine hydrolases. 1. Basic features of the association and dissociation processes

The S-adenosyl-l-homocysteine (AdoHcy) hydrolases catalyze the reversible conversion of AdoHcy to adenosine and homocysteine, making use of a catalytic cycle in which a tightly bound NAD+ oxidizes the 3-hydroxyl group of the substrate at the beginning of the cycle, activating the 4-CH bond for elimination of homocysteine, followed by Michael addition of water to the resulting intermediate and a final reduction by the tightly bound NADH to give adenosine. The equilibrium and kinetic properties of the association and dissociation of the cofactor NAD+ from the enzymes of Homo sapiens (Hs-SAHH) and Trypanosoma cruzi (Tc-SAHH) are qualitatively similar but quantitatively distinct. Both enzymes bind NAD+ in a complex scheme. The four active sites of the homotetrameric apoenzyme appear to divide into two numerically equal classes of active sites. One class of sites binds cofactor weakly and generates full activity very rapidly (in less than 1 min). The other class binds cofactor more strongly but generates activity only slowly (>30 min). In the case of Tc-SAHH, the final affinity for NAD+ is roughly micromolar and this affinity persists as the equilibrium affinity. In the case of Hs-SAHH, the slow-binding phase terminates in micromolar affinity also, but over a period of hours, the dissociation rate constant decreases until the final equilibrium affinity is in the nanomolar range. The slow binding of NAD+ by both enzymes exhibits saturation kinetics with respect to the cofactor concentration; however, binding to Hs-SAHH has a maximum rate constant around 0.06 s-1, while the rate constant for binding to Tc-SAHH levels out at 0.006 s-1. In contrast to the complex kinetics of association, both enzymes undergo dissociation of NAD+ from all four sites in a single first-order reaction. The equilibrium affinities of both Hs-SAHH and Tc-SAHH for NADH are in the nanomolar range. The dissociation rate constants and the slow-binding association rate constants for NAD+ show a complex temperature dependence with both enzymes; however, the cofactor always dissociates more rapidly from Tc-SAHH than from Hs-SAHH, the ratio being around 80-fold at 37 degrees C, and the cofactor binds more rapidly to Hs-SAHH than to Tc-SAHH above approximately 16 degrees C. These features present an opening for selective inhibition of Tc-SAHH over Hs-SAHH, demonstrated with the thioamide analogues of NAD+ and NADH. Both analogues bind to Hs-SAHH with approximately 40 nM affinities but much more weakly to Tc-SAHH (0.6-15 microM). Nevertheless, both analogues inactivated Tc-SAHH 60% (NAD+ analogue) or 100% (NADH analogue) within 30 min, while the degree of inhibition of Hs-SAHH approached 30% only after 12 h. The rate of loss of activity is equal to the rate of dissociation of the cofactor and thus 80-fold faster at 37 degrees C for Tc-SAHH.     

Projected changes in mineral soil carbon of European croplands and grasslands, 1990–2080

We present the most comprehensive pan‐European assessment of future changes in cropland and grassland soil organic carbon (SOC) stocks to date, using a dedicated process‐based SOC model and state‐of‐the‐art databases of soil, climate change, land‐use change and technology change. Soil carbon change was calculated using the Rothamsted carbon model on a European 10 × 10′ grid using climate data from four global climate models implementing four Intergovernmental Panel on Climate Change (IPCC) emissions scenarios (SRES). Changes in net primary production (NPP) were calculated by the Lund–Potsdam–Jena model. Land‐use change scenarios, interpreted from the narratives of the IPCC SRES story lines, were used to project changes in cropland and grassland areas. Projections for 1990–2080 are presented for mineral soil only. Climate effects (soil temperature and moisture) will tend to speed decomposition and cause soil carbon stocks to decrease, whereas increases in carbon input because of increasing NPP will slow the loss. Technological improvement may further increase carbon inputs to the soil. Changes in cropland and grassland areas will further affect the total soil carbon stock of European croplands and grasslands. While climate change will be a key driver of change in soil carbon over the 21st Century, changes in technology and land‐use change are estimated to have very significant effects. When incorporating all factors, cropland and grassland soils show a small increase in soil carbon on a per area basis under future climate (1–7 t C ha^?1 for cropland and 3–6 t C ha^?1 for grassland), but when the greatly decreasing area of cropland and grassland are accounted for, total European cropland stocks decline in all scenarios, and grassland stocks decline in all but one scenario. Different trends are seen in different regions. For Europe (the EU25 plus Norway and Switzerland), the cropland SOC stock decreases from 11 Pg in 1990 by 4–6 Pg (39–54%) by 2080, and the grassland SOC stock increases from 6 Pg in 1990 to 1.5 Pg (25%) under the B1 scenario, but decreases to 1–3 Pg (20–44%) under the other scenarios. Uncertainty associated with the land‐use and technology scenarios remains unquantified, but worst‐case quantified uncertainties are 22.5% for croplands and 16% for grasslands, equivalent to potential errors of 2.5 and 1 Pg SOC, respectively. This is equivalent to 42–63% of the predicted SOC stock change for croplands and 33–100% of the predicted SOC stock change for grasslands. Implications for accounting for SOC changes under the Kyoto Protocol are discussed.     

mr1e, a conotoxin from Conus marmoreus with a novel disulfide pattern

Conotoxins are well known for their highly variable structures and functions. Here we report the identification of a novel conotoxin named mr1e from Conus marmoreus . mr1e is composed of 11 amino acid residues cross-linked by two disulfide bonds (CCHSSWCKHLC). The spacing of intercysteine loops in mr1e is exactly the same as that in α4/3 conotoxins. However, the native mr1e peptide co-eluted on reverse-phase HPLC with the regioselectively synthesized ribbon disulfide linkage isomer (C¹-C⁴, C²-C³) but not the globular linkage isomer (C¹-C³, C²-C⁴). Although this peptide has the same disulfide connectivity as the χ-conotoxins, their sequences do not share significant homology. Thus, mr1e could be defined as a novel conotoxin family. By intracranial injection into mice, mr1e showed an excitatory effect. The characterization of mr1e certainly enriches our understanding of conotoxins, and also opens an avenue for further structural and functional investigation.     

不同生境下空心莲子草响应模拟昆虫采食的生长和化学防御策略

外来植物往往可以入侵多种生境并受到多种昆虫的采食,而不同生境条件将可能会影响这些入侵植物对昆虫采食的防御策略。以入侵我国的克隆植物——空心莲子草为研究对象,分别选择生长在水生生境、水陆两栖生境和陆生生境中的无性个体(分株),通过50%去叶处理模拟昆虫采食,分析不同生境下空心莲子草对模拟昆虫采食处理的生长及化学防御响应的差异。模拟昆虫采食处理显著抑制了陆生生境、水陆两栖生境以及水生生境下空心莲子草的根、茎、叶和总生物量,但对3种生境下空心莲子草的生物量分配(根冠比、根生物量分配、茎生物量分配和叶生物量分配)均无显著影响。陆生生境下空心莲子草根、茎和总生物量显著高于水陆两栖生境和水生生境,根冠比显著低于水陆两栖生境和水生生境。模拟昆虫采食处理显著降低了空心莲子草的木质素含量,而对单宁和总酚含量影响不显著。生境对木质素含量无显著影响,但陆生生境下空心莲子草单宁含量显著高于水陆两栖生境和水生生境,且总酚含量显著高于水陆两栖生境,表明陆生生境中空心莲子草具有更强的防御能力。空心莲子草木质素含量与总生物量无显著相关性,但在模拟采食情况下,其总酚含量与总生物量呈显著负相关,而无论模拟昆虫采食处理存在与否,空心莲子草单宁含量与总生物量均呈显著正相关。因此,空心莲子草存在昆虫介导的生长和化学防御之间的权衡,在昆虫采食的情况下可通过减少生长来增加对化学防御物质的投入,但生境对空心莲子草这种生长-防御权衡的影响十分有限。