Copper blocks V-ATPase activity and SNARE complex formation to inhibit yeast vacuole fusion.

The accumulation of copper in organisms can lead to altered functions of various pathways and become cytotoxic through the generation of reactive oxygen species. In yeast, cytotoxic metals such as Hg+ , Cd2+ and Cu2+ are transported into the lumen of the vacuole through various pumps. Copper ions are initially transported into the cell by the copper transporter Ctr1 at the plasma membrane and sequestered by chaperones and other factors to prevent cellular damage by free cations. Excess copper ions can subsequently be transported into the vacuole lumen by an unknown mechanism. Transport across membranes requires the reduction of Cu2+ to Cu+ . Labile copper ions can interact with membranes to alter fluidity, lateral phase separation and fusion. Here we found that CuCl2 potently inhibited vacuole fusion by blocking SNARE pairing. This was accompanied by the inhibition of V-ATPase H+ pumping. Deletion of the vacuolar reductase Fre6 had no effect on the inhibition of fusion by copper. This suggests that Cu2+ is responsible for the inhibition of vacuole fusion and V-ATPase function. This notion is supported by the differential effects of chelators. The Cu2+ -specific chelator triethylenetetramine rescued fusion, whereas the Cu+ -specific chelator bathocuproine disulfonate had no effect on the inhibited fusion.

Antioxidant Properties of the Vam3 Derivative of Resveratrol.

A considerable number of studies has shown that many constituents of foods derived from plants are effective and safe antioxidants. This explains the growing interest in natural antioxidants in food applications. The goal of this investigation was to evaluate the antioxidant properties of the Vam3, a resveratrol derivative, firstly isolated from ethanol extracts of Vitis amurensis Rupr as a secondary product, and to carry out a comparison with resveratrol and other phenolic compounds which are currently in the limelight all over the world due to their beneficial effects on the human body. The potential of Vam3 as an antioxidant was determined through the evaluation of some key thermodynamic parameters which are commonly used for this purpose and describe the antioxidant activity quite well. Various mechanisms through which antioxidants usually can carry out their action were also explored both in water and in apolar environment. The results indicated that Vam3 is an excellent candidate as a natural antioxidant.

Brassinosteroids regulate vacuolar morphology in root meristem cells of Arabidopsis thaliana.

Brassinosteroids (BRs) are plant hormones that regulate plant development and environmental response. Brz-insensitive-long hypocotyl4 (BIL4) was identified as a positive regulator of BR signaling that interacts with the BR receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), and inhibits vacuolar degradation of BRI1 in Arabidopsis thaliana. Although BIL4 also localizes to the vacuolar membrane, the possible vacuolar function of BIL4 remains unknown. Here, we studied the effect of BIL4 and BR signaling on vacuole shape in root meristem cells using genetic and pharmacological approaches. In BIL4-deficient plants, vacuoles assumed a smaller luminal structure. Treatment with brassinolide (BL), the most active BR, resulted in visibly larger vacuoles, whereas treatment with the BR biosynthesis inhibitor Brz resulted in substantially smaller luminal vacuolar structures. In the bri1 mutant, vacuolar shapes exhibited small and fragmented structures. Our results suggest that BR signaling impacts vacuolar shape.

Vam3 ameliorates total body irradiation-induced hematopoietic system injury partly by regulating the expression of Nrf2-targeted genes.

Vam3, a resveratrol dimer, has been implicated in the regulation of chronic obstructive pulmonary disease. However, the effect of Vam3 on total body irradiation (TBI)-induced hematopoietic progenitor cells (HPCs), and hematopoietic stem cells (HSCs) injury is unknown. In this study, we examined whether Vam3could ameliorate hematopoietic system injury induced by TBI. Our results indicated that Vam3 alleviated TBI-induced injury by improving the self-renewal and differentiation of HPCs, and HSCs. Vam3 decreased the intracellular ROS levels in irradiated mice HPCs/HSCs or c-kit positive cells and inhibited apoptosis and DNA damage in LSKs and HPCs after TBI. Vam3 up-regulated the expression of Nrf2 and related genes and proteins in irradiated c-kit positive cells in vitro. However, Vam3 did not increase the cell viability or the number of CFU-GM c-kit positive cells in irradiated Nrf2-/- mice but decreased the cellular ROS level. The above data showed that Vam3 ameliorates total body irradiation-induced hematopoietic system injury and that Nrf2 is essential in mediating Vam3's protective effect on the proliferation of c-kit positive cells after irradiation but not its ability to scavenge for free radicals.

Pharmacokinetic analysis and tissue distribution of Vam3 in the rat by a validated LC-MS/MS method.

Vam3 is a potential pharmacologically active ingredient isolated from Vitis amurensis Rupr. A rapid, simple and sensitive method to determine Vam3 levels in rat plasma and tissue was developed based on LC-MS/MS. Vam3 and an internal standard (IS) were chromatographed on a C18 short column with acetonitrile-0.1% formic acid in water by gradient elution. MS detection was performed by electrospray ionization in negative ion multiple reaction-monitoring modes. This method monitored the transitions m/z 451.0→345.0 and m/z 301.0→164.0 for Vam3 and IS, respectively. The calibration curve was linear over a concentration range of 1.64-1000 ng/mL. The inter-day and intra-day variabilities in precision was less than 12.8%, while the inter-day and intra-day accuracies ranged from -10.60% to 9.08% in plasma and tissue homogenates. This method was applied to investigate the pharmacokinetics and tissue distribution of Vam3 in rats. The results indicated that Vam3 had poor absorption into systemic circulation and extensive tissue distribution after oral administration, and the absolute bioavailability was low (0.79%). Vam3 had a relatively long terminal elimination half-life in lung, and the highest concentration was found in small intestinal tissue. The developed method and the pharmacokinetic data can provide a basis for further studies on the bioactivity of Vam3.

Pharmacokinetic analysis and tissue distribution of Vam3 in the rat by a validated LC-MS/MS method

Vam3 is a potential pharmacologically active ingredient isolated from Vitis amurensis Rupr. A rapid, simple and sensitive method to determine Vam3 levels in rat plasma and tissue was developed based on LC-MS/MS. Vam3 and an internal standard (IS) were chromatographed on a C18 short column with acetonitrile-0.1% formic acid in water by gradient elution. MS detection was performed by electrospray ionization in negative ion multiple reaction-monitoring modes. This method monitored the transitions m/z 451.0→345.0 and m/z 301.0→164.0 for Vam3 and IS, respectively. The calibration curve was linear over a concentration range of 1.64-1000 ng/mL. The inter-day and intra-day variabilities in precision was less than 12.8%, while the inter-day and intra-day accuracies ranged from -10.60% to 9.08% in plasma and tissue homogenates. This method was applied to investigate the pharmacokinetics and tissue distribution of Vam3 in rats. The results indicated that Vam3 had poor absorption into systemic circulation and extensive tissue distribution after oral administration, and the absolute bioavailability was low (0.79%). Vam3 had a relatively long terminal elimination half-life in lung, and the highest concentration was found in small intestinal tissue. The developed method and the pharmacokinetic data can provide a basis for further studies on the bioactivity of Vam3.

A dynamin homolog promotes the transition from hemifusion to content mixing in intracellular membrane fusion.

The convergence of the antagonistic reactions of membrane fusion and fission at the hemifusion/hemifission intermediate has generated a captivating enigma of whether Soluble N-ethylmaleimide sensitive factor Attachment Protein Receptor (SNAREs) and dynamin have unusual counter-functions in fission and fusion, respectively. SNARE-mediated fusion and dynamin-driven fission are fundamental membrane flux reactions known to occur during ubiquitous cellular communication events such as exocytosis, endocytosis and vesicle transport. Here we demonstrate the influence of the dynamin homolog Vps1 (Vacuolar protein sorting 1) on lipid mixing and content mixing properties of yeast vacuoles, and on the incorporation of SNAREs into fusogenic complexes. We propose a novel concept that Vps1, through its oligomerization and SNARE domain binding, promotes the hemifusion-content mixing transition in yeast vacuole fusion by increasing the number of trans-SNAREs.

Influence of dihydroxy-stilbene compound Vam3 on ATP-induced inflammatory response in macrophages and the underlying mechanisms / 中国药理学通报

Aim To investigate the effects of Vam3 on ATP-induced inflammatory response in macrophages and the underlying mechanisms. Methods LPS primed mouse peritoneal macrophages were stimulated with ATP,and IL-1βlevel in supernatants was meas-ured by ELISA.Activity of caspase 1 was measured u-sing caspase 1 activity assay kit.Reactive oxygen spe-cies (ROS )level was detected with fluorescent probe DCFH-DA.MTT assay was used to detect cell prolifer-ation,and intracellular Ca2+concentration was meas-ured using laser scanning confocal microscope.Results Extracellular ATP led to increase in IL-1βrelease, caspase 1 activity and ROS production.It also led to rapid increase in intracellular Ca2+concentration and induced cell death.These effects were inhibited by Vam3 .Conclusion Vam3 inhibits ATP-induced in-flammatory response in macrophages,which may sug-gest the blocking effect of Vam3 on caspase 1 ~IL-1βinflammatory signaling pathway in macrophages.