WSF-P-1, a novel AMPK activator,promotes adiponectin multimerization in 3T3-L1 adipocytes

Adiponectin, an adipokine with insulin-sensitizing effect, is secreted from adipocytes into circulation as high, medium, and low molecular weight forms (HMW, MMW, and LMW). The HMW adiponectin oligomers possess the most potent insulin-sensitizing activity. WSF-P-1(N-methyl-1,2,3,4,5,6-hexahydro-1,1,5,5-tetramethyl-7H-2,4α-methanonaphthalen-7-amine) is derived from natural sesquiterpene longifolene by chemical modifications. We found that WSF-P-1 activates AMPK in both 3T3-L1 adipocytes and 293T cells in this study. Activation of AMPK by WSF-P-1 promotes the assembly of HMW adiponectin and increases the HMW/total ratio of adiponectin in 3T3-L1 adipocytes. We demonstrated that the Ca²⁺-dependent CaMKK signaling pathway is involved in WSF-P-1-induced AMPK activation and adiponectin multimerization. WSF-P-1 also activates GLUT1-mediated glucose uptake in 3T3-L1 adipocytes, making it a potential drug candidate for the treatment of type 2 diabetes, obesity, and other obesity-related metabolic diseases.     

Calcium-independent calmodulin binding and two-metal-ion catalytic mechanism of anthrax edema factor

Edema factor (EF), a key anthrax exotoxin, has an anthrax protective antigen-binding domain (PABD) and a calmodulin (CaM)-activated adenylyl cyclase domain. Here, we report the crystal structures of CaM-bound EF, revealing the architecture of EF PABD. CaM has N- and C-terminal domains and each domain can bind two calcium ions. Calcium binding induces the conformational change of CaM from closed to open. Structures of the EF-CaM complex show how EF locks the N-terminal domain of CaM into a closed conformation regardless of its calcium-loading state. This represents a mechanism of how CaM effector alters the calcium affinity of CaM and uncouples the conformational change of CaM from calcium loading. Furthermore, structures of EF-CaM complexed with nucleotides show that EF uses two-metal-ion catalysis, a prevalent mechanism in DNA and RNA polymerases. A histidine (H351) further facilitates the catalysis of EF by activating a water to deprotonate 3'OH of ATP. Mammalian adenylyl cyclases share no structural similarity with EF and they also use two-metal-ion catalysis, suggesting the catalytic mechanism-driven convergent evolution of two structurally diverse adenylyl cyclases.     

Effects of a biocontrol agent and methyl jasmonate on postharvest diseases of peach fruit and the possible mechanisms involved

AIMS: To investigate effects of application of 200 micromol l(-1) methyl jasmonate [MeJA (200)] and Cryptococcus laurentii alone or in combination against postharvest diseases (Monilinia fructicola and Penicillium expansum) in peach fruit stored at 25 and 0 degrees C, and to evaluate the possible mechanisms involved. METHODS AND RESULTS: The efficacy of controlling postharvest diseases by resistance induced in peach fruit treated with MeJA (200) and C. laurentii alone or in combination and the relationship between activities of defence-related enzymes in peach fruit and lesions caused by M. fructicola and P. expansum were examined. At the same time, the effects of MeJA (200) on the population of C. laurentii in the peach wounds and on the mycelial growth of M. fructicola and P. expansumin vitro were investigated. The results indicated that treatment of peach fruit with C. laurentii at 1 x 10(8) CFU ml(-1) alone, or combining C. laurentii at 5 x 10(7) CFU ml(-1) with MeJA (200) all resulted in a lower lesion diameter of brown rot and blue mould caused by M. fructicola and P. expansum compared with the controls in peach fruit. MeJA (200) enhanced the population of C. laurentii, and inhibited mycelial growth of P. expansum. However, it had a little effect on M. fructicolain vitro. MeJA and C. laurentii alone or in combination induced higher activities of Chitinase, beta-1,3-glucanase, phenylalanine ammonia-lyase and peroxidase (POD) than applying the yeast alone at both 25 and 0 degrees C. CONCLUSIONS: MeJA (200) not only directly inhibited mycelial spread of postharvest pathogens, but also increased population of C. laurentii, which induced stronger disease resistance in fruit than MeJA or yeast alone, and resulted in a lower lesion diameter of brown rot and blue mould caused by M. fructicola and P. expansum. SIGNIFICANCE AND IMPACT OF THE STUDY: MeJA (200) in combination with C. laurentii was beneficial for controlling brown rot and blue mould caused by M. fructicola and P. expansum in peach fruit. The inhibitory mechanism was mainly because of resistance induced in peach fruit by MeJA and C. laurentii. In addition, direct inhibition of MeJA on P. expansum also played a role in controlling blue mould.     

Evidence for dissociation of chlorophyll b from the main light-harvesting complex in the oligomerization state isolated from marine alga, Bryopsis corticulans

We investigated the composition and organization of chlorophylls in monomers, trimers and oligomers (small aggregates) of the main light-harvesting complex (LHC II) isolated from marine alga, Bryopsis corticulans, using a combination of measurements with reversed-phase high performance liquid chromatography (RP-HPLC) and steady-state spectroscopy of absorption, circular dichroism (CD) and low temperature fluorescence. The composition and organization of the chlorophylls in monomeric and trimeric LHC II were essentially identical to those of LHC II from higher plants. For LHC II oligomers, a large decrease of chlorophyll (Chl) b absorption and of CD signals corresponding to Chl b was consistent with the quantitative analysis of Chl b by RP-HPLC, indicating that oligomerization of the LHC II proteins significantly influenced spectroscopic properties and led to the dissociation of Chl b molecules from LHC II. Our data strongly suggested that protein oligomerization constitutes a structural basis for the decrease of Chl b molecules in LHC II of B. corticulans. The LHC II of B. corticulans might play a photoprotective role with the reduction of the ability of light absorption via alteration of its own structural conformation.     

Effects of sequential deletions of residues from the N- or C-terminus on the functions of epsilon subunit of the chloroplast ATP synthase

Ten truncated mutants of chloroplast ATP synthase epsilon subunit from spinach (Spinacia oleracea), which had sequentially lost 1-5 amino acid residues from the N-terminus and 6-10 residues from the C-terminus, were generated by PCR. These mutants were overexpressed in Escherichia coli, reconstituted with soluble and membrane-bound CF(1), and the ATPase activity and proton conductance of thylakoid membrane were examined. Deletions of as few as 3 amino acid residues from the N-terminus or 6 residues from the C-terminus of epsilon subunit significantly affected their ATPase-inhibitory activity in solution. Deletion of 5 residues from the N-terminus abolished its abilities to inhibit ATPase activity and to restore proton impermeability. Considering the consequence of interaction of epsilon and gamma subunit in the enzyme functions, the special interactions between the epsilon variants and the gamma subunit were detected in the yeast two-hybrid system and in vitro binding assay. In addition, the structures of these mutants were modeled through the SWISS-MODEL Protein Modeling Server. These results suggested that in chloroplast ATP synthase, both the N-terminus and C-terminus of the epsilon subunit show importance in regulation of the ATPase activity. Furthermore, the N-terminus of the epsilon subunit is more important for its interaction with gamma and some CF(o) subunits, and crucial for the blocking of proton leakage. Compared with the epsilon subunit from E. coli [Jounouchi, M., Takeyama, M., Noumi, T., Moriyama, Y., Maeda, M., and Futai, M. (1992) Arch. Biochem. Biophys. 292, 87-94; Kuki, M., Noumi, T., Maeda, M., Amemura, A., and Futai, M. (1988) J. Biol. Chem. 263, 4335-4340], the chloroplast epsilon subunit is more sensitive to N-terminal or C-terminal truncations.     

Cyclic strain induces expression of specific smooth muscle cell markers in human endothelial cells

The objective of this study was to determine whether cyclic strain could promote human umbilical vein endothelial cells (HUVECs) to express markers in common with the mature smooth muscle cell (SMC) phenotype, suggesting endothelial cell to SMC transdifferentiation. HUVECs were cultured on stretched membranes at 10% stretch and 60 cycles/min for 24-96 hr, and demonstrated elongation with enhanced and organized F-actin distribution. By using real-time polymerase chain reaction analysis, the mRNA levels of five specific SMC markers, SM22-alpha, alpha-smooth muscle actin (alpha-SMA), caldesmon-1, smooth muscle myosin heavy chain (SMMHC), and calponin-1 were significantly increased in cyclic strain-treated HUVECs as compared with those in static control cells. Protein levels of SM22-alpha and alpha-SMA were also substantially increased by Western blot and immunofluorescence staining. In addition, two specific endothelial markers, von Willebrand factor (vWF) and vascular endothelial growth factor receptor-2 (VEGFR-2), showed a reduction in mRNA expression. In addition, cyclic strain-induced increase of SM22-alpha and alpha-SMA expression were reversible when cells were cultured back to the static condition. These results demonstrate a possible endothelial cell to SMC transdifferentiation in response to cyclic strain. Hemodynamic forces in modulating endothelial phenotype may play an important role in the vascular system.     

Knockdown of dual specificity phosphatase 4 enhances the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin

BackgroundBreast cancer is the major cause of cancer-related deaths in females world-wide. Doxorubicin-based therapy has limited efficacy in breast cancer due to drug resistance, which has been shown to be associated with the epithelial-to-mesenchymal transition (EMT). However, the molecular mechanisms linking the EMT and drug resistance in breast cancer cells remain unclear. Dual specificity phosphatase 4 (DUSP4), a member of the dual specificity phosphatase family, is associated with cellular proliferation and differentiation; however, its role in breast cancer progression is controversial.MethodsWe used cell viability assays, Western blotting and immunofluorescent staining, combined with siRNA interference, to evaluate chemoresistance and the EMT in MCF-7 and adriamycin-resistant MCF-7/ADR breast cancer cells, and investigate the underlying mechanisms.ResultsKnockdown of DUSP4 significantly increased the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin, and MCF-7/ADR cells which expressed high levels of DUSP4 had a mesenchymal phenotype. Furthermore, knockdown of DUSP4 reversed the EMT in MCF-7/ADR cells, as demonstrated by upregulation of epithelial biomarkers and downregulation of mesenchymal biomarkers, and also increased the chemosensitivity of MCF-7/ADR cells to doxorubicin.ConclusionsDUSP4 might represent a potential drug target for inhibiting drug resistance and regulating the process of the EMT during the treatment of breast cancer.