Determination of triacylglycerol composition in vegetable oils using high-performance liquid chromatography: a collaborative study.

Triacylglycerol (TAG) composition in vegetable oils was collaboratively analyzed in 9 laboratories using high-performance liquid chromatography (HPLC) equipped with a refractive index detector. Dococyl (C22) and triacontyl (C30) silica columns were used for analysis. The TAG molecular species in soybean, rapeseed, and palm oils were individually separated on the chromatogram. The collaborative study demonstrated that TAG composition in vegetable oils could be analyzed based on partition numbers (PN) between 38 and 50. In conclusion, the HPLC method using C22 and C30 silica columns would be useful for determining the TAG composition (%) in vegetable oils.

Trm2p-dependent derepression is essential for methanol-specific gene activation in the methylotrophic yeast Candida boidinii.

We identified a gene, designated TRM2, responsible for methanol-inducible gene expression in the methylotrophic yeast Candida boidinii. The encoded protein Trm2p contains two C(2)H(2)-type zinc finger motifs near the N terminus and shows high similarity to Saccharomyces cerevisiae Adr1p and Pichia pastoris Mxr1p. A C. boidinii gene-disrupted strain (trm2Delta) could not grow on methanol or oleate, but could grow on glucose or ethanol. Trm2p was necessary for the activation of five methanol-inducible promoters tested. Trm2p was localized to the nucleus during growth on nonfermentable carbon sources, but to the cytosol during growth on glucose. A chromatin immunoprecipitation assay revealed that Trm2p specifically bound to the promoters of the alcohol oxidase gene (AOD1) and the dihydroxyacetone synthase gene in cells grown on methanol or oleate, but did not bind to these promoters in cells grown on glucose. The derepressed level of expression of AOD1, which was observed in the trm1Delta strain (the TRM1 gene encodes a transcription factor responsible for methanol-specific gene activation), was decreased in the trm1Deltatrm2Delta strain to a level similar to that observed in the trm2Delta strain. These results suggest that Trm2p-dependent derepression is essential for the Trm1p-dependent methanol-specific gene activation in C. boidinii.

Interaction of endothelial cell-selective adhesion molecule and MAGI-1 promotes mature cell-cell adhesion via activation of RhoA.

Endothelial cell-selective adhesion molecule (ESAM) is a member of the immunoglobulin superfamily and mediates homophilic adhesion between endothelial cells. ESAM has been shown to bind to membrane-associated guanylate kinase (MAGUK) with inverted domain structure 1 (MAGI-1), but the interaction between these molecules remains unknown. We investigated the role of ESAM in the subcellular localization of MAGI-1 and cell adhesion by means of transfection experiments using Chinese hamster ovary (CHO) cells. Overexpression of ESAM recruited MAGI-1 to the cell-cell contact area. The intracellular domain of ESAM was necessary for the recruitment of MAGI-1 to the cell contact area, but did not participate in the initial cell-cell adhesion. Cell dissociation assays revealed that colocalization of ESAM and MAGI-1 promoted actin polymerization through the postsynaptic density 95/discs large/zonula occludens-1 (PDZ) domain and resulted in firm cell-cell adhesion, which was inhibited by an actin polymerization inhibitor. When the cells attach to each other, colocalization of ESAM and MAGI-1 can lead to the actin polymerization at intracellular contacts. Interaction of ESAM with MAGI-1 activated RhoA, and ESAM-mediated MAGI-1 recruitment to the cell membrane and mature cell adhesion were inhibited by a RhoA inhibitor. These findings suggest that ESAM may regulate MAGI-1 recruitment to the cell contacts, and subsequently promote actin polymerization and mature cell-cell adhesion through a RhoA-dependent mechanism.

Anti-tumor effect of pegylated interferon in the rat hepatocarcinogenesis model.

Interferon (IFN) is a multifunctional cytokine which works as a suppressor of hepatocarcinogenesis. Pegylated interferon (PEG-IFN) is a modified form of IFN with different pharmacokinetics. We evaluated the anti-tumor effect of PEG-IFN using a rat hepatocarcinogenesis model. Male Fisher Rats were treated using the Solt and Faber model to induce liver cancer. IFN and PEG-IFN were administered from chemical initiation, and pre-neoplastic foci and neoplastic hepatocellular carcinoma (HCC) were examined at 4 and 40 weeks after chemical initiation, respectively. Apoptosis-related molecules such as p53 and Fas-L, proliferating cell nuclear antigen (PCNA), and oxidative stress-related molecules such as 8-hydroxydeoxyguanosine (8-OHdG) and thioredoxin (TRX) were assessed by immunohistochemical analysis and reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of Notch-1, a molecule related to the regenerative and oncogenic processes was also examined. The generation of foci and HCC were significantly suppressed in IFN and PEG-IFN groups compared with the control group. Whereas PCNA and Notch-1 were strongly expressed in the foci and HCC, Fas-L was mainly detected in the surrounding hepatocytes. 8-OHdG and TRX were also detected in the foci. Although PCNA and Notch-1 were down-regulated in IFN- and PEG-IFN-treated groups, Fas-L was up-regulated in those groups. The activation of Notch-1 signaling and the accumulation of oxidative stress in the pre-neoplastic foci might be associated with the progression of HCC in the DEN-induced hepatocarcinogenesis model. The inhibitory effect of the PEG-IFN and IFN on hepatocarcinogenesis was almost the same, and this might be induced by the Fas-related apoptosis in the surrounding tissues.

Phosphorylation and up-regulation of diacylglycerol kinase gamma via its interaction with protein kinase C gamma.

Diacylglycerol (DAG) acts as an allosteric activator of protein kinase C (PKC) and is converted to phosphatidic acid by DAG kinase (DGK). Therefore, DGK is thought to be a negative regulator of PKC activation. Here we show molecular mechanisms of functional coupling of the two kinases. gammaPKC directly associated with DGKgamma through its accessory domain (AD), depending on Ca2+ as well as phosphatidylserine/diolein in vitro. Mass spectrometric analysis and mutation studies revealed that gammaPKC phosphorylated Ser-776 and Ser-779 in the AD of DGKgamma. The phosphorylation by gammaPKC resulted in activation of DGKgamma because a DGKgamma mutant in which Ser-776 and Ser-779 were substituted with glutamic acid to mimic phosphorylation exhibited significantly higher activity compared with wild type DGKgamma and an unphosphorylatable DGKgamma mutant. Importantly, the interaction of the two kinases and the phosphorylation of DGKgamma by gammaPKC could be confirmed in vivo, and overexpression of the AD of DGKgamma inhibited re-translocation of gammaPKC. These results demonstrate that localization and activation of the functionally correlated kinases, gammaPKC and DGKgamma, are spatio-temporally orchestrated by their direct association and phosphorylation, contributing to subtype-specific regulation of DGKgamma and DAG signaling.

Activation and translocation of PKCdelta is necessary for VEGF-induced ERK activation through KDR in HEK293T cells.

VEGF-KDR/Flk-1 signal utilizes the phospholipase C-gamma-protein kinase C (PKC)-Raf-MEK-ERK pathway as the major signaling pathway to induce gene expression and cPLA2 phosphorylation. However, the spatio-temporal activation of a specific PKC isoform induced by VEGF-KDR signal has not been clarified. We used HEK293T (human embryonic kidney) cells expressing transiently KDR to examine the activation mechanism of PKC. PKC specific inhibitors and human PKCdelta knock-down using siRNA method showed that PKCdelta played an important role in VEGF-KDR-induced ERK activation. Myristoylated alanine-rich C-kinase substrate (MARCKS) translocates from the plasma membrane to the cytoplasm depending upon phosphorylation by PKC. Translocation of MARCKS-GFP induced by VEGF-KDR stimulus was blocked by rottlerin, a PKCdelta specific inhibitor, or human PKCdelta siRNA. VEGF-KDR stimulation did not induce ERK phosphorylation in human PKCdelta-knockdown HEK293T cells, but co-expression of rat PKCdelta-GFP recovered the ERK phosphorylation. Y311/332F mutant of rat PKCdelta-GFP which cannot be activated by tyrosine-phosphorylation but activated by DAG recovered the ERK phosphorylation, while C1B-deletion mutant of rat PKCdelta-GFP, which can be activated by tyrosine-phosphorylation but not by DAG, failed to recover the ERK phosphorylation in human PKCdelta-knockdown HEK293T cell. These results indicate that PKCdelta is involved in VEGF-KDR-induced ERK activation via C1B domain.

Mammalian Sprouty4 suppresses Ras-independent ERK activation by binding to Raf1.

The signalling cascade including Raf, mitogen-activated protein kinase (MAPK) kinase and extracellular-signal-regulated kinase (ERK) is important in many facets of cellular regulation. Raf is activated through both Ras-dependent and Ras-independent mechanisms, but the regulatory mechanisms of Raf activation remain unclear. Two families of membrane-bound molecules, Sprouty and Sprouty-related EVH1-domain-containing protein (Spred) have been identified and characterized as negative regulators of growth-factor-induced ERK activation. But the molecular functions of mammalian Sproutys have not been clarified. Here we show that mammalian Sprouty4 suppresses vascular epithelial growth factor (VEGF)-induced, Ras-independent activation of Raf1 but does not affect epidermal growth factor (EGF)-induced, Ras-dependent activation of Raf1. Sprouty4 binds to Raf1 through its carboxy-terminal cysteine-rich domain, and this binding is necessary for the inhibitory activity of Sprouty4. In addition, Sprouty4 mutants of the amino-terminal region containing the conserved tyrosine residue, which is necessary for suppressing fibroblast growth factor signalling, still inhibit the VEGF-induced ERK pathway. Our results show that receptor tyrosine kinases use distinct pathways for Raf and ERK activation and that Sprouty4 differentially regulates these pathways.

Importance of C1B domain for lipid messenger-induced targeting of protein kinase C.

The molecular mechanisms by which arachidonic acid (AA) and ceramide elicit translocation of protein kinase C (PKC) were investigated. Ceramide translocated epsilonPKC from the cytoplasm to the Golgi complex, but with a mechanism distinct from that utilized by AA. Using fluorescence recovery after photobleaching, we showed that, upon treatment with AA, epsilonPKC was tightly associated with the Golgi complex; ceramide elicited an accumulation of epsilonPKC which was exchangeable with the cytoplasm. Stimulation with ceramide after AA converted the AA-induced Golgi complex staining to one elicited by ceramide alone; AA had no effect on the ceramide-stimulated localization. Using point mutants and deletions of epsilonPKC, we determined that the epsilonC1B domain was responsible for the ceramide- and AA-induced translocation. Switch chimeras, containing the C1B from epsilonPKC in the context of deltaPKC (delta(epsilonC1B)) and vice versa (epsilon(deltaC1B)), were generated and tested for their translocation in response to ceramide and AA. delta(epsilonC1B) translocated upon treatment with both ceramide and AA; epsilon(deltaC1B) responded only to ceramide. Thus, through the C1B domain, AA and ceramide induce different patterns of epsilonPKC translocation and the C1B domain defines the subtype specific sensitivity of PKCs to lipid second messengers.