Triterpene-enriched extracts from Ganoderma lucidum inhibit growth of hepatoma cells via suppressing protein kinase C,activating mitogen-activated protein kinases and G2-phase cell cycle arrest

The medicinal mushroom Ganoderma lucidum (G. lucidum) has been used in the Orient for the prevention and treatment of various diseases including cancer. Except for the immune enhancing properties of its polysaccharide constituent, very little is known about the anticancer activity of another major constituent, triterpenes. In this report, we studied the anticancer mechanism of triterpene-enriched extracts from G. lucidum. The triterpene-enriched fraction, WEES-G6, was prepared from mycelia of G. lucidum by sequential hot water extraction, removal of ethanol-insoluble polysaccharides and then gel-filtration chromatography. We found that WEES-G6 inhibited growth of human hepatoma Huh-7 cells, but not Chang liver cells, a normal human liver cell line. Treatment with WEES-G6 caused a rapid decrease in the activity of cell growth regulative protein, PKC, and the activation of JNK and p38 MAP kinases. The changes in these molecules resulted in a prolonged G2 cell cycle phase and strong growth inhibition. None of these effects were seen in the normal liver cells. Our findings suggest that the triterpenes contained in G. lucidum are potential anticancer agents.     

Immunogene therapy of tumors with vaccine based on xenogeneic epidermal growth factor receptor

The breaking of immune tolerance against self epidermal growth factor receptor (EGFr) should be a useful approach for the treatment of receptor-positive tumors with active immunization. To test this concept, we constructed a plasmid DNA encoding extracellular domain of xenogeneic (human) EGFr (hEe-p) or corresponding control mouse EGFr (mEe-p) and empty vector (c-p). Mice immunized with hEe-p showed both protective and therapeutic antitumor activity against EGFr-positive tumor. Sera isolated from the hEe-p-immunized mice exhibited positive staining for EGFr-positive tumor cells in flow cytometric analysis and recognized a single 170-kDa band in Western blot analysis. Ig subclasses responded to rEGFr proteins were elevated in IgG1, Ig2a, and Ig2b. There was the deposition of IgG on the tumor cells. Adoptive transfer of the purified Igs showed the antitumor activity. The increased killing activity of CTL against EGFr-positive tumor cells could be blocked by anti-CD8 or anti-MHC class I mAb. In vivo depletion of CD4(+) T lymphocytes could completely abrogate the antitumor activity, whereas the depletion of CD8(+) cells showed partial abrogation. The adoptive transfer of CD4-depleted (CD8(+)) or CD8-depleted (CD4(+)) T lymphocytes isolated from mice immunized with hEe-p vaccine showed the antitumor activity. In addition, the increase in level of both IFN-gamma and IL-4 was found. Taken together, these findings may provide a new vaccine strategy for the treatment of EGFr-positive tumors through the induction of the autoimmune response against EGFr in a cross-reaction between the xenogeneic homologous and self EGFr.     

Sulindac sulfide is a noncompetitive gamma-secretase inhibitor that preferentially reduces Abeta 42 generation

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been known to reduce risk for Alzheimer's disease. In addition to the anti-inflammatory effects of NSAIDs to block cylooxygenase, it has been shown recently that a subset of NSAIDs selectively inhibits the secretion of highly amyloidogenic Abeta42 from cultured cells, although the molecular target(s) of NSAIDs in reducing the activity of gamma-secretase for Abeta42 generation (gamma(42)-secretase) still remain unknown. Here we show that sulindac sulfide (SSide) directly acts on gamma-secretase and preferentially inhibits the gamma(42)-secretase activity derived from the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate-solubilized membrane fractions of HeLa cells, in an in vitro gamma-secretase assay using recombinant amyloid beta precursor protein C100 as a substrate. SSide also inhibits activities for the generation of Abeta40 as well as for Notch intracellular domain at higher concentrations. Notably, SSide displayed linear noncompetitive inhibition profiles for gamma(42)-secretase in vitro. Our data suggest that SSide is a direct inhibitor of gamma-secretase that preferentially affects the gamma(42)-secretase activity.     

A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly

The activated form of Ran (Ran-GTP) stimulates spindle assembly in Xenopus laevis egg extracts, presumably by releasing spindle assembly factors, such as TPX2 (target protein for Xenopus kinesin-like protein 2) and NuMA (nuclear-mitotic apparatus protein) from the inhibitory binding of importin-alpha and -beta. We report here that Ran-GTP stimulates the interaction between TPX2 and the Xenopus Aurora A kinase, Eg2. This interaction causes TPX2 to stimulate both the phosphorylation and the kinase activity of Eg2 in a microtubule-dependent manner. We show that TPX2 and microtubules promote phosphorylation of Eg2 by preventing phosphatase I (PPI)-induced dephosphorylation. Activation of Eg2 by TPX2 and microtubules is inhibited by importin-alpha and -beta, although this inhibition is overcome by Ran-GTP both in the egg extracts and in vitro with purified proteins. As the phosphorylation of Eg2 stimulated by the Ran-GTP-TPX2 pathway is essential for spindle assembly, we hypothesize that the Ran-GTP gradient established by the condensed chromosomes is translated into the Aurora A kinase gradient on the microtubules to regulate spindle assembly and dynamics.     

Role of Synaptophysin in Exocytotic Release of Dopamine from Xenopus Oocytes Injected with Rat Brain mRNA

1. The role of synaptophysin in the exocytotic release of dopamine (DA) was examined in Xenopus laevis oocytes injected with rat brain mRNA.2. The mRNA-injected oocytes showed DA uptake which depended on the incubation time and external DA concentrations.3. Stimulation with KCl (10–50 mM) of mRNA-injected oocytes preloaded with DA evoked external Ca²⁺-dependent release of DA. The noninjected and water-injected oocytes did not produce uptake of DA and stimulation-evoked release of DA.4. The high-KCl (50 mM)-stimulated release of DA decreased in the oocytes injected with rat brain mRNA together with antibody to synaptophysin.5. Immunoblot analysis demonstrated that synaptophysin was expressed in the brain mRNA-injected oocytes but not in the noninjected and water-injected oocytes.6. Thus, uptake and release machinery similar to native dopaminergic nerve terminals was expressed in Xenopus oocytes by injecting mRNA-extracted from the rat brain, and synaptophysin may play a role in the exocytotic release of DA.     

Cis-N-(p-Coumaroyl)serotonin from Konnyaku, Amorphophallus konjac K. Koch

Tobiko is produced as a by-product in the manufacture of konnyaku and comes to about 45% of dried konnyaku-imo. For a possible use of this powder, we studied its components. Serotonin, trans-N-(p-coumaroyl) serotonin, and a new compound, cis-N-(p-coumaroyl)serotonin, were characterized from this powder.     

Polygodial, a potent attachment-inhibiting substance for the blue mussel, Mytilus edulis galloprovincialis from Tasmannia lanceolata

A highly potent attachment-inhibitor, polygodial, was isolated from a hexane extract of the leaves of Tasmannia lanceolata. The attachment-inhibiting activity of polygodial against the blue mussel was increased 4-fold when used in combination with sorbic acid, anethole, and indole.     

Purification and identification of a growth-stimulating peptide for Bifidobacterium bifidum from natural rubber serum powder

Natural rubber serum powder, which is a by-product obtained in the production of latex rubber, has a strong growth-stimulating activity for Bifidobacterium bifidum JCM 1254. The retained fraction obtained by ultrafiltration (molecular weight cutoff 1000) showed a growth-stimulating activity in a dose-dependent manner on B12 assay medium with ammonium sulfate. One of the growth stimulators was purified from the retained fraction by acetone precipitation, solid-phase extraction with a hydrophobic pretreatment column, and multistage reversed-phase HPLC. An increase of 53-fold in the specific activity, and a recovery of 1.3% were obtained. The amino acid composition and N-terminal sequence analysis of this growth stimulator provided the structure of Ala-Thr-Pro-Glu-Lys-Glu-Glu-Pro-Thr-Ala. The molecular mass was 1075 by MALDI-TOF MS analysis. These results showed that this growth stimulator was a decapeptide with the sequence shown above. This is the first report that clarified the structure of an active peptide for the growth of Bifidobacterium.     

The SP1 sites of the human apoCIII enhancer are essential for the expression of the apoCIII gene and contribute to the hepatic and intestinal expression of the apoA-I gene in transgenic mice

We have generated transgenic mice carrying wild-type and mutant forms of the apolipoprotein (apo)A-I/apoCIII gene cluster. Mutations were introduced either in one or in three SP1 binding sites of the apoCIII enhancer. In mice carrying the wild-type transgene, major sites of apoA-I mRNA synthesis were liver and intestine and minor sites were kidney and, to a lesser extent, other tissues. The major site of chloramphenicol acetyl transferase (CAT) activity (used as a reporter for the apoCIII gene) was liver and minor sites intestine and kidney. A mutation in one SP1 binding site reduced the expression of the apoA-I gene to ~23 and 19% in the liver and intestine, respectively, as compared to the control wild-type. The hepatic expression of the CAT gene was not affected whereas the intestinal expression was nearly abolished. Mutations in three SP1 binding sites reduced the hepatic and intestinal expression of the apoA-I and CAT genes to 14 and 4%, respectively, as compared to the wild-type control, and abolished CAT expression in all tissues. The findings suggest that the SP1 sites of the apoCIII enhancer are required for the expression of the apoCIII gene and also contribute significantly to the hepatic and intestinal expression of the apoA-I gene in vivo.