Induction of F9 cell differentiation by transient exposure to retinoic acid.

The F9 cell is a mouse embryonal teratocarcinoma which can be induced to differentiate into visceral endoderm by treatment with retinoic acid (RA). Treatment with RA in conventional studies was carried out in the constant presence of RA. Here we demonstrate that treatment with RA can be as short as 3 hrs to induce differentiation of F9 cells. Morphology, alpha-fetoprotein gene activity, and temporal patterns of F9 cell differentiation are the same with both short- and long-term treatment with RA.     

Molecular cloning and primary structure of rat testes metalloendopeptidase EC 3.4.24.15

The complete amino acid sequence of rat testes metalloendopeptidase (EC 3.4.24.15) was deduced from the nucleotide sequence of a cDNA clone isolated by screening a rat testes library with a polyclonal antibody raised against a homogeneous preparation of the rat testes enzyme. The correctness of the sequence was verified by N-terminal amino acid sequence analysis of the isolated enzyme and by partial amino acid sequence analysis of three tryptic peptides located near the N-terminus, the middle, and C-terminus of the native protein. The enzyme is composed of 645 amino acids with a molecular weight of 72,985. This value is close to that of the purified rat testes and brain enzyme as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions and by molecular sieving chromatography. The enzyme contains the putative active-site sequence -H-E-F-G-H- that is homologous to the sequence in the active site of thermolysin and several other related bacterial enzymes, as well as to active-site sequences of several mammalian zinc metallopeptidases. No amino acid sequence homology, beyond this active site, was found with thermolysin, a bacterial zinc metalloendopeptidase, nor with several mammalian zinc metallopeptidases. Northern blot hybridization analyses showed the presence of mRNA encoding the enzyme in rat testes, but not in other rat tissues in spite of the finding that enzyme activity is widely distributed in all tissues and that relatively high activities are present in rat brain and pituitary.     

Choice of hydrogen uptake (Hup) status in legume‐rhizobia symbioses

The H₂ is an obligate by-product of N-fixation. Recycling of H₂ through uptake hydrogenase (Hup) inside the root nodules of leguminous plants is often considered an advantage for plants. However, many of the rhizobium-legume symbioses found in nature, especially those used in agriculture are shown to be Hup⁻, with the plants releasing H₂ produced by nitrogenase activity from root nodules into the surrounding rhizosphere. Recent studies have suggested that, H₂ induces plant-growth-promoting rhizobacteria, which may explain the widespread of Hup⁻ symbioses in spite of the low energy efficiency of such associations. Wild legumes grown in Nova Scotia, Canada, were surveyed to determine if any plant-growth characteristics could give an indication of Hup choice in leguminous plants. Out of the plants sampled, two legumes, Securigera varia and Vicia cracca, showed Hup⁺ associations. Securigera varia exhibited robust root structure as compared with the other plants surveyed. Data from the literature and the results from this study suggested that plants with established root systems are more likely to form the energy-efficient Hup⁺ symbiotic relationships with rhizobia. Conversely, Hup⁻ associations could be beneficial to leguminous plants due to H₂-oxidizing plant-growth-promoting rhizobacteria that allow plants to compete successfully, early in the growing season. However, some nodules from V. cracca tested Hup⁺, while others were Hup⁻. This was similar to that observed in Glycine max and Pisum sativum, giving reason to believe that Hup choice might be affected by various internal and environmental factors.     

Autophagy induction by xanthoangelol exhibits anti‐metastatic activities in hepatocellular carcinoma

Xanthoangelol (XAG), a prenylated chalcone isolated from the Japanese herb Angelica keiskei Koidzumi, has been reported to exhibit antineoplastic properties. However, the specific anti‐tumor activity of XAG in human hepatocellular carcinoma (HCC), and the relevant mechanisms are not known. Herein, we evaluated the effect of XAG against HCC in vitro and in vivo. Although XAG treatment did not significantly reduce the viability of the Hep3B and Huh7 cell lines, it suppressed cell migration, invasion, and EMT. This anti‐metastatic effect of XAG was due to induction of autophagy, because treatment with the autophagy inhibitor 3‐methyadenine (3‐MA) or knockdown of the pro‐autophagy Beclin‐1 effectively abrogated the XAG‐induced suppression of metastasis. Mechanistically, XAG induced autophagy via activation of the AMPK/mTOR signaling pathway, and XAG treatment dramatically increased the expression of p‐AMPK while decreasing p‐mTOR expression. In addition, blocking AMPK/mTOR axis with compound C abrogated the autophagy‐mediated inhibition of metastasis. The murine model of HCC metastasis also showed that XAG effectively reduced the number of metastatic pulmonary nodules. Taken together, our results revealed that autophagy via the activation of AMPK/mTOR pathway is essential for the anti‐metastatic effect of XAG against HCC. These findings not only contribute to our understanding of the anti‐tumor activity of XAG but also provide a basis for its clinical application in HCC. Before this study, evidence of XAG on HCC was purely anecdotal; present study provides the first comprehensive assessments of XAG on HCC metastasis and investigates its underlying mechanism. Results suggest that XAG exerts anti‐metastatic properties against HCC through inducing autophagy which is mediated by the activation of AMPK/mTOR signaling pathway. This research extends our knowledge about the antineoplastic properties of XAG and suggests that induction autophagy may represent future treatment strategies for metastatic HCC.     

Rotenone Directly Induces BV2 Cell Activation via the p38 MAPK Pathway

Parkinson’s disease (PD) is the second most common neurodegenerative disease. Although its pathogenesis is still unclear, increasing evidence suggests that mitochondrial dysfunction induced by environmental toxins, such as mitochondrial complex I inhibitors, plays a significant role in the disease process. The microglia in PD brains are highly activated, and inflammation is also an essential element in PD pathogenesis. However, the means by which these toxins activate microglia is still unclear. In the present study, we found that rotenone, a mitochondrial complex I inhibitor, could directly activate microglia via the nuclear factor kappa B (NF-κB) signaling pathway, thereby inducing significantly increased expression of inflammatory cytokines. We further observed that rotenone induced caspase-1 activation and mature IL-1β release, both of which are strictly dependent on p38 mitogen-activated protein kinase (MAPK). The activation of p38 is associated with the presence of reactive oxygen species (ROS) produced by rotenone. Removal of these ROS abrogated the activation of the microglia. Therefore, our data suggest that the environmental toxin rotenone can directly activate microglia through the p38 MAPK pathway.