Some dinophycean red tide plankton species generate a superoxide scavenging substance

Recent studies indicate that some raphidophycean red tide flagellates produce substances able to scavenge superoxide, whereas there have been no reports on superoxide scavenger production by dinophycean red tide flagellates. In this study, we examined the superoxide-scavenging activity of aqueous extracts from dinophycean red tide flagellates, Gymnodinium spp., Scrippsiella trochoidea, and Karenia sp., by a luminol analog L-012-dependent chemiluminescence (CL) method and an electron spin resonance (ESR)-spin trapping method, and compared the activity to that of raphidophycean red tide flagellates, Chattonella spp., Heterosigma akashiwo, and Fibrocapsa japonica. In the experiment applying the L-012-dependent CL method, only the aqueous extracts from raphidophycean red tide flagellates showed superoxide-scavenging activity. On the other hand, applying the ESR-spin trapping method, we found that the aqueous extracts from dinophycean red tide flagellates also showed superoxide-scavenging activity. This is the first report on the production of a superoxide-scavenger by dinophycean red tide flagellates.     

Antioxidant properties of aqueous extracts from red tide plankton cultures

The antioxidant properties of aqueous extracts from the dinophycean flagellates Gymnodinium impudicum and Alexandrium affine and the raphidophycean flagellate Chattonella ovata were examined. An electron spin resonance (ESR)-spin trapping method coupled with steady state kinetic analysis showed that all of the extracts directly scavenge superoxide, and that the superoxide scavenging potential of any of the extracts was comparable to that of L-ascorbic acid. As for hydroxyl radical scavenging, the Fenton reaction and the method of ultraviolet radiation to hydrogen peroxide were used as hydroxyl radical generation systems. All of extracts reduced the level of hydroxyl radicals in both of the systems, indicating that the extracts also directly scavenge hydroxyl radicals. Since the levels of phenolic compounds did not correlate with the antioxidant activities of the extracts, substances other than phenolic compounds also appeared to be attributable to the activities. It is of our interest that the scavenging activities of extract from G. impudicum against superoxide and hydroxyl radicals were increased by heat exposure at 100 degrees C and 200 degrees C respectively. Although the reason for the increased activities of the aqueous extract from G. impudicum is not clear, the heat-resistance of the extract from G. impudicum might make it a desirable antioxidant.     

JNK1-Dependent Phosphorylation of GAP-43 Serine 142 is a Novel Molecular Marker for Axonal Growth

Mammalian axon growth has mechanistic similarities with axon regeneration. The growth cone is an important structure that is involved in both processes, and GAP-43 (growth associated protein-43 kDa) is believed to be the classical molecular marker. Previously, we used growth cone phosphoproteomics to demonstrate that S96 and T172 of GAP-43 in rodents are highly phosphorylated sites that are phosphorylated by c-jun N-terminal protein kinase (JNK). We also revealed that phosphorylated (p)S96 and pT172 antibodies recognize growing axons in the developing brain and regenerating axons in adult peripheral nerves. In rodents, S142 is another putative JNK-dependent phosphorylation site that is modified at a lower frequency than S96 and T172. Here, we characterized this site using a pS142-specific antibody. We confirmed that pS142 was detected by co-expressing mouse GAP-43 and JNK1. pS142 antibody labeled growth cones and growing axons in developing mouse neurons. pS142 was sustained until at least nine weeks after birth in mouse brains. The pS142 antibody could detect regenerating axons following sciatic nerve injury in adult mice. Comparison of amino acid sequences indicated that rodent S142 corresponds to human S151, which is predicted to be a substrate of the MAPK family, which includes JNK. Thus, we confirmed that the pS142 antibody recognized human phospho-GAP-43 using activated JNK1, and also that its immunostaining pattern in neurons differentiated from human induced pluripotent cells was similar to those observed in mice. These results indicate that the S142 residue is phosphorylated by JNK1 and that the pS142 antibody is a new candidate molecular marker for axonal growth in both rodents and human.

     

Up-regulation of adipogenin, an adipocyte plasma transmembrane protein, during adipogenesis

Until now, the various proteins highly expressed in adipose tissues have been identified and characterized by traditional gene cloning techniques. However, methods of computer analysis have been developed to compare the levels of expression among various tissues, and genes whose expression levels differ significantly between tissues have been found. Among these genes, we report on the possible function of a new adipose-specific gene, showed higher expression in adipose tissue through ‘Search Expression’ on Genome Institute of Norvartis Research Foundation (GNF) SymAtlas v0.8.0. This database has generated and analyzed gene expression of each gene in diverse samples of normal tissues, organs, and cell lines. This newly discovered gene product was named adipogenin because of its role in stimulating adipocyte differentiation and development. Adipogenin mRNA was highly expressed in four different fat depots, and exclusively expressed in adipocytes isolated from adipose tissues. The level of adipogenin mRNA was up-regulated in the subcutaneous and visceral adipose tissues of mice fed a high-fat diet compared to those on the control diet. The expression of adipogenin mRNA is dramatically elevated during adipocyte differentiation of 3T3-L1 cells. Troglitazone, which up-regulated peroxisome proliferators-activated receptor γ2 (PPAR-γ2) expression, increased adipogenin mRNA expression, although this gene was down-regulated by retinoic acid. Confocal image analyses of green-fluorescent protein-adipogenin (pEGFP-adipogenin) transiently expressed in 3T3-L1 adipocytes showed that adipogenin was strictly localized to membranes and was absent from the cytosol. Moreover, small interfering RNA (siRNA) mediated a reduction of adipogenin mRNA in 3T3-L1 cells and blocked the process of adipocyte differentiation. These results indicate that adipogenin, an adipocyte-specific membrane protein, may be involved with adipogenesis, as one of the regulators of adipose tissue development.Yeon-Hee Hong and Daisuke Hishikawa contributed equally to this work     

The orexinergic synaptic innervation of serotonin- and orexin 1-receptor-containing neurons in the dorsal raphe nucleus

Orexin/hypocretin has been well demonstrated to excite the serotonergic neurons in the dorsal raphe nucleus (DRN). We studied the morphological relationships between orexin-containing axon terminals and serotonin- as well as orexin-receptor-containing neurons in the dorsal raphe nucleus. Using immunohistochemical techniques at the light microscopic level, orexin A (OXA)-like immunoreactive neuronal fibers in the DRN were found to make close contact with serotonergic neurons, while some of the serotonergic neurons also expressed the orexin 1 receptor (OX1R). At the electron microscopic level, double-immunostaining experiments showed that the orexin A-like immunoreactive fibers were present mostly as axon terminals that made synapses on the serotonin- and orexin 1-receptor-containing neurons. While only axodendritic synapses between orexin A-containing axon terminals and serotonergic neurons were detected, the synapses made by orexin A-containing axon terminals on the orexin 1-receptor-containing neurons were both axodendritic and axosomatic. The present study suggests that excitation effect of orexin A on dorsal raphe serotonergic neurons is via synaptic communication through orexin 1 receptor.     

Bioavailability and antioxidant effect of epigallocatechin gallate administered in purified form versus as green tea extract in healthy individuals

Tea polyphenols have strong in vitro antioxidant activity. Due to their limited bioavailability, however, their contribution to in vivo antioxidant activity may depend on the form of administration. A human intervention study was performed to evaluate the bioavailability and antioxidant capacity of (-)-epigallocatechin-3-gallate (EGCG) administered as a single large dose in the form of either purified EGCG or as green tea extract (Polyphenon E). Plasma concentrations of tea polyphenols were determined by high-performance liquid chromatography (HPLC) analysis combined with coulometric array electrochemical detection (ECD). We found no differences in plasma EGCG concentrations and trolox equivalents determined by the trolox equivalent antioxidant capacity assay after administration of either form of EGCG. However, we found that the plasma antioxidant activity was significantly affected by changes in the plasma urate concentration, which may have interfered with the effect of tea polyphenols on the antioxidant activity. In addition, lymphocyte 8-hydroxydeoxyguanosine to deoxyguanosine (8-OHdG/10(6)dG) ratios were determined by HPLC with ECD. The 8-OHdG/10(6)dG ratios did not change significantly during the 24 h following both EGCG interventions but correlated significantly within individuals determined during the two interventions separated by 1 week. In summary, changes in plasma uric acid due to dietary intake were significantly correlated to the plasma antioxidant activity and exerted a stronger influence on the plasma antioxidant activity compared with the EGCG intervention. In future studies of dietary effects on the plasma antioxidant capacity, changes in plasma uric acid will need to be closely monitored.     

EGCG inhibits activation of the insulin-like growth factor-1 receptor in human colon cancer cells

The IGF/IGF-1R system, which includes the IGF, IGF-1R, and IGFBPs proteins, plays an important role in the development and growth of colorectal cancer. We previously reported that in the HT29 human colon cancer cell line EGCG, the major biologically active component of green tea, inhibits activation of the RTKs EGFR, HER2, and HER3, and that this is associated with inhibition of multiple downstream signaling pathways. Since IGF-1R is also a RTK, in this study we examined the effects of EGCG on the activity of IGF/IGF-1R system in human colon cancer cells. We found that the colon cancer cell lines Caco2, HT29, SW837, and SW480 express high levels of the IGF-1R receptor, and that both SW837 and SW480 cells display constitutive activation of this receptor. Treatment of SW837 cells with 20 microg/ml of EGCG (the IC50 concentration for growth inhibition) caused within 6 h a decrease in the phosphorylated (i.e., activated) form of the IGF-1R protein. At 12 h, there was a decrease in the levels of both IGF-1 protein and mRNA and within 3-6 h there was an increase in the levels of both IGFBP-3 protein and mRNA. The increased expression of the latter protein was sustained for at least 48 h. When SW837 cells were treated with EGCG for a longer time, i.e., 96 h, a very low concentration (1.0 microg/ml) of EGCG also caused inhibition of activation of IGF-1R, a decrease in the IGF-1 protein, and an increase in the IGFBP-3 protein. EGCG also caused a decrease in the levels of mRNAs that encode MMPs-7 and -9, proteins that proteolyze IGFBP-3. In addition, treatment with EGCG caused a transient increase in the expression of TGF-beta2, an inducer of IGFBP-3 expression. These findings expand the roles of EGCG as an inhibitor of critical RTKs involved in cell proliferation, providing further evidence that EGCG and related compounds may be useful in the chemoprevention or treatment of colorectal cancer.     

Osteopontin as a positive regulator in the osteoclastogenesis of arthritis

We examined the role of osteopontin (OPN) in the osteoclastogenesis of arthritis using collagen-induced arthritis (CIA). Cells from arthritic joints of wild-type (OPN +/+) mice spontaneously developed bone-resorbing osteoclast-like cells (OCLs). The cultured cells showed an enhanced expression of receptor activator of nuclear factor kappaB ligand (RANKL) and a decreased expression of osteoprotegerin (OPG). The addition of OPG reduced the number of OCLs, indicating that the osteoclastogenesis depends on the RANK/RANKL/OPG system. The cells also produced OPN abundantly and anti-OPN neutralizing antibodies suppressed the development of OCLs. Moreover, the addition of OPN increased the expression of RANKL and augmented differentiation of OCLs from OPN-deficient (OPN -/-) cells. OPN, like the combination of 1alpha,25-dihydroxyvitamin D(3) and dexamethasone, also enhanced the RANKL expression and decreased OPG expression in a stromal cell line, ST2. These results suggest that OPN acts as a positive regulator in the osteoclastogenesis of arthritis through the RANK/RANKL/OPG system.