A highly bioactive lignophenol derivative from bamboo lignin exhibits a potent activity to suppress apoptosis induced by oxidative stress in human neuroblastoma SH-SY5Y cells

Approaches to protection against neurodegenerative diseases, in which oxidative stress and inflammation are implicated, should be based on the current concept on the etiology of these diseases. Recently, a new therapeutic strategy has been proposed to protect neurons from cell death by attenuating the apoptotic signal transduction. Lignin, a durable aromatic network polymer second to cellulose in abundance, was able to be converted into highly active lignophenol derivatives with antioxidant activity by using our newly developed phase-separation technique. These lignophenol derivatives were found to show the potent neuroprotective activity against oxidative stress. Among the compounds examined, a lignocresol derivative from bamboo (lig-8) exhibited the most potent neuroprotective activity against hydrogen peroxide (H(2)O(2))-induced apoptosis in human neuroblastoma cell line SH-SY5Y by preventing the caspase-3 activation via either caspase-8 or caspase-9. Furthermore, it was found that lig-8 exerted the antiapoptotic effect by inhibiting dissipation of the mitochondrial membrane permeability transition induced by H(2)O(2) or by the peripheral benzodiazepin receptor ligand PK11195. Lig-8 was also shown to be potent in the antioxidant activity in the cells exposed to H(2)O(2), as assessed by flow cytometry using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate and in vitro reactive oxygen species-scavenging potency. These data suggest that lig-8 is a promising neuroprotector, which affects the signaling pathway of neuronal cell death and that it would be of benefit to delay the progress of neurodegenerative diseases.     

Soluble branched beta-(1,4)glucans from Acetobacter species show strong activities to induce interleukin-12 in vitro and inhibit T-helper 2 cellular response with immunoglobulin E production in vivo

An extracellular polysaccharide, AC-1, produced by Acetobacter polysaccharogenes is composed of beta-(1,4)glucan with branches of glucosyl residues. We found that AC-1 showed a strong activity to induce production of interleukin-12 P40 and tumor necrosis factor-alpha by macrophage cell lines in vitro. Cellulase treatment completely abolished the activity of AC-1 to induce tumor necrosis factor-alpha production by macrophages, whereas treatment of AC-1 with polymyxin B or proteinase did not affect the activity. Results of experiments using toll-like receptor (TLR) 4-deficient mice and TLR4-transfected human cell line indicated that TLR4 is involved in pattern recognition of AC-1. In vivo administration of AC-1 significantly reduced the serum levels of ovalbumin (OVA)-specific IgE and interleukin-4 production by T cells in response to OVA in mice immunized with OVA. AC-1, a soluble branched beta-(1,4)glucan may be useful in prevention and treatment of allergic disorders With IgE production.     

The protozoa dinoflagellate Oxyrrhis marina contains selenoproteins and the relevant translation apparatus

In the phylogenetic tree, selenoproteins and the corresponding translation machinery are found in Archaea, Eubacteria, and animals, but not in fungi and higher plants. As very little is known about Protozoa, we searched for the presence of selenoproteins in the primitive dinoflagellate Oxyrrhis marina, belonging to the Protoctista kingdom. Four selenoproteins could be obtained from O. marina cells cultured in the presence of 75Se. Using O. marina or bovine liver cytosolic extracts, we could serylate and selenylate in vitro total O. marina tRNAs. Moreover, the existence of a tRNA(Sec) could be deduced from in vivo experiments. Lastly, an anti-serum against the specialized mammalian translation elongation factor mSelB reacted with a protein of 48-kDa molecular mass. Altogether, our data showed that O. marina contains selenoproteins and suggests that the corresponding translation machinery is related to that found in animals.     

DNA damage by ethylbenzenehydroperoxide formed from carcinogenic ethylbenzene by sunlight irradiation

Ethylbenzene, widely used in human life, is a non-mutagenic carcinogen. Sunlight-irradiated ethylbenzene caused DNA damage in the presence of Cu2+, but unirradiated ethylbenzene did not. A Cu+ -specific chelator bathocuproine inhibited DNA damage and catalase showed a little inhibitory effect. The scopoletin assay revealed that peroxides and H(2)O(2) were formed in ethylbenzene exposed to sunlight. These results suggest that Cu+ and alkoxyl radical mainly participate in DNA damage, and H(2)O(2) partially does. When catalase was added, DNA damage at thymine and cytosine was inhibited. Ethylbenzenehydroperoxide, identified by GC/MS analysis, induced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and caused DNA damage at consecutive guanines, as observed with cumenehydroperoxide. Equimolar concentrations of H(2)O(2) and acetophenone were produced by the sunlight-irradiation of 1-phenylethanol, a further degraded product of ethylbenzene. These results indicate a novel pathway that oxidative DNA damage induced by the peroxide and H(2)O(2) derived from sunlight-irradiated ethylbenzene may lead to expression of the carcinogenicity.     

cDNA microarray analysis of altered gene expression in Ara-C-treated leukemia cells

The acute lymphoblastic leukemia cell line CCRF-CEM is sensitive to Ara-C and undergoes apoptosis. In contrast, the chronic myelogenous leukemia (CML) cell line K562 is highly resistant to Ara-C, which causes the cells to differentiate into erythrocytes before undergoing apoptosis. We used cDNA microarrays to monitor the alterations in gene expression in these two cell lines under conditions leading to apoptosis or differentiation. Ara-C-treated CCRF-CEM cells were characterized by a cluster of down-regulated chaperone genes, whereas Ara-C-treated K562 cells were characterized by a cluster of up-regulated hemoglobin genes. In K562 cells, Ara-C treatment induced significant down-regulation of the asparagine synthetase gene, which is involved in resistance to L-asparaginase. Sequential treatment with Ara-C and L-asparaginase had a synergistic effect on the inhibition of K562 cell growth, and combination therapy with these two anticancer agents may prove effective in the treatment of CML, which cannot be cured by either drug alone.     

Vanadate protects human neuroblastoma SH-SY5Y cells against peroxynitrite-induced cell death

We investigated the effect of vanadate, a tyrosine phosphatase inhibitor, on cell death induced by peroxynitrite in human neuroblastoma SH-SY5Y cells. Vanadate prevented cell death induced by 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor; whereas SIN-1-induced cell death was not prevented by neither okadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, nor cyclosporin A, an inhibitor of serine/threonine phosphatase 2B. Vanadate did not prevent cell death induced by N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine, a nitric oxide donor. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), did not block the protective effect of vanadate, suggesting that the protective effect of vanadate is independent on PI3-kinase. Vanadate increased tyrosine phosphorylation of several proteins including the focal adhesion protein p130 Crk-associated substrate (p130(cas)). By the treatment with SIN-1, the endogenous association of p130(cas) and Crk was disrupted, and the association was restored by vanadate treatment. These results suggest that disruption of tyrosine phosphorylation signaling may be critical for peroxynitrite-induced cell death, and that vanadate prevents cell death at least in part through the enhancement in tyrosine phosphorylation of the proteins including p130(cas).     

Effects of Rho-kinase inhibitor on vasopressin-induced chronic myocardial damage in rats

The aim of this study was to develop a new model of vasopressin-induced chronic myocardial damage based on sustained ST-segment depression in electrocardiogram (ECG) with progression of myocardial fibrosis in rats. Furthermore, using this model, we examined the prophylactic potential of fasudil, a Rho-kinase inhibitor, against myocardial damage induced by vasopressin. In 10-week old male Donryu rats, intravenous administration of arginine vasopressin (0.5 iu/kg) induced significant ST-segment depression. Two days and one week after the administration of vasopressin, ST-segment depression was -0.19 +/- 0.02 and -0.14 +/- 0.02 mV, respectively. Fasudil (10 and 30 mg/kg, p.o.) significantly attenuated the ST-segment depression induced by vasopressin. One week after the administration of vasopressin, the percent area of myocardial fibrosis in control animals (0.42 +/- 0.11%, p < 0.01) was significantly greater than that in normal animals (0.05 +/- 0.01%). Fasudil (10 and 30 mg/kg) significantly prevented the development of the fibrosis. We present a new model of chronic myocardial damage based on sustained ST-segment depression with progression of myocardial fibrosis in rats, and suggest that this model may be useful to investigate the treatment of chronic angina. Inhibition of Rho-kinase is efficacious in preventing the ECG change and development of fibrosis induced by vasopressin in this model.     

Mouse Apg10 as an Apg12-conjugating enzyme: analysis by the conjugation-mediated yeast two-hybrid method

Autophagosome formation is a central event in macroautophagy. The Apg12-Apg5 conjugate, which is essential in this process, is generated by a ubiquitin-like protein conjugation system. In yeast, Apg12, following activation by the E1-like Apg7, forms a thioester with Apg10 (E2-like). Apg12 is finally conjugated to Apg5 via an isopeptide bond. The possible requirement of an E3-like protein for the conjugation, however, has not yet been confirmed. The Apg12 system is conserved among eukaryotes, although a mammalian counterpart of Apg10 has not yet been identified. Here, we report the identification and characterization of the mouse Apg10 ortholog. A yeast two-hybrid screen using the mouse Apg5 (mApg5) as bait identified a novel protein with 19% identity to yeast Apg10. We designated this protein mouse Apg10 (mApg10). We demonstrated by a modified yeast two-hybrid assay that mApg10 mediates the conjugation of mApg12 and mApg5. The in vivo interaction of mApg12 with mApg10 in HeLa cells suggests that mApg10 is an Apg12-conjugating enzyme, likely serving as an Apg5-recognition molecule in the Apg12 system. This novel two-hybrid method, which we have named 'conjugation-mediated yeast two-hybrid', proves to be a simple and useful technique with which to analyze protein-protein conjugation.     

Amphioxus homologs of Go-coupled rhodopsin and peropsin having 11-cis- and all-trans-retinals as their chromophores

Because of low contents in the native organs and failure of the expression in cultured cells, the chromophore configurations of the pigments in Go-coupled opsin and peropsin groups in the opsin family are unknown. Here we have succeeded in expression of the amphioxus homologs of these groups in HEK293s cells and found that they can be regenerated with 11-cis- and all-trans-retinals, respectively. Light isomerized the chromophores of these opsins into the all-trans and 11-cis forms, respectively. The results strongly suggest that the physiological function of peropsin would be a retinal photoisomerase, while 11-cis configuration is necessary for the Go-coupled opsin groups.     

Distinct physiological roles of fructokinase isozymes revealed by gene-specific suppression of Frk1 and Frk2 expression in tomato

There are two divergent fructokinase isozymes, Frk1 and Frk2 in tomato (Lycopersicon esculentum Mill.) plants. To investigate the physiological functions of each isozyme, the expression of each fructokinase mRNA was independently suppressed in transgenic tomato plants, and the respective phenotypes were evaluated. Suppression of Frk1 expression resulted in delayed flowering at the first inflorescence. Suppression of Frk2 did not effect flowering time but resulted in growth inhibition of stems and roots, reduction of flower and fruit number, and reduction of seed number per fruit. Localization of Frk1 and Frk2 mRNA accumulation by in situ hybridization in wild-type tomato fruit tissue indicated that Frk2 is expressed specifically in early tomato seed development. Fruit hexose and starch content were not effected by the suppression of either Frk gene alone. The results collectively indicate that flowering time is specifically promoted by Frk1 and that Frk2 plays specific roles in contributing to stem and root growth and to seed development. Because Frk1 and Frk2 gene expression was suppressed individually in transgenic plants, other significant metabolic roles of fructokinases may not have been observed if Frk1 and Frk2 play, at least partially, redundant metabolic roles.