(-)-Epigallocatechin-3-gallate suppresses growth of AZ521 human gastric cancer cells by targeting the DEAD-box RNA helicase p68

(-)-Epigallocatechin-3-gallate (EGCG), the most abundant and biologically active polyphenol in green tea, induces apoptosis and suppresses proliferation of cancer cells by modulating multiple signal transduction pathways. However, the fundamental mechanisms responsible for these cancer-preventive effects have not been clearly elucidated. Recently, we found that EGCG can covalently bind to cysteine residues in proteins through autoxidation and subsequently modulate protein function. In this study, we demonstrate the direct binding of EGCG to cellular proteins in AZ521 human gastric cancer cells by redox-cycle staining. We comprehensively explored the binding targets of EGCG from EGCG-treated AZ521 cells by proteomics techniques combined with the boronate-affinity pull-down method. The DEAD-box RNA helicase p68, which is overexpressed in a variety of tumor cells and plays an important role in cancer development and progression, was identified as a novel EGCG-binding target. Exposure of AZ521 cells to EGCG lowered the p68 level dose dependently. The present findings show that EGCG inhibits AZ521 cell proliferation by preventing β-catenin oncogenic signaling through proteasomal degradation of p68 and provide a new perspective on the molecular mechanism of EGCG action.     

N-haloacetylimino neonicotinoids: potency and molecular recognition at the insect nicotinic receptor

This structure-activity relationship study for neonicotinoids with an N-haloacetylimino pharmacophore identifies several candidate compounds showing outstanding insecticidal potency and consequently leads to establishing their molecular recognition at an insect nicotinic receptor structural model, wherein the neonicotinoid halogen atoms (fluorine, chlorine, bromine, and iodine) variously interact with the receptor loops C-D interfacial niche via H-bonding and/or hydrophobic interactions.     

Schistosomicidal and antifecundity effects of oral treatment of synthetic endoperoxide N-89

1,2,6,7-Tetraoxaspiro[7.11]nonadecane (N-89) is a chemically synthesized compound with good efficacy against malaria parasites. We observed strong anti-schistosomal activities of N-89 both in vitro and in vivo. In a murine model with experimental infection of Schistosoma mansoni, orally administered N-89 at the dose of 300 mg/kg resulted in a significant reduction in worm burden (63%) when mice were treated at 2-weeks postinfection. Strong larvicidal effects of N-89 were confirmed in vitro; schistosomula of S. mansoni were killed by N-89 at an EC50 of 16 nM. In contrast, no significant reduction in worm burden was observed when N-89 was administered at 5 weeks postinfection in vivo. However, egg production was markedly suppressed by N-89 treatment at that time point. On microscopic observation, the intestine of N-89-treated female worms seemed to be empty compared with the control group, and the mean body length was significantly shorter than that of controls. Nutritional impairment in the parasite due to N-89 treatment was possible, and therefore quantification of hemozoin was compared between parasites with or without N-89 treatment. We found that the hemozoin content was significantly reduced in N-89 treated parasites compared with controls (P < 0.001). The surface of adult worms was observed by scanning and transmission electron microscopy, but there were no apparent changes. Taken together, these observations suggested that N-89 has strong antischistosomal effects, probably through a unique mode of drug efficacy. As N-89 is less toxic to mammalian host animals, it is a possible drug candidate against schistosomiasis.     

Mieap, a p53-inducible protein, controls mitochondrial quality by repairing or eliminating unhealthy mitochondria

Maintenance of healthy mitochondria prevents aging, cancer, and a variety of degenerative diseases that are due to the result of defective mitochondrial quality control (MQC). Recently, we discovered a novel mechanism for MQC, in which Mieap induces intramitochondrial lysosome-like organella that plays a critical role in the elimination of oxidized mitochondrial proteins (designated MALM for Mieap-induced accumulation of lysosome-like organelles within mitochondria). However, a large part of the mechanisms for MQC remains unknown. Here, we report additional mechanisms for Mieap-regulated MQC. Reactive oxygen species (ROS) scavengers completely inhibited MALM. A mitochondrial outer membrane protein NIX interacted with Mieap in a ROS-dependent manner via the BH3 domain of NIX and the coiled-coil domain of Mieap. Deficiency of NIX also completely impaired MALM. When MALM was inhibited, Mieap induced vacuole-like structures (designated as MIV for Mieap-induced vacuole), which engulfed and degraded the unhealthy mitochondria by accumulating lysosomes. The inactivation of p53 severely impaired both MALM and MIV generation, leading to accumulation of unhealthy mitochondria. These results suggest that (1) mitochondrial ROS and NIX are essential factors for MALM, (2) MIV is a novel mechanism for lysosomal degradation of mitochondria, and (3) the p53-Mieap pathway plays a pivotal role in MQC by repairing or eliminating unhealthy mitochondria via MALM or MIV generation, respectively.     

Morphological examination during in vitro cartilage formation by human mesenchymal stem cells

The formation of the skeleton through endochondral ossification is one of the most complex processes in development. One approach to resolving this complexity is to examine simplified systems. In vitro cartilage formation by mesenchymal stem cells (MSCs) is observed when the cells are cultured as a micromass. Several studies have confirmed the molecular events, showing the usefulness of these cells as a differentiation model. We have elucidated the process of cartilage formation in MSCs from the morphological point of view by light and transmission electron microscopy and immunohistochemical examination. The morphology of the MSCs changed from spherical to spindle-shaped, and the cells aggregated and formed junctional complexes during Day 1. At Day 7, three layers were observed. The superficial zone consisted of several layers of elongated cells with junctional complexes. The middle zone was composed of apoptotic bodies, and the deep zone was occupied by chondrocyte-like cells excreting extracellular matrices. At Day 14, the middle zone had disappeared, and the chondrocyte-like cells in the deep zone were detected within cartilage lacuna. They were covered by cartilage matrices containing collagen types I, II, and X and chondroitin sulfate. By Day 21, the outer layer consisting of spindle-shaped cells had disappeared in places. As the pellet grew, the outer layer seemed to be unable to stretch to maintain a constant covering around the pellet. Our findings have thus revealed that MSCs change their morphology depending upon their microenvironment during differentiation. In vitro cartilage formation by MSCs makes it possible to clarify the detailed morphological events that occur during chondrogenesis. S. Ichinose and I. Sekiya contributed equally to this study     

Sister group relationship of turtles to the bird-crocodilian clade revealed by nuclear DNA-coded proteins

The phylogenetic position of turtles is a currently controversial issue. Recent molecular studies rejected a traditional view that turtles are basal living reptiles (Hedges, S. B., and L. L. Poling. 1999. A molecular phylogeny. Science 83:998-1001; Kumazawa, Y., and M. Nishida. 1999. Complete mitochondrial DNA sequences of the green turtle and blue-tailed mole skink, statistical evidence for archosaurian affinity of turtles. Mol. Biol. Evol. 16:784-792). Instead, these studies grouped turtles with birds and crocodiles. The relationship among turtles, birds, and crocodiles remained unclear to date. To resolve this issue, we have cloned and sequenced two nuclear genes encoding the catalytic subunit of DNA polymerase alpha and glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide formyltransferase from amniotes and an amphibian. The amino acid sequences of these proteins were subjected to a phylogenetic analysis based on the maximum likelihood method. The resulting tree showed that turtles are the sister group to a monophyletic cluster of archosaurs (birds and crocodiles). All other possible tree topologies were significantly rejected.     

Competitive inhibition and selectivity enhancement by Ca in the uptake of inorganic elements (Be, Na, Mg, K, Ca, Sc, Mn, Co, Zn, Se, Rb, Sr, Y, Zr, Ce, Pm, Gd, Hf) by carrot (Daucus carota cv. U.S. harumakigosun)

We investigated the uptake of inorganic elements (Be, Na, Mg, K, Ca, Sc, Mn, Co, Zn, Se, Rb, Sr, Y, Zr, Ce, Pm, Gd, and Hf) and the effect of Ca on their uptake in carrots (Daucus carota cv. U.S. harumakigosun) by the radioactive multitracer technique. The experimental results suggested that Na, Mg, K, and Rb competed for the functional groups outside the cells in roots with Ca but not for the transporter-binding sites on the plasma membrrane of the root cortex cells. In contrast, Y, Ce, Pm, and Gd competed with Ca for the transporters on the plasma membrane. The selectivity, which was defined as the value obtained by dividing the concentration ratio of an elemental pair, K/Na, Rb/Na, Be/Sr, and Mg/Sr, in the presence of 0.2 and 2 ppm Ca by that of the corresponding elemental pair in the absence of Ca in the solution was estimated. The selectivity of K and Rb in roots was increased in the presence of Ca. The selectivity of Be in roots was not affected, whereas the selectivity of Mg was increased by Ca. These observations suggest that the presence of Ca in the uptake solution enhances the selectivity in the uptake of metabolically important elements against unwanted elements.     

A novel mutagen, 2-(5-hydroxy-4,6-dinitroindolyl) ethanol, formed in the reaction between 5-hydroxytryptamine and nitrite under acid conditions, especially in the presence of L-cysteine

We examined the mutagenic activity of each of 29 amino acids mixed under acidic conditions with 5-hydroxytryptamine (5-HT) and nitrite using Salmonella typhimurium strain TA 100 with or without a metabolic activation system (S9 mix). The reaction mixture containing L-cysteine was strongly mutagenic without S9 mix. We subjected an ethyl acetate extract of the reaction mixture to HPLC, isolated a mutagenic component, and investigated its chemical structure by LC-mass spectrometry (MS), high-resolution fast atom bombardment (HRFAB)-MS, and 1H and 13C NMR. We identified the mutagen as 2-(5-hydroxy-4,6-dinitro-3-indolyl) ethanol (2HDIE). We injected 8 mg/kg 2HDIE i.p. into male ICR mice and found that the compound increased the frequency of micronuclei in peripheral reticulocytes. Our results suggest that 2HDIE might be formed in vivo by consumption of 5-HT, nitrite and L-cysteine in foods, and might act as a mutagen.     

Frequent retention of heterozygosity for point mutations in p53 and Ikaros in N-ethyl-N-nitrosourea-induced mouse thymic lymphomas

In agreement with Knudson's two-hit theory, recent findings indicate that the inactivation of tumor suppressor genes is not only mediated by the loss of function but also by the dominant-negative or gain-of-function activity. The former generally accompanies loss of a wild-type allele whereas in the latter a wild-type allele is retained. N-Ethyl-N-nitrosourea (ENU), which efficiently induces point mutations, reportedly leads to the development of tumors by activating ras oncogenes. Little is known about how ENU affects tumor suppressor genes and, therefore, we examined ENU-induced mutations of p53 and Ikaros in thymic lymphomas and compared these with mutations of Kras. In addition, loss of heterozygosity was examined for chromosome 11 to which both p53 and Ikaros were mapped. The frequency of point mutations in p53 and Ikaros was 30% (8/27) and 19% (5/27), respectively, comparable to that observed in Kras (33%: 9/27). In total, 14 of the 27 thymic lymphomas examined (52%) harbored mutations in at least one of these genes. One Ikaros mutation was located at the splice donor site, generating a novel splice isoform lacking zinc finger 3, Ik (F3del). Interestingly, 90% (10/11) of the tumors with point mutations retained wild-type alleles of p53 and Ikaros. Sequence analysis revealed that the most common nucleic acid substitutions were T>A (4/8) in p53, T>C (4/5) in Ikaros and G>A/T (8/9) in Kras, suggesting that the spectrum of mutations was gene dependent. These results suggest that point mutations in tumor suppressor genes without loss of the wild-type allele play an important role in ENU-induced lymphomagenesis.     

Transformation of Escherichia coli mediated by natural phospholipids

Transformation system for Escherichia coli based upon introduction of plasmid DNA by natural phospholipids has been developed. Transformants are easily obtained by treatment with natural phospholipids such as phosphatidylethanolamine, phosphatidylcoline, and phosphatidylserine, where the presence of MgCl2 or CaCl2 is essential. This method of transformation is applicable not only for small plasmid pHSG399 (2.3 Kb) but also for giant plasmid R6K (100 Kb).