Black tea theaflavins suppress dioxin-induced transformation of the aryl hydrocarbon receptor

Dioxins cause various adverse effects through transformation of aryl hydrocarbon receptor (AhR). In this study, we investigated whether black tea extract and its components, theaflavins, suppress AhR transformation in vitro. First, we confirmed that black tea extract strongly suppressed AhR transformation compared to green and oolong tea, although the catechin contents did not change significantly among the extracts. Then we isolated four theaflavins as active compounds from black tea leaves. They suppressed 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AhR transformation in a dose-dependent manner. The IC(50) values of theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'-digallate (Tfdg) were 4.5, 2.3, 2.2, and 0.7 muM, respectively. The suppressive effect of Tfdg was observed not only by pre-treatment but also by post-treatment. This suggests that theaflavins inhibit the binding of TCDD to the AhR and also the binding of the transformed AhR to the specific DNA-binding site as putative mechanisms.     

Effects of serum deprivation on expression of proteolytic-related genes in chick myotube cultures

We previously reported that serum deprivation stimulates myofibrillar proteolysis in chick myotubes. In the present study, we examined the effect of serum deprivation on expression of the proteolytic-related genes (ubiquitin, proteasome, calpains, and cathepsin B) by real-time PCR of cDNA in chick myotubes. Myotubes were incubated with serum-free medium for 24 h. Ubiquitin and proteasome subunits (C1 and C2) and calpains (m-, mu-, and p94/calpain-3) but not cathepsin B mRNA expression were increased by serum deprivation. These results indicate that serum deprivation stimulates ubiquitin-proteasome and calpain proteolytic pathways, resulting in an increase in myofibrillar proteolysis in chick myotubes.     

Muscle oxidative metabolism accelerates with mild acidosis during incremental intermittent isometric plantar flexion exercise

BACKGROUND: It has been thought that intramuscular ADP and phosphocreatine (PCr) concentrations are important regulators of mitochondorial respiration. There is a threshold work rate or metabolic rate for cellular acidosis, and the decrease in muscle PCr is accelerated with drop in pH during incremental exercise. We tested the hypothesis that increase in muscle oxygen consumption (o2mus) is accelerated with rapid decrease in PCr (concomitant increase in ADP) in muscles with drop in pH occurs during incremental plantar flexion exercise. METHODS: Five male subjects performed a repetitive intermittent isometric plantar flexion exercise (6-s contraction/4-s relaxation). Exercise intensity was raised every 1 min by 10% maximal voluntary contraction (MVC), starting at 10% MVC until exhaustion. The measurement site was at the medial head of the gastrocnemius muscle. Changes in muscle PCr, inorganic phosphate (Pi), ADP, and pH were measured by 31P-magnetic resonance spectroscopy. o2mus was determined from the rate of decrease in oxygenated hemoglobin and/or myoglobin using near-infrared continuous wave spectroscopy under transient arterial occlusion. Electromyogram (EMG) was also recorded. Pulmonary oxygen uptake (o2pul ) was measured by the breath-by-breath gas analysis. RESULTS: EMG amplitude increased as exercise intensity progressed. In contrast, muscle PCr, ADP, o2mus, and o2pul did not change appreciably below 40% MVC, whereas above 40% MVC muscle PCr decreased, and ADP, o2mus, and o2pul increased as exercise intensity progressed, and above 70% MVC, changes in muscle PCr, ADP, o2mus, and o2pul accelerated with the decrease in muscle pH (~6.78). The kinetics of muscle PCr, ADP, o2mus, and o2pul were similar, and there was a close correlation between each pair of parameters (r = 0.969~0.983, p < 0.001). CONCLUSION: With decrease in pH muscle oxidative metabolism accelerated and changes in intramuscular PCr and ADP accelerated during incremental intermittent isometric plantar flexion exercise. These results suggest that rapid changes in muscle PCr and/or ADP with mild acidosis stimulate accelerative muscle oxidative metabolism.     

Fucoxanthin induces cell cycle arrest at G0/G1 phase in human colon carcinoma cells through up-regulation of p21WAF1/Cip1

Fucoxanthin, a natural carotenoid, has been reported to have antitumorigenic activity in mouse colon, skin and duodenum models. The present study was designed to evaluate the molecular mechanisms of fucoxanthin against colon cancer using the human colon adenocarcinoma cell lines. Fucoxanthin reduced the viability of WiDr cells in a dose-dependent manner accompanied by the induction of cell cycle arrest during the G0/G1 phase at 25 microM and apoptosis at 50 microM. Fucoxanthin at 25 microM inhibited the phosphorylation of the retinoblastoma protein (pRb) at Ser780 and Ser807/811 24 h after treatment without changes in the protein levels of the D-types of cyclin and cyclin-dependent kinase (cdk) 4, whose complexes are responsible for the phosphorylation of pRb at these sites. A cdk inhibitory protein, p21WAF1/Cip1 increased 24 h after the treatment with 25 microM of fucoxanthin, but not p27Kip1. In addition, the mRNA of p21WAF1/Cip1 also increased in a dose-dependent manner. According to the experiments using the isogenic human colon adenocarcinoma cell lines, fucoxanthin failed to induce G0/G1 arrest in the p21-deficient HCT116 cells, but not in HCT116 wild-type cells. All of these findings showed that fucoxanthin inhibited proliferation of colon cancer cells. The inhibitory mechanism is due to the cell cycle arrest during the G0/G1 phase mediated through the up-regulation of p21WAF1/Cip1, which may be related to the antitumorigenic activity.     

Odorant binding initially occurring at the central pocket in bovine odorant-binding protein

Why bovine odorant-binding protein (OBPb), among OBP family, assumes a dimeric structure has been unclear. Here we clarified, by measuring the fluorescence of intrinsic tryptophan and tyrosine residues of intact OBPb and OBPb whose C-terminal 10 amino acids were deleted, that odorant enters the central pocket formed by the dimerization when OBPb first encounters odorant, and odorant with high affinity with OBPb subsequently enters the internal cavity (suggested binding site), releasing the pre-bound odorant. The internal cavity-bound odorant can be released by the binding of other odorants at another internal cavity or at the central pocket, depending on the binding odorants. Due to this mechanism enabled by the dimerization, OBPb is more reactive than other monomeric OBPs.     

Possible involvement of group I mGluRs in neuroprotective effect of theanine

We investigated the molecular mechanism underlying the neuroprotective effect of theanine, a green tea component, using primary cultured rat cortical neurons, focusing on group I metabotropic glutamate receptors (mGluRs). Theanine and a group I mGluR agonist, DHPG, inhibited the delayed death of neurons caused by brief exposure to glutamate, and this effect of theanine was abolished by group I mGluR antagonists. Although the administration of glutamate alone decreased the neuronal expression of phospholipase C (PLC)-beta1 and -gamma1, which are linked to group I mGluRs, their expression was equal to the control levels on cotreatment with theanine. Treatment with theanine or DHPG alone for 5-7 days resulted in increased expression of PLC-beta1 and -gamma1, and the action of theanine was completely abolished by group I mGluR antagonists. These findings indicate that group I mGluRs might be involved in neuroprotective effect of theanine by increasing the expression levels of PLC-beta1 and -gamma1.     

Human chromosome 21q22.2-qter carries a gene(s) responsible for downregulation of mlc2a and PEBP in Down syndrome model mice

Congenital heart disease (CHD) is a major clinical manifestation of Down syndrome (DS). We recently showed that chimeric mice containing a human chromosome 21 (Chr 21) exhibited phenotypic traits of DS, including CHD. Our previous study showed that myosin light chain-2a (mlc2a) expression was reduced in the hearts of chimeric mice and DS patients. We found that phosphatidylethanolamine binding protein (PEBP) was also downregulated in Chr 21 chimeras in this study. As mlc2a is involved in heart morphogenesis, and PEBP controls the proliferation and differentiation of different cell types, these genes are candidates for involvement in DS-CHD. The DS-CHD candidate region has been suggested to span between PFKL and D21S3, which is the STS marker near the ETS2 loci. To identify gene(s) or a gene cluster on Chr 21 responsible for the downregulation of mlc2a and PEBP, we fragmented Chr 21 at the EST2 loci, by telomere-directed chromosome truncation in homologous recombination-proficient chicken DT40 cells. The modified Chr 21 was transferred to mouse ES cells by microcell-mediated chromosome transfer (MMCT), via CHO cells. We used ES cell lines retaining the Chr 21 truncated at the ETS2 locus (Chr 21E) to produce chimeric mice and compared overall protein expression patterns in hearts of the chimeras containing the intact and the fragmented Chr 21 by two-dimensional electrophoresis. While mouse mlc2a and PEBP expression was downregulated in the chimeras containing the intact Chr 21, the expression was not affected in the Chr 21E chimeras. Therefore, we suggest that Chr 21 gene(s) distal from the ETS2 locus reduce mouse mlc2a and PEBP expression in DS model mice and DS. Thus, this chromosome engineering technology is a useful tool for identification or mapping of genes that contribute to the DS phenotypes.