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By comprehensively measuring changes in metabolites in the hippocampus of stress-loaded mice, we investigated the reasons for stress vulnerability and the effect of theanine, i.e., an abundant amino acid in tea leaves, on the metabolism. Stress sensitivity was higher in senescence-accelerated mouse prone 10 (SAMP10) mice than in normal ddY mice when these mice were loaded with stress on the basis of territorial consciousness in males. Group housing was used as the low-stress condition reference. Among the statistically altered metabolites, depression-related kynurenine and excitability-related histamine were significantly higher in SAMP10 mice than in ddY mice. In contrast, carnosine, which has antidepressant-like activity, and ornithine, which has antistress effects, were significantly lower in SAMP10 mice than in ddY mice. The ingestion of theanine, an excellent antistress amino acid, modulated the levels of kynurenine, histamine, and carnosine only in the stress-loaded SAMP10 mice and not in the group-housing mice. Depression-like behavior was suppressed in mice that had ingested theanine only under stress loading. Taken together, changes in these metabolites, such as kynurenine, histamine, carnosine, and ornithine, were suggested to be associated with the stress vulnerability and depression-like behavior of stressed SAMP10 mice. It was also shown that theanine action appears in the metabolism of mice only under stress loading.

Chronic stress can impair the health of human brains. An important strategy that may prevent the accumulation of stress may be the consumption of functional foods. When senescence-accelerated mice prone 10 (SAMP10), a stress-sensitive strain, were loaded with stress using imposed male mouse territoriality, brain volume decreased. However, in mice that ingested theanine (6 mg/kg), the main amino acid in tea leaves, brain atrophy was suppressed, even under stress. On the other hand, brain atrophy was not clearly observed in a mouse strain that aged normally (Slc:ddY). The expression level of the transcription factor Npas4 (neuronal PAS domain protein 4), which regulates the formation and maintenance of inhibitory synapses in response to excitatory synaptic activity, decreased in the hippocampus and prefrontal cortex of stressed SAMP10 mice, but increased in mice that ingested theanine. Lipocalin 2 (Lcn2), the expression of which increased in response to stress, was significantly high in the hippocampus and prefrontal cortex of stressed SAMP10 mice, but not in mice that ingested theanine. These data suggest that Npas4 and Lcn2 are involved in the brain atrophy and stress vulnerability of SAMP10 mice, which are prevented by the consumption of theanine, causing changes in the expression of these genes.

The young leaves of green tea become lighter in color than usual when protected from sunlight by a shading net for about two weeks while growing. These leaves are called &ldquo, shaded white leaf tea&rdquo, or SWLT. In the eluate of SWLT, the amount of amino acids (361 mg/L) was significantly higher than that in regular tea (53.5 mg/L). Since theanine and arginine, the first and second most abundant amino acids in SWLT, have significant antistress effects, we examined the antistress effect of SWLT on humans. SWLT or placebo green tea (3 g) was eluted with room-temperature water (500 mL). Participants consumed the tea for one week prior to pharmacy practice and continued for 10 days in the practice period. The state-trait anxiety inventory, an anxiety questionnaire, tended to be scored lower in the SWLT group than the placebo, but other stress markers showed no differences. The effect of the difference in SWLT components examined with mice showed that aspartic acid and asparagine, which are abundant in SWLT, counteracted the antistress effects of theanine and arginine. Large amounts of caffeine also interfered with SWLT&rsquo, s antistress effect. Thus, SWLT, which is high in caffeine and amino acids, suppressed depressant behavior in mice.

Dimerization in signal transduction is a dynamically regulated process and a key regulatory mechanism. Signal transducer and activator of transcription 3 (STAT3) dimerizes after tyrosine phosphorylation upon cytokine stimulation. Because only the STAT3 dimer possesses the trans-activation activity, dimerization is an indispensable process for cytokine signaling. Here we report the detection of dynamic STAT3 dimerization in living cells using the homoFluoppi system. This method allowed us to validate the presence of an intact Src homology 2 domain and STAT3 Tyr705 phosphorylation, which facilitate puncta formation and homodimerization. Puncta formation was reversible, as determined by a decreased punctate signal after washout of oncostatin M. We analyzed STAT3 mutants, which have been reported in patients with hyper IgE syndrome and inflammatory hepatocellular adenoma (IHCA). Analysis of the IHCA mutants using homoFluoppi revealed constitutive activity independent of cytokine stimulation and novel insight into kinetics of dimer dissociation process. Next, we used homoFluoppi to screen for inhibitors of STAT3 dimerization, and identified 3,4-methylenedioxy-β-nitrostyrene as a novel inhibitor. The results of this study show that homoFluoppi is a useful research tool for the analysis of proteins like STAT3 that dynamically dimerize, and is applicable for the screening of dimerization modulators.

Bendamustine (BENDA), which bears the bis(2-chloroethyl)amino moiety, is an alkylating agent that stops the growth of cancer cells by binding to DNA and interfering with its replication. However, the mechanism of action underlying its excellent clinical efficacy remains unclear. In this work, we report that BENDA inhibits signal transducer and activator of transcription 3 (STAT3). In an AlphaScreen-based biochemical assay using recombinant human STAT3, binding of STAT3-Src homology 2 (SH2) to the phosphotyrosine (pTyr, pY) peptide was inhibited by BENDA but not by the inactive metabolite dihydroxy bendamustine (HP2). When a single point mutation of C550A or C712A was introduced into recombinant human STAT3, its sensitivity to BENDA was substantially reduced, suggesting that these cysteine residues are important for BENDA to inhibit STAT3. Furthermore, BENDA suppressed the function of cellular STAT3 as a transcriptional activator in a human breast cancer cell line, MDA-MB-468, with constitutively activated STAT3. A competitive pull-down assay using biotinylated BENDA (Bio-BENDA) revealed that BENDA bound tightly to cellular STAT3, presumably through covalent bonds. Therefore, our results suggest that the anticancer effects of BENDA may be associated, at least in part, with its inhibitory effect on the SH2 domain of STAT3.

Many biological activities of green tea have been attributed to a major constituent, (minus;)-epigallocatechin gallate (EGCG). We previously reported that EGCG and an EGCG-free fraction derived from green tea modulated the gene expression of gluconeogenic enzymes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, in the mouse liver. EGCG is also known to affect the gene expression of enzymes related to lipid metabolism. However, it remains to be examined whether or not a constituent other than EGCG contributes to the change in gene expression of these enzymes. In this study, we prepared an EGCG-free water-soluble fraction (GT-W), and examined its effects on the hepatic gene expression of lipogenic enzymes in mice. The results of quantitative real-time PCR assays indicated that the dietary administration of GT-W for 4 weeks reduced the hepatic gene expression of lipogenic enzymes: fatty acid synthase, hydroxymethylglutaryl coenzyme A reductase, and acetyl-coenzyme A carboxylase alpha. Also, the gene expression of sterol regulatory element-binding transcription factor (Srebf)1 and/or Srebf2 was reduced, suggesting that the reduction of Srebfs contributed to the down-regulation of the lipogenic enzymes, since these transcription factors bind the promoter region to enhance their expression. The plasma levels of triglycerides and cholesterol were reduced with statistical significance in the group given a diet containing GT-W. These results suggest that in addition to EGCG, green tea contains some component(s) which may help to prevent arteriosclerosis and obesity.

Galactosamine is known to induce hepatic injury in rats and the galactosamine-induced hepatitis is believed to be similar to viral hepatitis both morphologically and functionally. In the present study, we examined how drinking green tea affects the gene expression of inflammatory cytokines which may be up-regulated in galactosamine-induced hepatitis. As has been reported, galactosamine caused hepatic injury in rats as evidenced by an increase in serum transaminase activities and histological observations of the liver. The results of the reverse transcription and polymerase chain reaction indicated an increased gene expression of inflammatory cytokines, tumor necrosis factor-alpha and interleukin-1beta, in the injured liver and the enzyme linked immunoassay showed an increase in the serum levels of these cytokines. Oral administration of green tea rich in catechins (Healthya green tea) restored these biomarkers in the galacotsamine-treated rats to near the control levels. These results suggest that the drinking of green tea with a high catechin content may help to prevent and/or attenuate the development of a certain type of hepatitis.