DNA Mutagenicity of Hydroxyhydroquinone in Roasted Coffee Products and Its Suppression by Chlorogenic Acid, a Coffee Polyphenol, in Oxidative-Damage-Sensitive SAMP8 Mice.

Hydroxyhydroquinone (HHQ) is an oxidative component produced by roasting coffee beans and has been reported to generate relatively large amounts of reactive oxygen species (ROS). In this study, we used senescence-accelerated mouse prone 8 (SAMP8) mice to determine whether HHQ consumption increases oxidative-stress-induced injury, because in SAMP8 mice, the activity of 8-oxoguanine DNA glycosylase 1, which repairs oxidative modifications in DNA, is decreased. The results showed that two out of twelve (16.7%) HHQ-treated mice presented polyuria and glucosuria around 2 months after the start of treatment, indicating that HHQ may act as a mutagen against SAMP8 mice, which is sensitive to oxidative damage. No abnormalities were observed in the chlorogenic acid (coffee polyphenol, CPP)-treated group. The concentration of hydrogen peroxide in the serum of SAMP8 mice was significantly higher than that in SAMR1 (senescence-resistant) control mice, and the concentration was further increased in the HHQ-treated group. CPP, when coexisting with HHQ at the rate contained in roasted coffee, decreased the amount of hydrogen peroxide in the serum of SAMP8 mice. Although CPP can act both oxidatively and antioxidatively as a polyphenol, CPP acts more antioxidatively when coexisting with HHQ. Thus, the oxidative effect of HHQ was shown to be counteracted by CPP.

Stress Reduction Potential in Mice Ingesting DNA from Salmon Milt.

The functionality of food-derived nucleotides is revealed when nucleotide components are ingested in emergency situations, such as during stress loading, though it is difficult to elucidate the physiological function of dietary nucleotide supplementation. Using a stress load experimental system utilizing territoriality among male mice, we evaluated whether DNA sodium salt derived from salmon milt (DNA-Na) has stress-relieving effects. It was found that stress was reduced in mice fed a diet containing a 1% concentration of DNA-Na, but this was insignificant for yeast-derived RNA. Next, we attempted to elucidate the anti-stress effects of DNA-Na using another experimental system, in which mice were subjected to chronic crowding stress associated with aging: six mice in a cage were kept until they were 7 months of age, resulting in overcrowding. We compared these older mice with 2-month-old mice that were kept in groups for only one month. The results show that the expression of genes associated with hippocampal inflammation was increased in the older mice, whereas the expression of these genes was suppressed in the DNA-Na-fed group. This suggests that dietary DNA intake may suppress inflammation in the brain caused by stress, which increases with age.

Mouse Models with SGLT2 Mutations: Toward Understanding the Role of SGLT2 beyond Glucose Reabsorption.

The sodium-glucose cotransporter 2 (SGLT2) mainly carries out glucose reabsorption in the kidney. Familial renal glycosuria, which is a mutation of SGLT2, is known to excrete glucose in the urine, but blood glucose levels are almost normal. Therefore, SGLT2 inhibitors are attracting attention as a new therapeutic drug for diabetes, which is increasing worldwide. In fact, SGLT2 inhibitors not only suppress hyperglycemia but also reduce renal, heart, and cardiovascular diseases. However, whether long-term SGLT2 inhibition is completely harmless requires further investigation. In this context, mice with mutations in SGLT2 have been generated and detailed studies are being conducted, e.g., the SGLT2-/- mouse, Sweet Pee mouse, Jimbee mouse, and SAMP10-ΔSglt2 mouse. Biological changes associated with SGLT2 mutations have been reported in these model mice, suggesting that SGLT2 is not only responsible for sugar reabsorption but is also related to other functions, such as bone metabolism, longevity, and cognitive functions. In this review, we present the characteristics of these mutant mice. Moreover, because the relationship between diabetes and Alzheimer's disease has been discussed, we examined the relationship between changes in glucose homeostasis and the amyloid precursor protein in SGLT2 mutant mice.

Theanine, a Tea-Leaf-Specific Amino Acid, Alleviates Stress through Modulation of Npas4 Expression in Group-Housed Older Mice.

Group rearing is a common housing condition, but group-housed older mice show increased adrenal hypertrophy, a marker of stress. However, the ingestion of theanine, an amino acid unique to tea leaves, suppressed stress. We aimed to elucidate the mechanism of theanine's stress-reducing effects using group-reared older mice. The expression of repressor element 1 silencing transcription factor (REST), which represses excitability-related genes, was increased in the hippocampus of group-reared older mice, whereas the expression of neuronal PAS domain protein 4 (Npas4), which is involved in the regulation of excitation and inhibition in the brain, was lower in the hippocampus of older group-reared mice than in same-aged two-to-a-house mice. That is, the expression patterns of REST and Npas4 were found to be just inversely correlated. On the other hand, the expression levels of the glucocorticoid receptor and DNA methyltransferase, which suppress Npas4 transcription, were higher in the older group-housed mice. In mice fed theanine, the stress response was reduced and Npas4 expression tended to be increased. These results suggest that Npas4 expression was suppressed by the increased expression of REST and Npas4 downregulators in the group-fed older mice, but that theanine avoids the decrease in Npas4 expression by suppressing the expression of Npas4 transcriptional repressors.

Improvement of Depressed Mood with Green Tea Intake.

Being in a prolonged depressed state increases the risk of developing depression. To investigate whether green tea intake is effective in improving depression-like moods, we used an experimental animal model of depression with lipopolysaccharide (LPS) and clarified the effects of green tea on the biological stress response and inflammation in the brain. Regarding the stress reduction effect of green tea, we found that the sum of caffeine (C) and epigallocatechin gallate (E) relative to the sum of theanine (T) and arginine (A), the major components of green tea, or the CE/TA ratio, is important. The results showed that depression-like behavior, adrenal hypertrophy as a typical stress response, and brain inflammation were suppressed in mice fed green tea components with CE/TA ratios of 2 to 8. In addition, the expression of Npas4, which is reduced in anxiety and depression, was maintained at the same level as controls in mice that consumed green tea with a CE/TA ratio of 4. In clinical human trials, the consumption of green tea with CE/TA ratios of 3.9 and 4.7 reduced susceptibility to subjective depression. These results suggest that the daily consumption of green tea with a CE/TA ratio of 4-5 is beneficial to improving depressed mood.

Coffee Polyphenol, Chlorogenic Acid, Suppresses Brain Aging and Its Effects Are Enhanced by Milk Fat Globule Membrane Components.

Mice feed with coffee polyphenols (CPP, chlorogenic acid) and milk fat globule membrane (MFGM) has increased survival rates and helps retain long-term memory. In the cerebral cortex of aged mice, CPP intake decreased the expression of the proinflammatory cytokine TNF-α, and lysosomal enzyme cathepsin B. The suppression of inflammation in the brain during aging was thought to result in the suppression of the repressor element 1-silencing transcription factor (REST) and prevention of brain aging. In contrast, CPP increased the expression of REST, cAMP-responsive element binding (CREB) and transforming growth factor ß1 (TGF-ß1) in the young hippocampus. The increased expression of these factors may contribute to the induction of neuronal differentiation and the suppression of memory decline with aging. Taken together, these results suggest that CPP increases CREB in the young hippocampus and suppresses inflammation in the old brain, resulting in a preventive effect on brain aging. The endotoxin levels were not elevated in the serum of aged mice. Although the mechanism of action of MFGM has not yet been elucidated, the increase in survival rate with both CPP and MFGM intake suggests that adding milk to coffee may improve not only the taste, but also the function.

Theanine, Antistress Amino Acid in Tea Leaves, Causes Hippocampal Metabolic Changes and Antidepressant Effects in Stress-Loaded Mice.

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.

Mutation in Sodium-Glucose Cotransporter 2 Results in Down-Regulation of Amyloid Beta (A4) Precursor-Like Protein 1 in Young Age, Which May Lead to Poor Memory Retention in Old Age.

Senescence-accelerated mouse prone 10 (SAMP10) exhibits cerebral atrophy and depression-like behavior. A line of SAMP10 with spontaneous mutation in the Slc5a2 gene encoding the sodium-glucose cotransporter (SGLT) 2 was named SAMP10/TaSlc-Slc5a2slc (SAMP10-ΔSglt2) and was identified as a renal diabetes model. In contrast, a line of SAMP10 with no mutation in SGLT2 (SAMP10/TaIdrSlc, SAMP10(+)) was recently established under a specific pathogen-free condition. Here, we examined the mutation effect in SGLT2 on brain function and longevity. No differences were found in the survival curve, depression-like behavior, and age-related brain atrophy between SAMP10-ΔSglt2 and SAMP10(+). However, memory retention was lower in SAMP10-ΔSglt2 mice than SAMP10(+). Amyloid beta (A4) precursor-like protein 1 (Aplp1) expression was significantly lower in the hippocampus of SAMP10-ΔSGLT2 than in SAMP10(+) at 2 months of age, but was similar at 12 months of age. CaM kinase-like vesicle association (Camkv) expression was remarkably lower in SAMP10(+). These genes have been reported to be involved in dendrite function. Amyloid precursor proteins have been reported to involve in maintaining homeostasis of glucose and insulin. These results suggest that mutation in SGLT2 results in down-regulation of Aplp1 in young age, which can lead to poor memory retention in old age.

Antidepressant Effect of Shaded White Leaf Tea Containing High Levels of Caffeine and Amino Acids.

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 "shaded white leaf tea" 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's antistress effect. Thus, SWLT, which is high in caffeine and amino acids, suppressed depressant behavior in mice.

Green Tea Catechins Trigger Immediate-Early Genes in the Hippocampus and Prevent Cognitive Decline and Lifespan Shortening.

Senescence-accelerated mouse prone 10 (SAMP10) mice, after ingesting green tea catechins (GT-catechin, 60 mg/kg), were found to have suppressed aging-related decline in brain function. The dose dependence of brain function on GT-catechin indicated that intake of 1 mg/kg or more suppressed cognitive decline and a shortened lifespan. Mice that ingested 1 mg/kg GT-catechin had the longest median survival, but the dose was less effective at suppressing cognitive decline. The optimal dose for improving memory acquisition was 60 mg/kg, and memory retention was higher in mice that ingested 30 mg/kg or more. To elucidate the mechanism by which cognitive decline is suppressed by GT-catechin, changes in gene expression in the hippocampus of SAMP10 mice one month after ingesting GT-catechin were analyzed. The results show that the expression of immediate-early genes such as nuclear receptor subfamily 4 (Nr4a), FBJ osteosarcoma oncogene (Fos), early growth response 1 (Egr1), neuronal PAS domain protein 4 (Npas4), and cysteine-rich protein 61 (Cyr61) was significantly increased. These results suggest that GT-catechin suppresses age-related cognitive decline via increased expression of immediate-early genes that are involved in long-term changes in plasticity of synapses and neuronal circuits.

Theanine, the Main Amino Acid in Tea, Prevents Stress-Induced Brain Atrophy by Modifying Early Stress Responses.

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.

Visualization and quantification of dynamic STAT3 homodimerization in living cells using homoFluoppi.

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-ß-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.

New sulfurated derivatives of cinnamic acids and rosmaricine as inhibitors of STAT3 and NF-κB transcription factors.

A set of new sulfurated drug hybrids, mainly derived from caffeic and ferulic acids and rosmaricine, has been synthesized and their ability to inhibit both STAT3 and NF-κB transcription factors have been evaluated. Results showed that most of the new hybrid compounds were able to strongly and selectively bind to STAT3, whereas the parent drugs were devoid of this ability at the tested concentrations. Some of them were also able to inhibit the NF-κB transcriptional activity in HCT-116 cell line and inhibited HCT-116 cell proliferation in vitro with IC50 in micromolar range, thus suggesting a potential anticancer activity. Taken together, our study described the identification of new derivatives with dual STAT3/NF-κB inhibitory activity, which may represent hit compounds for developing multi-target anticancer agents.

Inhibition of STAT3 by Anticancer Drug Bendamustine.

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.

Discovery of N-{5-[3-(3-hydroxypiperidin-1-yl)-1,2,4-oxadiazol-5-yl]-4-methyl-1,3-thiazol-2-yl}acetamide (TASP0415914) as an orally potent phosphoinositide 3-kinase γ inhibitor for the treatment of inflammatory diseases.

Class I phosphoinositide 3-kinases (PI3Ks), particularly PI3Kγ, have become attractive drug targets for inflammatory and autoimmune disorders such as rheumatoid arthritis. Herein, we describe the synthesis and the structure-activity relationships (SAR) of a series of 2-amino-5-oxadiazolyl thiazoles, culminating in the identification of 8j (TASP0415914), an orally potent inhibitor of phosphoinositide 3-kinase γ (PI3Kγ). TASP0415914 demonstrated good potency in a cell-based assay and, furthermore, exhibited in vivo efficacy in a collagen induced arthritis (CIA) model in mice after oral administration.

The clinical and immunomodulatory effects of green soybean extracts.

The present study was performed to investigate the immune-modulating activities of extracts from green soybean (Glycine max) in a 2,4-toluene diisocyanate (TDI)-inducing guinea pig rhinitis model and a human trial study for allergic rhinitis. Hot water extracts of green soybean were chosen for animal experimentation on the basis of their ability to regulate the production of B cell-activating factor of the TNF family and a proliferation-inducing ligand in mouse spleen cells. Green soybean extracts significantly decreased the levels of ovalubumin (OVA)-specific IgE in mice and significantly suppressed the TDI-induced nasal mucosa secretion. An open-label human pilot study was performed on 16 subjects, using Japanese cedar pollinosis. The symptom scores for Japanese cedar pollinosis were better in the long-term green soybean extracts intake group than in the withdrawal short-term intake group. Green soybean extracts had great potential as an orally active immune modulator for the treatment of various allergic diseases.

Effects of a catechin-free fraction derived from green tea on gene expression of enzymes related to lipid metabolism in the mouse liver.

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.

Effects of (-)-epigallocatechin-3-O-gallate on expression of gluconeogenesis-related genes in the mouse duodenum.

Green tea has been shown to have many beneficial health effects. We have previously reported that dietary (-)-epigallocatechin-3-O-gallate (EGCG), the major polyphenol in green tea, reduced gene expressions of gluconeogenic enzymes, glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), in the normal mouse liver. In the present study, we examined the effects of intragastrical administration of EGCG on the expression of gluconeogenesis-related genes in the mouse intestine. The results of experiments with the semi-quantitative reverse transcription-polymerase chain reaction indicated that EGCG at 0.6 mg/head caused a reduced expression of G6Pase, PEPCK, hepatocyte nuclear factor 1α (HNF1α), and HNF4α. Experiments using the quantitative real-time polymerase chain reaction confirmed these effects. We then examined the effects of EGCG using human colon carcinoma Caco-2 cells stimulated with dexamethasone and dibutyryl cAMP. The results were generally consistent with those from the experiments in vivo. The present findings suggest EGCG to contribute to the beneficial effects of green tea on diabetes, obesity, and cancer by modulating gene expression in the intestine.

DNA microarray analysis of changes in gene expression induced by 1,25-dihydroxyvitamin D3 in human promyelocytic leukemia HL-60 cells.

Using a DNA microarray, we analyzed about 16,600 genes for changes in expression associated with the differentiation of human promyelocytic leukemia HL-60 cells induced by 1alpha,25-dihydroxyvitamin D3 (DVD). Many of the up-regulated genes could be correlated to differentiation-associated changes toward a monocyte/macrophage lineage, and many down-regulated genes could be correlated to repressed cell growth. The present study revealed the down-regulated gene expression of importins and exportins 1, 5, 7, and exportin-tRNA. Thus, the present results confirmed our previous findings of down-regulation of exportin 1 and exportin-tRNA by DVD. Gene expression of exportin 6 is suggested to be regulated differently from that of exportins 1, 5, 7, and exportin-tRNA. The down-regulation of nuclear transport factors may be deeply associated with the differentiation of HL-60 cells induced by DVD.

Green tea with a high catechin content suppresses inflammatory cytokine expression in the galactosamine-injured rat liver.

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.