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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.

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.

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-&Delta, Sglt2) 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-&Delta, Sglt2 and SAMP10(+). However, memory retention was lower in SAMP10-&Delta, Sglt2 mice than SAMP10(+). Amyloid beta (A4) precursor-like protein 1 (Aplp1) expression was significantly lower in the hippocampus of SAMP10-&Delta, SGLT2 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A quarantine was performed on conventional Hartley guinea pigs free from Streptococcus zooepidemicus, Bordetella bronchiseptica and Salmonella spp., but infected with Eimeria caviae, which were purchased from twenty-five commercial breeders by the National Institute of Health. Physical examinations revealed an incidence of 0.88 per cent or 1,461 sick animals in 166, 050 guinea pigs quarantined during the period 1964 to 1982. The result obtained showed the following significant differences between the periods 1964 to 1971 and 1972 to 1982: Annual incidence of sick animals in the period 1972 to 1982 increased twice as many as 0.56 to 0.81 per cent during the 1964 to 1971, and monthly incidence showed bimodal occurrence at April and October in the former period but continual occurrence from November to April in the latter period. Major clinical signs in the former period were death and diarrhea, which occurred usually within a week after arrival of the guinea pigs at our institute and caused significant decrease of body weight, but those in the latter period were retarded growth and weakness which became detectable during 1-3 weeks after arrival of the animals. Discussions were made on possible reasons concerning the differences in incidence of sick animals during the two periods.

An epizootiological survey has been carried out on naturally occurring coccidiosis in Hartley guinea pigs (weight, 250g) purchased by the National Institute of Health, Tokyo during the period 1964 to 1982. Coccidial infections in breeding colonies of guinea pigs were observed very frequently in weaned animals but scarcely in adult and suckling animals. Oocysts of Eimeria caviae were detected in 53.8% of the 7,162 fecal samples collected from transportation boxes and coccidiosis occurred in 39% of the 1,461 dead or culled animals obtained during the routine one week quarantine period. In the period 1964 to 1971, particularly high rates of prevalence of oocysts, between 55-86%, and incidence of coccidiosis, between 55-76%, were observed. These rates were clearly reduced in the period 1972 to 1982, with a lower rate of isolation of oocysts ranging from 14-48% and les than 20% incidence of coccidiosis (except in 1981 and 1982). The monthly fluctuation of occurrence rates of oocysts and clinical coccidiosis differed over the period of study. From 1964 to 1971, the high prevalence of oocysts was consistently observed accompanied by a bimodal pattern of incidence of coccidiosis in April (85%) and October (78%). In the period 1972 to 1982, both parameters showed a single peak, for prevalence of oocysts in June (60.7%) and for incidence of coccidiosis in May (45%). Oocysts in feces disappeared in February and March and coccidiosis occurred irregularly in 1981 and 1982.(ABSTRACT TRUNCATED AT 250 WORDS)