Shigeru Yamamoto

Role of nucleosides and nucleotides in the immune system, gut reparation after injury, and brain function

Emerging evidence indicates the importance of nucleosides and nucleotides in the maintenance of functions of the bone marrow hematopoietic cells, intestinal mucosa, and the brain, which have limited de novo synthesis of purine and pyrimidine bases. We have found that nucleosides and nucleotides stimulate hemopoieses and increase peripheral neutrophil counts in mice treated with cyclophosphoamide. Intraperitoneal administration of nucleosides and nucleotides decreased bacterial translocation, the number of colony-forming units, and increased survival against methicillin-resistant Staphylococcus aureus. In vitro immune studies in mice showed that nucleosides and nucleotides increase the delayed-type cutaneous hypersensitivity and the popliteal lymph node blastogenic response to antigens, allogens, and mitogens. Both intraperitoneal and oral administration of nucleosides and nucleotides reduced endotoxin-induced bacterial translocation and improved injury to the gut in protein-deficient mice. However, oral administration of nucleosides and nucleotides in experimental colitis resulted in a worsening of colitic conditions and increased interleukin-8 and tumor necrosis factor-alpha concentrations in inflamed colonic portions, indicating the pro-inflammatory activities of nucleosides and nucleotides. Memory-deficient senescence-accelerated mice and mice with dementia showed improved memory with dietary nucleosides and nucleotides supplementation. These results indicate that supplementation with nucleosides and nucleotides is beneficial to the functions of the system and the brain. However, beneficial effects to the gut appear to depend on the type of damage sustained by the gut.

Nucleoside-nucleotide free diet protects rat colonic mucosa from damage induced by trinitrobenzene sulphonic acid.

BACKGROUND: Growing evidence suggests that intestinal recovery from injury induced by radiation, endotoxin, and protein deficiency is improved by the ingestion of nucleosides and nucleotides. AIM: This study examined the effect of dietary nucleosides and nucleotides supplementation on trinitrobenzene sulphonic acid induced colonic damage in experimental colitis. METHODS: Sprague-Dawley rats were randomised into two groups and fed nucleic acid free 20% casein diet (control) or this diet supplemented with 0.5% nucleoside-nucleotide mixture for four weeks. On the second week, colonic inflammation was induced in rats by intracolonic administration of 0.25 ml of 50% ethanol containing 25 mg of trinitrobenzene sulphonic acid. Additionally, other sets of rats were treated with 0.25 ml of 50% ethanol, 25 mg of trinitrobenzene sulphonic acid in 0.25 ml saline, or 0.25 ml of 0.9% saline. RESULTS: After two weeks, colon weight, macroscopic and microscopic damage scores, were significantly greater (p < 0.05) in the nucleoside-nucleotide supplemented group compared with the non-supplemented control groups. The same variables seen in the trinitrobenzene sulphonic acid-ethanol group fed nucleoside-nucleotide free diet were greater (p < 0.05) than in the rest of the groups fed nucleoside-nucleotide free diet and treated with ethanol, trinitrobenzene sulphonic acid in saline, or saline. Histologically, segmental ulceration and inflammation associated with significantly increased infiltration of polymorphonuclear leucocytes, macrophages, lymphocytes, fibroblasts were observed in the supplemented group compared with the controls. In the nucleoside-nucleotide supplemented group the epithelial damage, mucosal erosion, oedema, and coagulative necrosis of the muscularis propria was more extensive in comparison to the non-supplemented control groups. CONCLUSIONS: This study suggests that dietary nucleosides and nucleotides may aggravate colonic damage and inflammation in chemically induced experimental colitis in rats; and that nucleoside-nucleotide free diet combined with other pharmacological agents may offer a better response.

Modulation of age-related changes in immune functions of protein-deficient senescence-accelerated mice by dietary nucleoside-nucleotide mixture supplementation

In the present study we examined the immune-enhancing effect of a nucleoside-nucleotide mixture on the non-specific T-cell immune functions of senescence-accelerated mice (SAM) fed on a low-protein diet. The immune functions studied were in vitro thymic and splenic cell lymphoproliferative responses to phytohaemagglutinin, lipopolysaccharide and concanavalin A and their production of interleukin-2 (IL-2) and interferon-gamma (INF-gamma) in response to mitogen stimulation. SAMP8 mice aged 3 and 6 months were used. In each age group, mice were fed on diets containing either 50 g casein/kg, 50 g casein/kg supplemented with 5 g nucleoside-nucleotide mixture/kg or 200 g casein/kg for 3 weeks. The supplemented 3- and 6-month-old mice had higher (P < 0.05) thymic and splenic cell counts compared with the low-protein group. In both age groups of mice, concanavalin A induced higher (P < 0.05) total thymic and splenic lymphoproliferative responses for the nucleoside-nucleotide mixture-supplemented group compared with the 50 g casein/kg dietary groups. Thymic and splenic production of IL-2 was higher for the 3-month-old mice in both the supplemented and the 200 g casein/kg dietary groups. INF-gamma production in the supplemented 3-month-old group and the 6-month-old 200 g casein/kg dietary group was higher (P < 0.05) compared with the other groups. Overall the supplemented 3-month-old mice exhibited both higher lymphoproliferative responses and production of cytokines compared with the supplemented 6-month-old mice. The results indicate that early nucleoside-nucleotide mixture supplementation may enhance the immune response in protein-deprived SAMP8 mice.

Favorable effects of egg white protein on lipid metabolism in rats and mice

Abstract Egg is a cholesterol-rich food and has a strong hyper-cholesterolemic action. However, all the cholesterol is in egg yolk and egg white is cholesterol-free. The effect of egg white protein and its hydrolysates on the serum lipids were compared with casein and soybean protein in rats and mice. The animals were given 30% casein diet (Ca group) or diets of 15% casein plus 15% soybean protein isolate (SPI group), egg white protein (EW group) or egg white protein hydrolysates (EW-P group) for 3 (rats) or 2 (mice) weeks. Food intake and growth were very similar among the different dietary groups. Hypocholesterolemic effect was observed in SP, EW and EW-P groups in rats and EW group in mice. Prevention of the reduction of HDL-cholesterol was found in EW and EW-P groups in rats and EW-P group in mice. The result suggests the possibility of the use of egg white for the prevention and treatment of hyper-cholesteremia.

Effects of dietary phosphatidylcholine on memory in memory deficient mice with low brain acetylcholine concentration.

Data concerning the effect of phosphatidylcholine (PCh) administration on the improvement of memory in senile dementia of Alzheimer type are inconsistent, probably due to the different conditions under which studies were conducted. Animal studies provide a good model, but data on this is limited. We studied the effect of PCh on memory in memory deficient mice (Dull mice) with low brain acetylcholine (ACh) concentration and normal mice. Mice were fed 24% casein diet (control) or this diet supplemented with 2 or 8% egg yolk PCh from gestation (Experiment 1) and after weaning (Experiment 2). Memory acquisition and retention were studied by step-down type passive avoidance performance at 8 and 10 weeks old, respectively. Control group of Dull mice had poorer memories than that of the normal mice in Experiments 1 and 2. On the 2% PCh diet, Dull mice improved memory acquisition and retention in Experiment 1 and retention in Experiment 2. On the 8% PCh diet in Dull mice there was improvement of memory and retention in Experiment 1, but no effect was observed in Experiment 2 (P > 0.05). In the normal mice, the 2% PCh diet did not affect memory acquisition and retention, however on the 8% PCh diet, there was no or adverse effect. These results suggest that dietary supplementation of egg yolk PCh improves memory of Dull mice, particularly when given from gestation and that the 2% PCh diet elicits better response than the 8% PCh diet.

Nucleoside-Nucleotide-Free Diet Suppresses Cytokine Production and Contact Sensitivity Responses in Rats With Trinitrobenzene Sulphonic Acid-Induced Colitis

We examined the effects of dietary nucleoside-nucleotide mixture on synthesis of inflammatory cytokines, interleukin-8 and tumor necrosis factor-alpha, in sensitized and nonsensitized colitic rats. Sensitized and nonsensitized colitic rats that were fed a nucleoside-nucleotide mixture had greater colonic weight and macroscopic and microscopic damage scores than nucleoside-nucleotide-free sensitized and nonsensitized colitic rats. Increased colonic tumor necrosis factor-alpha and interleukin-8 concentrations were associated with increased colonic inflammation and ulceration in the nucleoside-nucleotide mixture-fed group. There was also increased ear thickness in the nucleoside-nucleotide mixture-fed sensitized and nonsensitized colitic rats, which correlated highly with increased tumor necrosis factor-alpha and interleukin-8 levels in the ear lobes. Nucleoside-nucleotide-free diets may suppress cytokine secretion, thereby reducing colonic damage and contact sensitivity responses in colitic rats.

Effects of dietary nucleoside-nucleotide mixture on memory in aged and young memory deficient mice

Intestinal mucosa, bone marrow hematopoietic cells and brain have limited capacity for the de novo synthesis of nucleosides (NSs) and nucleotides (NTs). Whereas the role of dietary NS and NT in the former two tissues is known, it is not known in the brain. Therefore we studied the effect of dietary NS and NT mixture on memory in aged mice (Experiment 1) and young memory deficient mice (Experiment 2). Memory retention was studied by step-through type passive avoidance performance (maximum 180 seconds). In Experiment 1 aged (7 month old) senescence accelerated mice (SAM) were fed 20% casein diet (control) or this diet supplemented with 0.5% NS/NT mixture for 12 weeks. Memory was studied 1, 2 and 3 days after the electric shock (punishment). In Experiment 2, young (1 month old) memory deficient mice (Dull mice) and normal mice (ddY mice) were fed the same diets as those in Experiment 1 for 12 weeks. Memory retention was studied 1 and 3 days after the punishment. In the aged SAM the average time of avoidance and also the percentages of successful memory 2 and 3 days after the punishment were significantly higher in the NS/NT diet group than the control diet group (P < 0.05). In the Dull mice percentage of successful memory was higher in the NS/NT diet group than in the control group 3 days after the punishment, however, such an effect was not observed in the normal mice. These results suggest that insufficient endogenous supply of NSs and NTs may be responsible for the factor of memory deficiency with aging or of genetical memory deficiency, which can be improved by the dietary administration of NSs and NTs.

EFFECT OF DIETARY PEPTIDES ON PLASMA LIPIDS AND ITS MECHANISM STUDIED IN RATS AND MICE

The present 4 experiments were planned to confirm our hypothesis that small quantity of intraluminal peptides enters into circulatory system and affects on lipid metabolism. In Experiments 1 and 2, rats and mice were fed diets of casein or two casein hydrolysate peptides for 2 and 3 weeks, respectively. Hypocholesterolemic effect in one of the peptides was observed in both animals. In Experiment 3, we administered small amount of the above peptides, amino acid mixture simulating casein pattern or saline to mice through tail vein daily for 10 days. Hypocholesterolemic effect was observed in the mice administered peptides but not in the mice administered saline and the amino acid mixture. In Experiment 4, entry of peptides into the circulatory system was shown with the everted sac of rat intestine. These results may indicate that small quantity of intraluminal peptides enter the circulatory system from lumen and affect on lipid metabolism.

Effect of Dietary Nucleosides and Nucleotides on Murine Allergic Rhinitis

Although there are studies that report the effects of dietary nucleoside and nucleotide mixtures on the immune response, none are concerned with the role in allergic disease. This study evaluated the effect of dietary nucleic acid mixture (NAM) on mice with a nasal allergy model. One group of mice was supplemented with a 0.5% NAM and the other two groups were fed with a nucleic acid-free diet with 20% casein that served as sensitized and nonsensitized controls. The mice of the NAM group and the sensitized control group were sensitized in two courses by 2 microl of 5% 2,4-toluene diisocyanate (TDI), whereas the nonsensitized control was given 2 microl of ethylacetate instead of TDI. On the 28th day, an allergy was provoked with 4 microl of 2.5% TDI and the allergic responses were observed for 10 minutes. Results showed that the NAM diet group had more severe symptoms of itching, rhinorrhea, snorting, and irritability compared with the controls; also observed were a high incidence of sneezing at 34.7 +/- 4.0 in NAM compared with 19.0 +/- 3.0 (P < 0.001) in sensitized controls and 2.8 +/- 0.7 in nonsensitized controls. From this study, it can be concluded that diets supplemented with nucleic acid mixture contribute to the severity of murine allergic rhinitis.

Comparative effect of amino acid mixtures with or without asparagine and glutamine on growth of rats

Abstract When proteins, peptides and amino acid mixtures are compared, some amino acid mixtures include asparagine and/or glutamine and others do not. Therefore in this experiment we studied the effect of the presence or absence of these amino acids on the growth and nutritional status of rats. Rats weighing about 65 g were divided into 4 groups and fed experimental diets for 21 days. The diets contained 11% casein (N=1.57%, group A), 10% amino acid mixture (N=1.57%, including asparagine and glutamine, group B), 10% amino acid mixture (N=1.25%, excluding asparagine and glutamine, group C), or 12.5% amino acid mixture (N=1.57%, excluding asparagine and glutamine, group D). In group D we increased amino acid concentration to 12.5% in order to make N concentration same as the diets of groups A and B. Daily food intake, body weight gain and hematologic values were measured. Most of the indicators of groups A, B and C were similar and lower than those of group D. The results suggest that amino acid mixtures excluding asparagine and glutamine have the same effect as protein and the mixture including these amino acids on growth and nutritional status of young rats but overestimates the effect when total N concentration is increased to the level of protein.