Keiko Yamauchi

ACTIVATION OF NUCLEAR TRANSCRIPTION FACTOR–κB IN MOUSE BRAIN INDUCED BY A SIMULATED MICROGRAVITY ENVIRONMENT

SummaryMicrogravity induces inflammatory responses and modulates immune functions that may increase oxidative stress. Exposure to a microgravity environment induces adverse neurological effects; however, there is little research exploring the etiology of these effects resulting from exposure to such an environment. It is also known that spaceflight is associated with increase in oxidative stress; however, this phenomenon has not been reproduced in land-based simulated microgravity models. In this study, an attempt has been made to show the induction of reactive oxygen species (ROS) in mice brain, using ground-based microgravity simulator. Increased ROS was observed in brain stem and frontal cortex with concomitant decrease in glutathione, on exposing mice to simulated microgravity for 7 d. Oxidative stress-induced activation of nuclear factor-kappaB was observed in all the regions of the brain. Moreover, mitogen-activated protein kinase kinase was phosphorylated equally in all regions of the brain exposed to simulated microgravity. These results suggest that exposure of brain to simulated microgravity can induce expression of certain transcription factors, and these have been earlier argued to be oxidative stress dependent.

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.

Altered cytokine expression in tissues of mice subjected to simulated microgravity.

Space flight is known to induce microgravity-associated immune dysfunction in humans, non-human primates and rodents. To understand the mechanism underlying these defects, several studies in rodents have been conducted in a ground-based antiorthostatic suspension (AOS) model that would mimic the effects of microgravity. In all these in vivo studies that showed the effects on cytokine profiles actually investigated the ex vivo production from culturing the cells isolated from whole organism that was exposed to space flight and/or microgravity. So, the purpose of the study was to examine the in vivo expression of cytokines in mice in immunologically important tissue environments of mice that were subjected to AOS. Cytokines such as Interleukin-1β (IL-1β), IL-2, IL-3, IL-6, Interferon-γ (IFN-γ) and Tumor Necrosis Factor-α (TNF-α) were measured by Enzyme Linked Immunosorbent Assay (ELISA) in the homogenates of spleen tissue, lymph nodes and also in serum of AOS mice and compared with that of control mice. AOS induced no change in the IL-3 levels, but IL-1β was increased significantly whereas IL-2 levels decreased in spleen, lymph nodes and serum. IL-6 levels did not differ in spleen but were significantly increased in lymph nodes and serum of AOS mice. IFN-γ levels in spleen did not change but showed nonsignificant reduction in lymph nodes and significant reduction in serum in response to AOS. TNF-α levels in spleen and serum were unchanged and increased in lymph nodes. This in vivo cytokine study confirms the earlier findings that microgravity-simulated conditions induce tissue-specific immune response (Mol Cell Biochem 266: 79–85, 2004)

Glutamine and arginine affect Caco-2 cell proliferation by promotion of nucleotide synthesis.

OBJECTIVE We tested our hypothesis that 1) the major effect of Gln is as a nitrogen donor, not an energy source, for nucleotides (NT) and 2) the supplementation of culture medium with arginine (Arg) decreases the flux of glutamine (Gln) for conversion to Arg, thus accelerating NT synthesis. METHODS Various concentrations of nucleosides (NS+NT) Gln, and glutamate (Glu) in culture were tested for their effect on Caco-2 cell proliferation. (Arg was tested in media with and without Gln to evaluate the Gln pathway. The incorporation of (15)N from L-[5-(15)N]-Gln into NTs of DNA was measured under different NS + NT and Arg concentrations.) RESULTS The proliferation of Caco-2 cells was increased by NS + NT and Gln supplementation, but not by Glu. The effective concentration of NS + NT was 100-fold smaller than that of Gln. An Arg effect was observed only in the presence of Gln. The NT synthesis from Gln, as indicated by (15)N incorporation from L-[5-(15)N]-Gln, was increased by Arg supplementation and decreased by NS + NT supplementation. CONCLUSION These results support our hypothesis that the effects of Gln and Arg on Caco-2 cell proliferation are by the promotion of NT synthesis and that the major role of Gln is not energy supply.

A COUNTERMEASURE TO AMELIORATE IMMUNE DYSFUNCTION IN IN VITRO SIMULATED MICROGRAVITY ENVIRONMENT: ROLE OF CELLULAR NUCLEOTIDE NUTRITION

SummaryConsiderable evidence suggests that space travelers are immunosuppressed, presumably by microgravity environmental stresses, putting them at risk for adverse effects, such as opportunistic infections, poor wound healing, and cancer. The purpose of this study was to examine the role and mechanisms of nucleotide (NT) supplementation as a countermeasure to obviate immunosuppression during space travel. The in vitro rotary cell culture system, a bioractor (BIO), was used to simulate the effect of microgravity and to isolate the neuroendocrine effects inherent to in vitro models. The splenocytes from normal mice were cultured in BIO and control tissue culture (TC) flasks with and without phytohemagglutinin (PHA) for mitogen assays. The culture medium was then supplemented with various concentrations of a nucleosides-nucleotides mixture (NS+NT), inosine, and uridine. Cytokines interleukin (IL)-1β, IL-2, IL-3, tumor necrosis factor-α, and interferon (IFN)-γ were measured from the supernatant by enzyme-linked immunosorbent assay. In the PHA-stimulated cultures the cellular proliferation in the BIO was significantly decreased as compared with the TC flask cells. BIO-cultured cells in the presence of NS+NT maintained mitogen responses similar to the control TC flask cells. The maintenance of the mitogen response in BIO was observed by the supplementation of uridine and not of inosine. These results are in aggreement with our earlier results from unit gravity experiments that showed that pyrimidines are more effective in pleiogenic immunoprotection to hosts. Cytokines IL-1β, IL-2, and IFN-γ in the BIO supernatants of cells cultured in the presence of NS+NT had a significantly higher response than the control vessel. Thus, supplemental NT, especially pyrimidines, can confer immune protection and enhance cytokine responses during space travel.

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.

Comparative effects of dietary nucleoside-nucleotide mixture and its components on endotoxin induced bacterial translocation and small intestinal injury in protein deficient mice.

BACKGROUND--Nucleoside-nucleotide mixture has been shown to improve gut morphology and reduce the incidence of bacterial translocation in protein deficient mice. AIMS--To compare the reparative effect of nucleoside-nucleotide mixture and their individual components on maintenance of gut integrity and bacterial translocation based on their differential metabolism and utilisation. METHODS--ICR (CD-1) mice were randomised into eight groups of 10 animals each and fed 20% casein diet (control), protein free diet, or protein free diet supplemented with 3 M cytidine, uridine, thymidine, inosine, guanosine monophosphate, or nucleoside-nucleotide mixture for four weeks. On the fourth week, each mouse was injected lipopolysaccharide intraperitoneally (50 micrograms/500 microliters) and the incidence of bacterial translocation, caecal bacterial populations, and the ileal histology, noted 48 hours later. RESULTS--The death rate in the control group was 40% compared with 10% in the nucleoside-nucleotide mixture and 20% each in the individual components groups, respectively. Bacterial translocation to the mesenteric lymph node did occur in 100% of the surviving mice fed the control diet in comparison with 44% (nucleoside-nucleotide), 50% (cytidine), 75% (thymidine), 75% (uridine), 63% (inosine), and 63% (guanosine monophosphate). Histologically, the damage to the gut was more distinct in the protein free diet group. Villous height, crypt depth, and wall thickness in the nucleoside-nucleotide mixture group mean (SEM) (5.01 (0.34); 0.87 (0.14); 0.33 (0.10)), were respectively, higher compared with the protein free diet (3.34 (0.34); 0.61 (0.03); 0.18 (0.04)) group. In the cytidine group, crypt depth (0.86) (0.08)), and wall thickness (0.30 (0.002)) were higher. The same measurements in the components groups tended to be higher than the protein free diet group. Caecal bacterial populations were, however, similar in all groups. CONCLUSIONS--These results suggest that dietary nucleosides and nucleotides are essential nutrients for intestinal repair; nucleotides or cytidine provide a better response.

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.

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.

Activation of activator protein-1 in mouse brain regions exposed to simulated microgravity

SummaryMicrogravity induces stress, and the brain is one of the targets that is more influenced in this environment. Alteration in transcription factors can have enormous effect because of discrepancy in the signaling process of the cells. Activator protein-1 (AP-1) is a stress-regulated transcription factor and is involved in the regulation of physiological and pathological stimuli that include cytokines, growth factors, and stress signals. In the present study, an attempt has been made to observe the effect of a microgravity environment on the activation of AP-1 in the mouse brain. Our results show tha0105 AP-1 transcription factor is activated in simulated microgravity conditions in different regions of the brain. The activation of the AP-1 is dependent upon the increased kinase activity of c-Jun NH-terminal2 kinase-1. These results suggest tha0105 microgravity stress in the brain can elicit AP-1 activity.