Improvement of the mismatch amplification mutation assay-PCR for discrimination between Streptococcus suis serotypes 2 and 1/2.

A mismatch amplification mutation assay (MAMA)-PCR, which detects a single-nucleotide polymorphism contributed to serological difference between Streptococcus suis serotypes 2 and 1/2, is used to discriminate between these serotypes. The present study reports unusual serotype 1/2 isolates untypable by the MAMA-PCR and improvement of the MAMA-PCR for typing such isolates.

The pharyngeal nervous system orchestrates feeding behavior in planarians.

Planarians exhibit traits of cephalization but are unique among bilaterians in that they ingest food by means of goal-directed movements of a trunk-positioned pharynx, following protrusion of the pharynx out of the body, raising the question of how planarians control such a complex set of body movements for achieving robust feeding. Here, we use the freshwater planarian Dugesia japonica to show that an isolated pharynx amputated from the planarian body self-directedly executes its entire sequence of feeding functions: food sensing, approach, decisions about ingestion, and intake. Gene-specific silencing experiments by RNA interference demonstrated that the pharyngeal nervous system (PhNS) is required not only for feeding functions of the pharynx itself but also for food-localization movements of individual animals, presumably via communication with the brain. These findings reveal an unexpected central role of the PhNS in the linkage between unique morphological phenotypes and feeding behavior in planarians.

Usefulness of multiple chalk-based food colorings for inducing better gene silencing by feeding RNA interference in planarians.

Planarians have become widely recognized as one of the major animal models for regeneration studies in invertebrates. To induce RNA interference (RNAi) by feeding in planarians, the widely accepted protocol is one in which animals undergo two or three feedings of food containing double-stranded RNA (dsRNA) plus visible food coloring (e.g., blood) for confirmation of feeding by individual animals. However, one possible problem is that incorporated food coloring is often retained within the gut for several days, which makes it difficult to confirm the success of each round of dsRNA feeding based on the difference of the color density within the gut before and after feeding. As a consequence, the difference of appetite levels among individuals undergoing dsRNA feeding leads to phenotypic variability among them due to insufficient knockdown. In our attempts to overcome this problem, we have developed a novel method for achieving robust confirmation of the success of dsRNA feeding in individuals fed multiple times by means of including a combination of three different colored chalks (pink, yellow and blue) as food coloring. Notably, we found that this method is superior to the conventional method for positively marking individuals that actively consumed the dsRNA-containing food during four times of once-daily feeding. Using these selected animals, we obtained stable and sufficiently strong RNAi-induced phenotypes. We termed this improved multi-colored chalk-spiked method of feeding RNAi "Candi" and propose its benefits for gene function analysis in planarians.

Successful endoscopic fragmentation of large hardened fecaloma using jumbo forceps.

We present a rare case of fecaloma, 7 cm in size, in the setting of systemic scleroderma. A colonoscopy revealed a giant brown fecaloma occupying the lumen of the colon and a colonic ulcer that was caused by the fecaloma. The surface of the fecaloma was hard, large and slippery, and fragmentation was not possible despite the use of various devices, including standard biopsy forceps, an injection needle, and a snare. However, jumbo forceps were able to shave the surface of the fecaloma and break it successfully by repeated biting for 6 h over 2 d. The ability of the jumbo forceps to collect large mucosal samples was also appropriate for achieving fragmentation of the giant fecaloma.

Therapy with granulocyte colony-stimulating factor in the chronic stage, but not in the acute stage, improves experimental autoimmune myocarditis in rats via nitric oxide.

We systematically investigated serial efficacy of granulocyte colony-stimulating factor (G-CSF) therapy upon experimental autoimmune myocarditis (EAM) in rats treated with and without the inhibition of nitric oxide (NO) with the analyses of tissue regeneration. G-CSF could mobilize multipotent progenitor cells of bone marrow into the peripheral blood and may improve ventricular function. A rat model of porcine myosin-induced EAM was used. After the immunization of myosin, G-CSF (10 microg/kg/day) or saline was injected intraperitoneally on days 0-21 in experiment 1 and on days 21-42 in experiment 2. Additional myosin-immunized rats were orally given 25 mg/kg/day of N(G)-nitro-L-arginine methylester (L-NAME), an inhibitor of nitric oxide synthase (NOS), in each experiment (each group; n=8-21). Serum cytokines and peripheral blood cell counts were measured in each group. In experiment 1, G-CSF treatment aggravated cardiac pathology associated with increased macrophage inflammatory protein-2 (MIP-2) and interleukin-6 (IL-6) levels and enhanced superoxide production. In experiment 2, G-CSF treatment reduced the severity of myocarditis with increased capillary density and improved left ventricular ejection fraction. In the rats with EAM treated with G-CSF associated with oral L-NAME treatment in experiment 2, the severity of myocarditis was not reduced. Myocardial c-kit(+) cells were demonstrated only in G-CSF-treated group in experiment 2 but not in other groups. G-CSF has differential effects on EAM in rats associated with the modulation of cytokine network. The overwhelming superoxide production by G-CSF administration in the acute stage may worsen the disease. G-CSF therapy improved cardiac function via NO system in a rat model of myocarditis in the chronic stage, but not in the acute stage, possibly through the myocardial regeneration and acceleration of healing process.

Naloxone, an opiate receptor antagonist, ameliorates acute experimental autoimmune myocarditis by reducing cytotoxic activities.

Little is known about effects of naloxone, an opiate receptor antagonist, upon experimental autoimmune myocarditis (EAM) in mice. The aim of the present study was to test the effects of naloxone upon EAM in mice. Four-week-old C57BL/6 mice were injected with porcine cardiac myosin. Naloxone 1 mg/kg/day was given intraperitoneally daily on days 0 to 21 in experiment I (acute stage) and on days 21 to 42 in experiment II (chronic stage). For the analysis of endogenous opiate and neurohumoral system, plasma beta-endorphin and cytokines were measured. The cytotoxic activity of lymphocytes was determined by Cr-release assay. Naloxone reversed hypotension produced by massive myocardial injury in experiment I. In experiment I, the severity of cardiac pathology and inflammatory cytokines were decreased in the naloxone-treated group associated with higher beta-endorphin level compared with the untreated group. In addition, naloxone induced a shift from Th1 cytokine toward Th2 cytokine balance. Thus, cytotoxic activities of lymphocytes against EL-4 tumor cells were lower in the treated group than in the untreated group in experiment I, but not in experiment II. Naloxone is beneficial for heart failure caused by EAM in the acute stage, but not in the chronic stage, due to decreasing cytotoxic activities of lymphocytes and to its neurohumoral modulating effects.

L-arginine ameliorates experimental autoimmune myocarditis by maintaining extracellular matrix and reducing cytotoxic activity of lymphocytes.

It was previously shown that administration of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) aggravated murine viral myocarditis by increasing myocardial virus titres. Experimental autoimmune myocarditis in mice and rats mimics human fulminant myocarditis. The effects of L-arginine, a precursor of nitric oxide, upon heart failure in experimental autoimmune myocarditis were evaluated. Dietary L-arginine (L-arginine group) and L-arginine plus N(G)-nitro-L-arginine methyl ester (L-arginine + l-NAME group) were administered to C57BL/6 mice immunized with porcine cardiac myosin over 3 weeks. An untreated myocarditis group was prepared. Cardiac damage was less in the L-arginine group compared with the other two groups, as was incidence of heart failure. In addition, extracellular matrix change was less prominent in the L-arginine group. Plasma concentrations of nitric oxide were elevated in the L-arginine group. Cytotoxic activities of lymphocytes were lower in L-arginine group than in other two groups. L-arginine treatment may be effective in preventing the development of heart failure in experimental myocarditis by maintaining extracellular matrix and reducing the cytotoxic activity of lymphocytes.

Exercise training reduces severity of atherosclerosis in apolipoprotein E knockout mice via nitric oxide.

BACKGROUND: Exercise training may protect against the development of atherosclerosis, although the precise mechanisms are still unknown. The present study assessed the hypothesis that exercise training would reduce the severity of experimental atherosclerosis in apolipoprotein-E (apoE)-deficient mice via nitric oxide (NO). METHODS AND RESULTS: ApoE-deficient mice fed a high-fat diet underwent exercise training (30 min swimming) 3 times per week for 8 weeks. The exercise group were also given oral N(G)-nitro-L-arginine methylester (L-NAME; 25 mg x kg (-1) x day(-1)), an inhibitor of NO synthase. Fatty streak plaque lesions developed in ApoE-deficient mice fed the high-fat diet, and were suppressed in the mice that underwent swimming training. In contrast, atherosclerotic lesions were not ameliorated in mice that had exercise training plus oral L-NAME treatment. Immunohistochemical analysis revealed that the expression of endothelial NO increased in mice undergoing exercise compared with the mice that did not exercise, and that the expression was suppressed in the mice having exercise plus oral L-NAME treatment. Differences in lesion area did not correlate with any significant alterations in serum lipid levels. CONCLUSION: Exercise training suppressed atherosclerosis via the NO system.

Swimming reduces the severity of atherosclerosis in apolipoprotein E deficient mice by antioxidant effects.

OBJECTIVE: It was shown that aerobic exercise training may protect against the development of atherosclerosis. However, the precise mechanisms are still unknown. We assessed the hypothesis that exercise training reduced the severity of experimental atherosclerosis in apolipoprotein (apo) E-deficient mice by antioxidant effects. METHODS: Exercise training (45 min swimming, 3 times/week) was conducted on apo E-deficient mice fed a high fat diet. Over 8 and 16 weeks on alternate days, mice were treated with and without exercise, and additional exercise-treated mice were orally given 25 mg/kg/day of NG-nitro-L-arginine methylester (L-NAME), an inhibitor of nitric oxide synthase (NOS). In addition, the effect of L-arginine against L-NAME was also tested. RESULTS: Fatty streak formation at 8 weeks and fibrofatty plaques at 16 weeks developed in apo E-deficient mice fed a high fat diet, and were suppressed in mice treated with swimming for 8 and 16 weeks. In contrast, atherosclerotic lesions were not ameliorated in mice treated with exercise training associated with oral L-NAME. However, in mice treated with swimming associated with L-NAME and L-arginine, the atherosclerotic lesions were reduced. Immunohistochemical analysis revealed that macrophage and CD4+ cell accumulation in the fatty streak lesions was suppressed in mice treated with exercise, but not in those treated with exercise associated with L-NAME administration. The severity of atherosclerotic lesions was inversely correlated with the endothelial NOS expression and the expression of an endogenous antioxidant protein, thioredoxin. Namely, the expression of thioredoxin in mice treated with exercise was suppressed compared with mice without exercise. Plasma thiobarbituric acid-reactive substance levels were significantly lower in groups with exercise than in those without exercise or with exercise associated with L-NAME administration, suggesting exercise-induced less lipid peroxidation. Differences in lesion area did not correlate with any significant alterations in serum lipid levels. The exercise load used in the current study did not affect energy metabolism efficacies in the hearts. CONCLUSION: Exercise training, in which the load did not affect energy metabolism efficacy of the heart, suppressed atherosclerosis by antioxidant effects via the vascular NO system.

MCI-186 (edaravone), a novel free radical scavenger, protects against acute autoimmune myocarditis in rats.

In this study, we tested the hypothesis that MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one; edaravone), a novel free radical scavenger, protects against acute experimental autoimmune myocarditis (EAM) in rats by the radical scavenging action associated with the suppression of cytotoxic myocardial injury. Recent evidence suggests that oxidative stress may play a role in myocarditis. We administered MCI-186 intraperitoneally at 1, 3, and 10 mg.kg(-1).day(-1) to rats with EAM for 3 wk. The results were compared with untreated rats with EAM. MCI-186 treatment did not affect hemodynamics. MCI-186 treatment (3 and 10 mg.kg(-1).day(-1)) reduced the severity of myocarditis as assessed by comparing the heart-to-body weight ratio and pathological scores. Myocardial interleukin-1beta (IL-1beta)-positive cells and myocardial oxidative stress overload with DNA damage in rats with EAM given MCI-186 treatment were significantly less compared with those of the untreated rats with EAM. In addition, MCI-186 treatment decreased not only the myocardial protein carbonyl contents but also the myocardial thiobarbituric acid reactive substance products in rats with EAM. The formation of hydroxyl radicals in MCI-186-treated heart homogenates was decreased compared with untreated heart homogenates. Furthermore, cytotoxic activities of lymphocytes of rats with EAM treated with MCI-186 were significantly lower compared with those of the untreated rats with EAM. Hydroxyl radicals may be involved in the development of myocarditis. MCI-186 protects against acute EAM in rats associated with scavenging hydroxyl free radicals, resulting in the suppression of autoimmune-mediated myocardial damage associated with reduced oxidative stress state.

Cardioprotective effects of 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), a novel free radical scavenger, on acute autoimmune myocarditis in rats.

BACKGROUND: Recent evidence suggests that oxidative stress may play a role in myocarditis. PURPOSE: To test the hypothesis that 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone or MCI-186), a novel free radical scavenger, protects against acute experimental autoimmune myocarditis (EAM) in rats. METHODS: MCI-186 was administered intraperitoneally (1 mg/kg/day, 3 mg/kg/day or 10 mg/kg/day) in rats with EAM for three weeks. The results were compared with those from untreated rats with EAM. RESULTS: MCI-186 treatment did not affect the hemodynamics of the rats, but did reduce the severity of myocarditis when the heart weight:body weight ratio and the pathological scores were compared. There were significantly fewer myocardial interleukin-1beta-positive cells in rats with EAM treated with MCI-186 (at 3 mg/kg/day and 10 mg/kg/day) than in the untreated rats with EAM. CONCLUSION: MCI-186 ameliorated acute EAM in rats, suggesting that free radicals may be involved in the development of myocarditis.