Randomized clinical trial: rikkunshito in the treatment of functional dyspepsia--a multicenter, double-blind, randomized, placebo-controlled study.

BACKGROUND: Rikkunshito, a standardized Japanese herbal medicine, is thought to accelerate gastric emptying and relieve dyspepsia, although no large-scale, randomized, placebo-controlled trials of rikkunshito have been conducted. This study aimed to determine the efficacy and safety of rikkunshito for treating functional dyspepsia (FD). METHODS: FD patients received 2.5 g rikkunshito or placebo three times a day for 8 weeks in this multicenter, randomized, placebo-controlled, parallel-group trial. The primary end point was the proportion of responders at 8 weeks after starting test drug, determined by global patient assessment (GPA). The improvement in four major dyspepsia symptoms severity scale was also evaluated. In addition, plasma ghrelin levels were investigated before and after treatment. KEY RESULTS: Two hundred forty-seven patients were randomly assigned. In the eighth week, the rikkunshito group had more GPA responders (33.6%) than the placebo (23.8%), although this did not reach statistical significance (p = 0.09). Epigastric pain was significantly improved (p = 0.04) and postprandial fullness tended to improve (p = 0.06) in the rikkunshito group at week 8. Rikkunshito was relatively more effective among Helicobacter pylori-infected participants (rikkunshito: 40.0% vs placebo: 20.5%, p = 0.07), and seemed less effective among H. pylori-uninfected participants (rikkunshito: 29.3% vs placebo: 25.6%, p = 0.72). Among H. pylori-positive individuals, acyl ghrelin levels were improved just in rikkunshito group. There were no severe adverse events in both groups. CONCLUSIONS & INFERENCES: Administration of rikkunshito for 8 weeks reduced dyspepsia, particularly symptoms of epigastric pain and postprandial fullness. (UMIN Clinical Trials Registry, Number UMIN000003954).

Prophylactic strategies in recurrent vulvovaginal candidiasis: a 2-year study testing a phytonutrient vs itraconazole.

The aim of the present study was to assess the clinical efficacy of a one week/month treatment with a phytocompound with antimycotic properties (K-712, with following 100 mg composition: 10 mg of oleoresin from Pseudowintera colorata at 30 percent concentration in Polygodial together with trace amounts of Olea europea) in recurrent vulvo-vaginal candidiasis (RVVC), as compared to once a week treatment with an azole drug for 24 months follow up. This prospective randomized study involving 122 women (19 to 63 years old) with a history of proven episodes of RVVC in the prior 12 months. Patients were allocated in two treatment groups of 61 patients each and given A) Itraconazole 200 mg orally once a week or B) 1 tab twice a day of K-712 for one week/month. Each treatment schedule was well tolerated with 19 patients in the azole group complaining of transient mild symptoms (nausea, abdominal discomfort, unpleasant taste), while only 3 patients on K-712 reported slight dyspepsia. The number of relapses was significantly lower in the K-712-treated group as compared to the itraconazole-group (22 vs 39, p less than 0.05). Moreover, the former group showed a significantly decreased number of cases resistant or dose-dependent susceptible as compared to group A (p less than 0.05 vs itraconazole) and the same occurred for the occurrence of non-albicans species (group A 64.1 percent vs group B 31.8 percent, p less than 0.05). The overall mycological cure at the end of the 2-year study showed a comparable benefit between the two groups. From these data it appears that the present antifungal phytonutrient is equally effective as itraconazole in the overall treatment of RVVC over a 2-year follow-up, but yielding a significantly better prophylactic effect and also maintenance benefit with lower relapse rate, antifungal susceptibility and growth of azole-resistant species.

Is there a potential application of a fermented nutraceutical in acute respiratory illnesses? An in-vivo placebo-controlled, cross-over clinical study in different age groups of healthy subjects.

The role of oxidants in viral diseases is fairly complex because it includes metabolic regulation both of host metabolism and viral replication. However, a role for reactive oxygen species (ROS) and reactive nitrogen species (RNS) as mediators of virus-induced lung damage is supported by studies and antioxidants can thus be expected to act at many different levels. The aim of the present pilot study was to test an antioxidant nutraceutical approach on some relevant immunological parameters known to be affected in common seasonal respiratory tract infection. The study population consisted of 90 sedentary healthy patients, previously selected as being GSTM1-positive, divided into three groups: A) 20-40 years; B) 41-65 years; B) over 65 years. Each patients was administered a life style and dietary questionnaire. Subjects were supplemented for 6 weeks with either 9g/day (4.5g twice a day sublingually) of a fermented papaya preparation (Osato Research Institute, Gifu, Japan) or placebo. After a further month period of wash out, subjects were treated again in a crossover manner. Parameters checked were as follows: routine blood tests with WBC formula, saliva flow rate and secretary IgA and lysozyme production and redox gene expression of Phase II enzyme and SOD from upper airways cells (from nasal lavage). Salivary secretion rate showed an age-related decline and was significantly increased by FPP supplementation only in the youngest age-group (p less than 0.05). Subjects treated with FPP showed a significantly higher lever of IgA and lisozyme production., irrespective of age group while their baseline production was significantly lower in the oldest age-group as compared to the youngest one (C vs A, p less than 0.05). FPP treatment brought about a significant upregulation of all phase II enzyme and SOD gene expression tested in nasal lavage cells. In conclusion, FPP supplementation during 1 month resulted in higher salivary IgA and increase in phase II and SOD enzyme expression, i.e the most important antioxidant in the respiratory tract. The biological significance of these effects i.e., whether it will help reducing the whole respiratory oxidative stress in the human airway and, hopefully, the incidence and/or severity of URTI remains to be demonstrated in longer clinical trials.

Redox balance signalling in occupational stress: modification by nutraceutical intervention.

There is increasing evidence that psychosocial stress can be viewed as a system-wide derangement of cellular homeostasis, with heightened oxidative stress and triggered proinflammatory mechanisms. The aim of this study is twofold: a) to replicate findings that psychological stress increases oxidative damage and b) to determine whether a fermented papaya preparation known to exert significant protective antioxidant properties could buffer such increases in nuclear DNA damage while also inducing epigenetic protective mechanisms. Twenty-eight sedentary men and women (age range: 28-52), who reported living a stressful lifestyle but with an overall positive attitude, were recruited for this study. Chronic diseases as well as severe burnout and use of drugs for anxiety constituted exclusion criteria. Subjects were supplemented for 1 month with 9 g/day (4.5 g twice a day) of a certified fermented papaya preparation. All subjects were given a stress and sleep quality questionnaire together with a diet and life style assessment. Blood was collected at 2 and 4 week, erythrocyte and leukocyte were separated to assess redox balance and heme oxygenase-1 (HO-1) gene expression while bilirubin oxidized metabolites (BOMs) were tested in the urine. Stressed individuals showed a significant abnormality of redox status with increased MDA of erythrocyte and increased level of 8-0HdG in leukocyte and BOMs excretion (p<0.05). Nutraceutical supplementation brought about a normalization of such values already at the 2 week observation (p<0.05) together with a significant upregulation of HO-1 (p<0.01). Taken together, the results of this study confirm that stressful occupational life per se, without any overt psychiatric illness, may be associated to increased oxidative stress. Supplementation with functional food affecting redox regulation may be part of the therapeutic armamentarium to be considered in this clinical setting.

Protective effect of an oral natural phytonutrient in recurrent vulvovaginal candidiasis: a 12-month study.

The aim of the present study is to assess the clinical efficacy of a phytocompound with antimicotic properties (K-712, with the following 100 mg composition: 10 mg of oleoresin from Pseudowintera colorata at 30 percent concentration in Polygodial together with trace amounts of Olea europea) in recurrent vulvo-vaginal candidiasis (RVVC) as compared to an azole drug during a 12-month period: 6 months of treatment followed by 6 months of observation. This prospective randomized study involved 82 women (19-61 years) with complaints of abnormal vaginal discharge and with a history of at least four proven episodes of RVVC in the previous 12 months. Patients were divided into two groups of treatment of 41 patients each and were given: A) Itraconazole 200 mg orally daily for 4 days, then 200 mg once weekly for 6 months or B) 1 tablet twice a day of a K-712 for 4 weeks and then for the first 2 weeks of each month for a total of 6 months. Both groups were then followed-up for further 6 months. Each treatment schedule was well tolerated with only 4 patients in the azole group complaining of transient mild symptoms (nausea, abdominal discomfort, unpleasant taste). Itraconazole reached an earlier symptomatic relief during the first two weeks of observation as compared with K-712 (p<0.05) but both treatments enabled a comparable benefit during the entire treatment study period, afterwards with comparable symptom/sign score (itraconazole vs K-712: 9 vs 11). At 6-month observation, mycological cure was reached by 83 percent in the itraconazole group and in 78 percent of the K-712-treated patients. During the further 6-month observation period without treatment, the itraconazole group showed significantly more relapses (65.7 vs 34.2 in K-712, p<0.05) and at the end of the whole 12-month study period the mycological cure was significantly higher in the K-712-treated patients (65.8 vs 34.3 percent, p<0.05). There was a non- significant trend increase of less drug-susceptible species in the itraconazole group. From these preliminary data it would appear that a natural antifungal phytocompound proves to be as good as itraconazole in the maintenance treatment of RVVC. Moreover, this approach seems to maintain a higher mycological success rate afterwards by reducing the number of relapses and probably of the growth of azole-resistant species.

Subclinical candiduria in patients with gastrointestinal malignancies: a preliminary study on the protective effect of a natural phytocompound.

There is a great concern for the increasing incidence of candidiasis in cancer patients following immune-suppressive, cytostatic or antibiotic treatment. There are cancer patients with repeat asymptomatic recovery of candida in the urine in whom the choice of treatment, if any, is still a matter of debate. The aim of the study is to test the efficacy and tolerability of a natural anti-fungal phytocompound in patients with tumors of the gastrointestinal tract with prior or ongoing candiduria. Thirty-nine patients with operated gastrointestinal malignancies (18 still under current chemotherapy) with a history of repeated candiduria were enrolled. Eleven patients showed candiduria on enrolment and were treated with K-712, a natural antifungal phytocompound. Genomic analysis was carried out on blood samples of all patients on a monthly basis for 6 months. Within 3 weeks all 11 treated patients had negative cultures in the urine (10 patients after 2 weeks), 7 patients remained free of candiduria throughout the study period while 4 required a new treatment course. Three patients had positive genomic tests for systemic candidiasis and were treated with fluconazole. Eighteen (64 percent) out of the 28 patients who were free of candiduria on enrolment, developed a urinary candida infection during the 6-month follow-up and all cases were successfully treated with K-712. Seven (38 percent) of these cases presented a further recurrence at a later stage and all responded to a new course of K-172. No positive genomic tests were observed during the follow-up period. These data suggest that a consistent part of patients, mostly with gastrointestinal malignancies develop urinary candida infection when following chemotherapy treatment. A therapeutic approach with a natural antifungal phytocompound seems a safe and effective measure and a tentative prophylactic approach might also be envisaged.

Effect of an isoflavones-containing red clover preparation and alkaline supplementation on bone metabolism in ovariectomized rats.

The aim of this study was to test the combined effect of a quality-controlled red clover extract (RCE) standardized to contain 40% isoflavones by weight (genistein, daidzein, biochanin A, and formononetin present as hydrolyzed aglycones) together with a modified alkaline supplementation on bone metabolic and biomechanical parameters in an experimental model of surgically-induced menopause. Sprague-Dawley female rats were maintained under controlled standard conditions of light and fed with conventional food of standard calcium content and no alfalfa or soybean components. Rats were randomized into four groups: Group A represented normal rats (sham operated) while three other groups were ovariectomized (OVX) and fed for three months as follows: standard food (group B), 6 mg/kg/day food mixed with RCE (Group C), or given 6 mg/kg/day of RCE plus a modified alkaline supplementation (BP) through a nasogastric tube at a dose of 16 mg (group D). The animals were killed 90 days after surgery. As compared to group B, RCE or RCE + BP treatments brought about significantly higher level of estradiol and mitigated the weight loss of the uterus and improved maximum load of the femoral neck. Osteocalcin level showed an over 65% increase in group B but both RCE and RCE + BP treatments prevented such abnormality with a significantly better result in RCE + BP group which virtually normalized such parameter as well as urinary excretion of DPD. Group C and D reduced the over 20% loss of bone mineral density and bone mineral content/body weight ratio observed in untreated post-ovariectomy group. Untreated ovariectomy caused about 48% decrease of cancellous bone mass in the femoral neck while this abnormality was prevented at similar extent by both RCE and RCE + BP treatments. Ovariectomy determined an over 80% increase of bone alkaline phosphatase (BALP) level but both RCE and RCE + BP treatments significantly mitigated such variable. The BALP decrease yielded by the combined RCE + BP treatment was statistically lower than RCE alone. Taken together these data show that red clover preparation in dosages amenable to clinical practice do improve OVX-induced osteoporosis while a mild metabolic alkalosis might further synergize some therapeutic aspects.

Nutraceutical strategy in aging: targeting heat shock protein and inflammatory profile through understanding interleukin-6 polymorphism.

The aging process is paralleled by two- to fourfold increases in plasma/serum levels of inflammatory mediators, such as cytokines and acute-phase proteins. In this study we assessed the inflammatory profile and polymorphism of healthy elderly subjects and the influence of a nutraceutical supplement. Forty elderly, generally healthy subjects were recruited, divided into two matched groups, and given either a fermented papaya preparation 9 g/day by mouth or the same amount of placebo. Treatments were carried out in a cross-over manner with a 3-month supplementation period followed by a 6-week washout period between treatments. Ten healthy young subjects served as controls. Interleukin-6 (IL-6) promoter -174 G/C polymorphism genotype was determined together with blood levels for redox status, proinflammatory cytokines, high sensitivity C-reactive protein, and serum 70 kDa heat shock protein (Hsp70) concentrations. Tumor necrosis factor-alpha and IL-6 were higher in elderly subjects (P < 0.05 versus young controls). The concentration of Hsp70 inversely correlated with markers of inflammation in -174 G/C-negative subjects (r = 0.62, P < 0.05). Nutraceutical intervention normalized the inflammatory parameters (P < 0.05) with a rise of Hsp70 (P < 0.05). This suggests that healthy elderly individuals may have a proinflammatory profile playing as a downregulating factor for inducible Hsp70, particularly if -174 G/C-negative. A nutraceutical intervention seems able to beneficially modulate such a phenomenon.

Hyperthermia ameliorates 2,4,6-trinitrobenzene sulphonic acid-induced colitis in rats: the role of heat shock proteins.

PURPOSE: Hyperthermia is known to protect against cellular injury through the expression of heat shock proteins. In this study, the therapeutic effects of hyperthermia on experimental colitis in the rat were evaluated. MATERIALS AND METHODS: Male Wistar rats were given a single intracolonic injection of 2,4,6-trinitrobenzene sulphonic acid (TNBS). Hyperthermia was induced in anesthetized rats by placing them in a temperature-controlled water bath. We started the hyperthermic treatment on the day after the enema. The severity of colitis was evaluated pathologically, and the activities of tissue myeloperoxidase were measured 6 days after the induction of colitis. Furthermore, cytokines, and hyperthermia-induced heat shock proteins in colonic mucosa were detected by enzyme-linked immunosorbent assay and Western blotting. We also investigated the effects of geranylgeranylacetone and zinc protoporphyrin IX on the therapeutic effect of hyperthermia. RESULTS: Hyperthermia significantly improved the macroscopic scores of colitis. The TNBS-induced increases in the activities of myeloperoxidase in the colonic tissue were blunted significantly in hyperthermia-treated animals. Furthermore, hyperthermia attenuated increases in cytokine-induced neutrophil chemoattractants-1 and tumor necrosis factor-alpha in the colon. Furthermore, hyperthermia induced the production of heat shock proteins in rat colonic mucosa, and the combination of geranylgeranylacetone with hyperthermia further induced the heat shock protein HSP70, which resulted in further improvement of TNBS-induced colitis. On the other hand, the combination of zinc protoporphyrin IX with hyperthermia attenuated the therapeutic effect of hyperthermia. CONCLUSIONS: Hyperthermia ameliorates TNBS-induced colitis in rats through the expression of HSP70 and HO-1. It is postulated that hyperthermia may be useful for the treatment of inflammatory bowel diseases.

A recovery from enhancement of activation in auditory cortex of patients with idiopathic sudden sensorineural hearing loss.

OBJECTIVE: We previously reported enhanced activation of auditory cortex in patients with bilateral chronic inner-ear hearing loss. To determine whether this enhancement can exhibit a short-term alteration, we measured auditory evoked magnetic fields (AEFs) in patients with idiopathic sudden sensorineural hearing loss (ISSHL) in the acute phase (AP) and recovery phases (RPs). METHODS: We recorded AEFs in two unilateral ISSHL patients at three time points (AP, RP1, and RP2) using a whole-head neuromagnetometer. Tone bursts of 1 kHz were presented monaurally to the affected and healthy ear at four different intensities (40-70 dB HL). RESULTS: Both patients showed the enhancement of N100 m moment at AP and not at RPs in response to the affected ear stimulation, and stronger N100 m moment in ipsilateral than contralateral hemisphere in response to the healthy ear stimulation at AP. CONCLUSIONS: Enhancement of N100 m amplitude occurs in ISSHL patients and disappears on the scale of days. Enhancement of activity in the auditory cortex derived from inner-ear hearing loss can thus exhibit short-term change. SIGNIFICANCE: The results of this study provide first evidence for a recovery from enhancement of activation in the auditory cortex following injury of peripheral hearing organ.

The expression of genes for transepithelial calcium-transporting proteins in the bovine duodenum.

Intestinal epithelial cells contain calcium-binding proteins and Ca2+-transporting adenosine triphosphatase (Ca2+-ATPase), which play important roles in intestinal Ca transport. However, the factors that affect the expression of these transepithelial Ca-transporting proteins in dairy cattle are unknown. In this study, a semi-quantitative reverse transcription polymerase chain reaction was used to determine the expression of the mRNAs for intestinal Ca-binding protein calbindin-D9k (CaBP9k), two isoforms of plasma membrane Ca2+-ATPase (PMCA1 and PMCA4), and vitamin D receptor (VDR) in duodenal tissue samples from 20 non-lactating, non-pregnant Holstein dairy cattle (0.4-135.9 months old). The correlations between the expressions of transepithelial Ca-transporting proteins, the ages of the cattle, and the presence of several plasma components were evaluated. The duodenal CaBP9k mRNA content had a significant negative correlation with age and positive correlations with plasma inorganic phosphorus (iP) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) concentrations. The PMCA1 mRNA content was negatively correlated with the plasma Ca concentration. The duodenal PMCA4 mRNA content was correlated negatively with the plasma iP. The VDR mRNA content had a positive correlation with the plasma magnesium concentration.

Heme oxygenase-1 (Hsp32) is involved in the protection of small intestine by whole body mild hyperthermia from ischemia/reperfusion injury in rat.

AIM: The aim of the present study was to explore whether heme oxygenase-1 (HO-1) is involved in the hyperthermia-provided protection of the small intestine from ischemia/reperfusion injury in rats. METHODS: Intestinal damage was induced in male Sprague-Dawley rats by clamping both the superior mesenteric artery and the celiac trunk for 30 min, followed by reperfusion. Whole-body hyperthermia was induced in anesthetized rats by placement in a temperature-controlled water bath. Whole-body hyperthermia to a core temperature of 42-43 degrees C for 15 min was followed by passive cooling. We started the hyperthermic treatment 6 h before the vascular clamping. The severity of the mucosal injury was evaluated by several biochemical markers and histological findings. Hyperthermia-induced heat-shock proteins were detected by Western blotting. We also investigated the effect of zinc protoporphyrin IX (an HO-1 inhibitor) on the protective effect of hyperthermia. RESULTS: The rats, which were killed after ischemia/reperfusion, had severe intestinal inflammation. Hyperthermia significantly induced the production of Hsp70 and HO-1 in intestinal mucosa and significantly reduced ischemia/reperfusion-induced mucosal injury. The combination of zinc protoporphyrin IX with hyperthermia extinguished the protective effects of hyperthermia on ischemia/reperfusion injury. CONCLUSION: Hyperthermia protects against ischemia/reperfusion injury in rat small intestine through the expression of heat-shock proteins, especially HO-1.

Preliminary study of autologous blood predonation in pediatric open-heart surgery impact of advance infusion of recombinant human erythropoietin.

We applied an autologous blood predonation protocol using recombinant human erythropoietin in pediatric open-heart surgery. The study included 69 children weighing 8 kg or more. Twice before operation, 8 ml/kg of blood was taken. At each donation, 100 (group 1; n = 20), 200 (group 2; n = 11), or 300 (group 3; n = 13) units/kg of erythropoietin was given subcutaneously. In group 4 (n = 25), 300 units/kg of erythropoietin was given 1 week prior to the first donation, followed by 300 units/kg given at each donation. No harmful events occurred during the donation period. During the donation period, the patients' hematocrit decreased but the hematocrit 1 day after the operation remained at 32.1 +/- 0.6% and was 33.3 +/- 0.6% 2 weeks later. The decrease in hematocrit was minimal in group 4 (39.0 +/- 0.6% before donation to 37.5 +/- 0.5% before operation) compared with that of the other three groups. Among those who completed the protocol, 58 patients (93.5%) were discharged without homologous blood transfusion. We consider our protocol of autologous blood predonation using erythropoietin to be safe and effective for avoiding homologous blood transfusion in pediatric patients. The early infusion of erythropoietin prior to the first donation minimizes the decrease in hematocrit level before operation.

Enhanced activation of the auditory cortex in patients with inner-ear hearing impairment: a magnetoencephalographic study.

OBJECTIVE: Injury of peripheral auditory organ often induces abnormality of loudness sensation such as loudness recruitment. However, objective evaluation of this phenomenon has rarely been performed. To elucidate this abnormal loudness sensation, cortical mechanisms were investigated by recording auditory evoked magnetic fields (AEFs). METHODS: We recorded AEFs in 8 patients suffering from inner-ear hearing impairment with loudness recruitment and in 14 healthy hearing controls using a 122-channel whole-head neuromagnetometer. Tone bursts of 1 kHz were presented monaurally at 4 different intensities (40, 50, 60, 70 dB HL) with a constant interstimulus interval of 1 s. RESULTS: In both groups, the 100 ms response (N100m) increased in amplitude and decreased in latency as a function of stimulus intensity in both hemispheres. Concerning the source strength, increment of dipole moment of N100m was more rapid according to the stimulus intensity in patients compared with that in healthy subjects. Source strength of N100m was enhanced at high stimulus intensity in patients, and its ratio to healthy subjects was 1.08 at 50 dB, 1.69 at 60 dB and 2.04 at 70 dB. CONCLUSIONS: In patients with inner-ear hearing impairment, enhanced activation of the auditory cortex was observed, and may help explain loudness recruitment.

Ubiquitin-proteasome inhibitor enhances tumour necrosis factor-alpha-induced apoptosis in rat gastric epithelial cells.

BACKGROUND: Tumour necrosis factor (TNF-alpha) is a candidate factor for involvement in inflammation-mediated gastric mucosal injury. However, the effect of this cytokine on gastric epithelial cells has been poorly investigated. In the present study, we examined whether gastric epithelial cells are resistant to TNF-alpha-induced apoptosis, and whether this resistance is related to ubiquitin-proteasome-associated nuclear factor-kappaB (NF-kappaB) activation. METHODS: The rat gastric mucosal cell line RGM-1 was grown in DMEM/F12 medium supplemented with 10% FCS. Confluent monolayers of cells were pretreated or not for 60 min with PSI, a peptide aldehyde known to specifically inhibit the chymotrypsin-like activity of 26S proteasome. Cells were subsequently stimulated with recombinant rat TNF-alpha and their viability was determined by WST-1 assay. Apoptosis was confirmed by fluorescence microscopy after staining with Hoechst 33342 and propidium iodide, and DNA fragmentation was determined by flow cytometry using an APO-BRDU kit. IkappaB-alpha and the p65 binding subunit of NF-kappaB were detected by Western blots. RESULTS: Twenty-four-hour incubation with TNF-alpha alone or PSI alone did not affect the cell viability of RGM-1 cells. Pretreatment with PSI significantly enhanced the level of apoptosis induced by TNF-alpha. In RGM-1 cells treated with TNF-alpha, cytoplasmic IkappaB-alpha decreased and p65 in nuclear extracts increased markedly 30 min after cytokine stimulation. Pretreatment with PSI at 12.5 micromol/L blocked these TNF-alpha-induced changes. CONCLUSION: PSI enhances TNF-alpha-induced apoptosis through inhibition of NF-kappaB activation in RGM-1 cells.

Differing effects of two nitric oxide synthase inhibitors on experimental colitis.

BACKGROUND/AIMS: The aim of this study was to investigate the effects of two inhibitors of nitric oxide synthase on a rat model of colitis. METHODOLOGY: Colitis was induced by administration of an enema containing trinitrobenzene sulfonic acid. This colitis was treated everyday for one week with NG-nitro-L-arginine (10 mg/kg, i.v.), which is a non-selective inhibitor of both constitutive nitric oxide synthase and inducible nitric oxide synthase, or aminoguanidine (10 mg/kg, i.v.) which is an inhibitor of inducible nitric oxide synthase. Exposure to the trinitrobenzene sulfonic acid enema inhibited the increase in body weight of rats, and markedly increased the colonic damage scores, wet weight, thiobarbituric acid-reactive substances and myeloperoxidase activity. RESULTS: The inhibition of weight increase caused by trinitrobenzene sulfonic acid was significantly reduced by aminoguanidine treatment, whereas weight loss tended to be aggravated by NG-nitro-L-arginine treatment. The increases in the colonic damage scores, wet weight, thiobarbituric acid-reactive substances and myeloperoxidase activity in trinitrobenzene sulfonic acid-colitis were significantly inhibited by aminoguanidine treatment, although they tended to be aggravated by NG-nitro-L-arginine treatment. CONCLUSIONS: These results suggest that an inhibitor of inducible nitric oxide synthase, but not of constitutive nitric oxide synthase, was effective in treating experimental colitis in rats.

Role of P-selectin and intercellular adhesion molecule-1 in TNB-induced colitis in rats.

It has been proposed that neutrophil-endothelial cell interactions mediated by adhesion molecules are involved in the pathogenesis of inflammatory bowel disease. The objective of the present study was to determine the effects of monoclonal antibodies (MAbs) directed against endothelial adhesion molecules, P-selectin and intercellular adhesion molecule-1 (ICAM-1), in rats with colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNB). Colonic inflammation was induced by administering an enema of TNB dissolved in 50% ethanol (120 mg/ml) to male Wistar rats (at a total volume of 0.25 ml per rat) after a 48-hour fast. Anti-P-selectin MAb or anti-ICAM-1 MAb was injected via the tail vein at a dose of 1 mg/kg after the induction of colitis. Rats in the control group received nonbinding mouse immunoglobulin G1. The plasma level of soluble P-selectin showed an increase within 48 h after the TNB enema. Colonic inflammation was assessed at 1 week after TNB administration. The colonic damage score and the wet weight of the colon were significantly decreased by treatment with either MAb. The increase of myeloperoxidase (MPO) activity, an index of neutrophil accumulation, and the increase of thiobarbituric acid-reactive substances (TBA-RS), an index of lipid peroxidation, in the colonic mucosa were inhibited by both MAbs. These results suggest that neutrophil-endothelial cell interactions via P-selectin and ICAM-1 play an important role in the development of TNB-induced colitis in rats.

The involvement of a cysteine proteinase in the nodule development in Chinese milk vetch infected with Mesorhizobium huakuii subsp. rengei.

Cys proteinases play important roles in plant cell development and senescence. A cDNA, AsNODf32, obtained by differential screening of a nodule cDNA library of the leguminous plant Chinese milk vetch (Astragalus sinicus), represents a nodule-specific Cys proteinase similar to that reported for the actinorhizal Alnus glutinosa-Flankia symbiosis. A characteristic feature of this proteinase is the presence of a putative vacuolar targetting signal, LQDA, within its propeptide. Expression of the AsNODf32 gene, which was studied on northern blots and in situ hybridization, showed good correlation with the onset of nodule senescence. In situ hybridization studies revealed that AsNODf32 was expressed in senescent-infected tissue at the base of the nodule, as well as in interzone II-III of the infected nodules. In addition to degrading old nodule tissues and bacteroids, AsNODf32 protein may be required as a component of tissue remodeling during nodule development.

Increase in blood pressure with enhanced Na+, K+ -ATPase activity in stroke-prone spontaneously hypertensive rats after 4-weeks intake of rapeseed oil as the sole dietary fat.

Twenty stroke-prone spontaneously hypertensive rats were divided into 2 groups of 10 animals each, and fed a defatted diet and orally administered rapeseed (canola) oil or soybean oil at 10 (w/w)% of the consumed diet once a day for 4 weeks. At the 4th week of administration, the systolic blood pressure in the canola oil group was higher (235 +/- 2 mmHg, mean +/- S.E.M., N=10) than that in the soybean oil group (225 +/- 4 mmHg, N=10, P<0.05). In isolated, perfused mesenteric bed from these rats, the increase in perfusion pressure by norepinephrine, ATP, arachidonic acid, endothelin-1, angiotensin II or serotonin showed no between-group differences. There were also no between-group differences in the production of thromboxane A2 and prostaglandin 12 in the outflow by arachidonic acid injection. On the other hand, in the isolated aortic ring from the canola oil group, developed tension in potassium-free solution was enhanced with activation of Na+, K+ -ATPase. These results suggest that canola oil intake as the sole dietary fat increases systolic blood pressure of stroke-prone spontaneously hypertensive rats. The changes in vascular responsiveness to vasoconstrictors and production of prostanoids are unlikely to have relevance to the elevation of blood pressure. However, altered Na+, K+ -ATPase activity may play a role in the promotion of blood pressure elevation.

Blood coagulation and osmolar tolerance of erythrocytes in stroke-prone spontaneously hypertensive rats given rapeseed oil or soybean oil as the only dietary fat.

The life-span of stroke-prone spontaneously hypertensive rats (SHRSP) has been reported to become shorter by ingestion of some vegetable oils, including rapeseed oil, when given as the sole dietary fat. The present study was undertaken to examine if the ingestion of rapeseed (canola) oil affects blood coagulating time and erythrocyte membranes. Namely, SHRSP were orally given canola oil or soybean oil as the only dietary fat (10% of diet) for 4 weeks. After the 4-week feeding, activated partial thromboplastin time (APTT) in the canola oil group (19.9+/-0.5 s, N=8) was significantly shorter than that in the soybean oil group (21.6+/-0.6 s, N=8, P<0. 05), though there were no between-group differences in plasma Ca(2+), platelet density and platelet aggregation. Erythrocytes from the canola oil group were less tolerant to low osmotic pressure than those from soybean oil group; the EC(50) values for NaCl concentration to cause hemolysis were 0.42+/-0.004 and 0.40+/-0.005% in the canola oil and the soybean oil groups, respectively (N=10, P<0.01). The canola oil-induced shortening of blood coagulation time and increased fragility in erythrocyte membranes may have relevance to the promotion of strokes in SHRSP.