Nrf2 Regulates the Risk of a Diesel Exhaust Inhalation-Induced Immune Response during Bleomycin Lung Injury and Fibrosis in Mice.

The present study investigated the effects of diesel exhaust (DE) on an experimental model of bleomycin (BLM)-induced lung injury and fibrosis in mice. BLM was intravenously administered to both Nrf2+/+ and Nrf2-/- C57BL/6J mice on day 0. The mice were exposed to DE for 56 days from 28 days before the BLM injection to 28 days after the BLM injection. Inhalation of DE induced significant inhibition of airway clearance function and the proinflammatory cytokine secretion in macrophages, an increase in neutrophils, and severe lung inflammatory injury, which were greater in Nrf2-/- mice than in Nrf2+/+ mice. In contrast, inhalation of DE was observed to induce a greater increase of hydroxyproline content in the lung tissues and significantly higher pulmonary antioxidant enzyme mRNA expression in the Nrf2+/+ mice than in Nrf2-/- mice. DE is an important risk factor, and Nrf2 regulates the risk of a DE inhalation induced immune response during BLM lung injury and fibrosis in mice.

EM, EM703 inhibit NF-kB activation induced by oxidative stress from diesel exhaust particle in human bronchial epithelial cells: importance in IL-8 transcription.

Diesel exhaust particle (DEP) is the major components of PM2.5, and much attention has focused on PM2.5 in relation to adverse health effects, and many pulmonary diseases. In the present study, we used a human bronchial epithelial cell (HBEC) line to investigate the anti-inflammatory effects of erythromycin (EM) and EM703 - a new derivative of erythromycin without antibacterial effects on the expressions of IL-8 caused by DEP exposure. DEP showed a dose-dependent stimulatory effect on IL-8 product in HBEC. Increases of IL-8 expression by DEP stimulation were significantly blocked by both EM and EM703 pretreatment. Furthermore, NF-κB and Nrf2 activation, the antioxidant enzymes such as HO-1, NQO-1 mRNA expression were increased by DEP exposure and these increases were blocked by both of EM and EM703 pretreatment. Our results suggest that, EM and EM703 may have an inhibitory effect on expression inflammatory cytokines in HBEC induced by DEP not only as an anti-inflammation but also an antioxidant drug. EM and EM703 might contribute to chemical prevention of the risk of pulmonary diseases induced by oxidative stress from environmental pollutant, such as DEP.

The effect of sleep restriction and psychological load on the diurnal metabolic changes in tryptamine-related compounds in human urine.

OBJECTIVE: The effect of a severely stressful situation (sleep restriction and psychological load) on the diurnal changes in novel tryptamine-related compounds (hydroxydiacetyltryptamine, sulphatoxymelatonin, and dihydromelatonin) was evaluated in human subjects for 16 days. METHODS: The subjects were allowed to sleep for 5 h on days three through 12 and for 8 h on the other days. On days three through 12, the subjects were asked to perform a psychological task. The first two and the last 4 days were viewed as control days. A performance test was administered to evaluate the extent of the subjects' fatigue. Total urine was sampled by collecting it into bottles three times a day [(1) during the sleeping period, (2) in the morning, and (3) in the afternoon]. Seven tryptamine-related compounds in urine were assayed using HPLC-fluorometry. RESULTS: The urine melatonin level was high at night and low during the day. In contrast, urinary levels of hydroxydiacetyltryptamine and sulphatoxydiacetyltryptamine were low at night and high during the day. Dihydromelatonin was undetectable in urine during the sleeping period. Sleep restriction and psychological load did not affect diurnal changes in urinary melatonin, hydroxydiacetyltryptamine, sulphatoxydiacetyltryptamine, or N-acetylserotonin levels. The concentrations of hydroxymelatonin and sulphatoxymelatonin in urine did not show diurnal changes and decreased gradually during the experimental days. A principal component analysis confirmed the diurnal changes and suggested two novel metabolic pathways: (1) N-acetylserotonin to sulphtoxydiacetyltryptamine via hydroxydiacetyltryptamine, and (2) melatonin to dihydromelatonin. CONCLUSION: Severely stressful situations did not affect diurnal changes in melatonin, hydroxydiacetyltryptamine, sulphatoxydiacetyltryptamine, or N-acetylserotonin levels in urine.

Relationship among lifestyles, aging and psychological wellbeing using the General Health Questionnaire 12-items in Japanese working men.

INTRODUCTION. The relationship among lifestyle, aging and psychological wellbeing was evaluated in Japanese working men. METHODS. Self-administered questionnaire on six lifestyle factors and the General Health Questionnaire 12-item version (GHQ12) were administered to 3306 male workers. Health practice index (HPI) was calculated as a desirable lifestyle score by summing up each binary lifestyle score (0, 1), ranging from 0 to 6. To check validity of the study outcome, the authors repeated twice with 1 year interval. HPI was categorised into three groups by the score of 0-2, 3-4 and 5-6. RESULTS. The number of subjects categorised by HPI was 532, 1967 and 807, respectively. The mean value of GHQ12 significantly decreased as the HPI increased by adjusting age. Multiple regression analysis was conducted to predict GHQ12 by six lifestyle scores, and age, sleep, night snacking and exercise were significantly related to GHQ12. Multiple logistic regression analysis was conducted and age in 50s, two-shift work, sleep, night snacking and exercise were significantly associated with GHQ12. CONCLUSION. Although cause-effect relationship cannot make clear, some of desirable health practices and aging were closely related to psychological wellbeing judged by GHQ12.

Acute effects of walking in forest environments on cardiovascular and metabolic parameters.

We previously found that forest environments reduced stress hormones such as adrenaline and noradrenaline and showed the relaxing effect both in male and female subjects. In the present study, we investigated the effects of walking under forest environments on cardiovascular and metabolic parameters. Sixteen healthy male subjects (mean age 57.4 ± 11.6 years) were selected after obtaining informed consent. The subjects took day trips to a forest park in the suburbs of Tokyo and to an urban area of Tokyo as a control in September 2010. On both trips, they walked for 2 h in the morning and afternoon on a Sunday. Blood and urine were sampled on the morning before each trip and after each trip. Blood pressure was measured on the morning (0800) before each trip, at noon (1300), in the afternoon (1600) during each trip, and on the morning (0800) after each trip. The day trip to the forest park significantly reduced blood pressure and urinary noradrenaline and dopamine levels and significantly increased serum adiponectin and dehydroepiandrosterone sulfate (DHEA-S) levels. Walking exercise also reduced the levels of serum N-terminal pro-B-type natriuretic peptide (NT-proBNP) and urinary dopamine. Taken together, habitual walking in forest environments may lower blood pressure by reducing sympathetic nerve activity and have beneficial effects on blood adiponectin and DHEA-S levels, and habitual walking exercise may have beneficial effects on blood NT-proBNP levels.

Increase in the prevalence of metabolic syndrome among workers according to age.

INTRODUCTION: Statistical information regarding the prevalence of metabolic syndrome among a wide age range of workers is insufficient. METHODS: A total of 4278 men between the ages of 20 and 59 years participated in the study. Metabolic syndrome was diagnosed according to the International Diabetes Federation (IDF) and the National Cholesterol Education Program (NCEP) III criteria. RESULTS: Overall, the prevalences of metabolic syndrome according to the IDF and NCEPIII criteria were 13.6% and 14.8%, respectively. The prevalence of metabolic syndrome according to the IDF (NCEPIII) criteria among workers in their 20s, 30s, 40s, and 50s were 4.8% (6.1%), 9.9% (12.2%), 18.4% (21.6%) and 25.8% (34.0%), respectively. A plot of the prevalence of metabolic syndrome according to the NCEPIII criteria versus age had a steep gradient and increased sharply for men in their 50s. In contrast, a plot of the prevalence of metabolic syndrome according to the IDF criteria versus age increased in a linear manner. CONCLUSION: The prevalence of metabolic syndrome increased among workers according to age, but the increasing trend and the absolute prevalence of metabolic syndrome differed according to the two sets of diagnostic criteria used in this study.

A cross-sectional study on the shift work and metabolic syndrome in Japanese male workers.

INTRODUCTION: Shift work has been reported to be associated with an increase in the metabolic syndrome (MetS). To clarify the association between the type of shift work and the risk of MetS, a cross-sectional field survey was conducted after adjusting for age and lifestyle factors. METHODS: The subjects were 3007 Japanese males, aged 34-64 years old, who were employees (1700 day and 1307 shift workers) of a car-manufacturing company. The standard Japanese criteria for the diagnosis of MetS was used. Age, smoking habit, drinking habit, sleeping habit and exercise habit were used as the independent variables. RESULTS: The prevalence of MetS in the day workers, two-shift workers, and three-shift workers were 13.8% (234/1700), 10.7% (120/1125) and 17.6% (32/182), respectively. There was a significant difference in the prevalence between the two-shift workers and the day workers. Estimation of the odds ratios (95% confidence intervals) of age, two-shift work and habitual exercise for MetS were 1.03 (1.01-1.04), 0.77 (0.61-0.98) and 0.64 (0.51-0.81), respectively. CONCLUSION: Two-shift work was associated with lower risk of MetS, which is not in accordance with past reports. This finding should therefore be re-analysed, including investigation of the job content in each group.

Nrf2 Lowers the Risk of Lung Injury via Modulating the Airway Innate Immune Response Induced by Diesel Exhaust in Mice.

In the present study, we investigated the role of Nrf2 in airway immune responses induced by diesel exhaust (DE) inhalation in mice. C57BL/6J Nrf2+/+ and Nrf2-/- mice were exposed to DE or clean air for 8 h/day and 6 days/week for 4 weeks. After DE exposure, the number of neutrophils and macrophage inflammatory protein (MIP)-2 level in bronchoalveolar lavage fluid (BALF) and interleukin (IL)-17 level in the lung tissue increased in Nrf2-/- mice compared with Nrf2+/+ mice; however, the lack of an increase in the level of tumor necrosis factor (TNF)-α in the lung tissue in Nrf2+/+ mice and mild suppression of the level of TNF-α in Nrf2-/- mice were observed; the level of granulocyte macrophage colony-stimulating factor (GM-CSF) in the lung tissue decreased in Nrf2-/- mice than in Nrf2+/+ mice; the number of DE particle-laden alveolar macrophages in BALF were larger in Nrf2-/- mice than in Nrf2+/+ mice. The results of electron microscope observations showed alveolar type II cell injury and degeneration of the lamellar body after DE exposure in Nrf2-/- mice. Antioxidant enzyme NAD(P)H quinone dehydrogenase (NQO)1 mRNA expression level was higher in Nrf2+/+ mice than in Nrf2-/- mice after DE exposure. Our results suggested that Nrf2 reduces the risk of pulmonary disease via modulating the airway innate immune response caused by DE in mice.

Impact of quadruple regimen of clarithromycin added to metronidazole-containing triple therapy against Helicobacter pylori infection following clarithromycin-containing triple-therapy failure.

BACKGROUND: The establishment of an optimal second-line regimen for Helicobacter pylori infection is required. Although quadruple therapy should overcome resistance to either clarithromycin or metronidazole, the effects of a quadruple regimen in second-line therapy are unknown. This study aims to evaluate the efficacy of triple therapy composed of proton pump inhibitor/amoxicillin plus metronidazole with the combined additive effects of clarithromycin as a second-line quadruple therapy against H. pylori infection. MATERIALS AND METHODS: Participants were 104 patients in whom first-line therapy containing proton pump inhibitor-amoxicillin-clarithromycin failed. Before starting second-line therapy, patients underwent endoscopy to obtain H. pylori strain for antibiotic susceptibility tests. Patients were randomized to receive rabeprazole (10 mg), amoxicillin (750 mg), and metronidazole (250 mg), either with clarithromycin (200 mg; RAMC group) or without (RAM group); all treatments were administered twice daily for 7 days. H. pylori eradication was confirmed by (13)C-urea breath tests performed 2 to 3 months post-therapy. RESULTS: As shown by intention-to-treat/per-protocol analyses, the cure rates for H. pylori infection were 88.5%/93.9% and 82.7%/84.3% for the RAMC and RAM groups. Although the study probably had an insufficient power to show a significant difference between the cure rates of the two regimens, the eradication rates showed a clear trend in favor of the RAMC group. There were no severe side-effects in any group. CONCLUSIONS: In Japan, the RAMC regimen is thought to be a promising alternative strategy for second-line eradication of H. pylori infection.

Patterns in self-rated health according to age and sex in a Japanese national survey, 1989-2004.

BACKGROUND: Perceived good health or good self-rated health is considered to be a predictor of longer survival and maintenance of good quality of life, which is a public health goal. OBJECTIVE: This study assessed trends in the percentage of self-rated poor health among Japanese residents, based on data from the National Comprehensive Survey of the Living Conditions of People on Health and Welfare. METHODS: Results of the survey (which is conducted in Japan every 3 years to determine the living conditions of people receiving health and welfare services) were analyzed using multistage and stratified cluster sampling of households. Self-rated health was measured by response to the question, "Recently, would you say that in general your health has been good, fairly good, fair, fairly poor, or poor?" The trend in fairly poor or poor health status during the period from 1989 through 2004 was stratified by sex and age group. RESULTS: The rates of response to the survey were 90.9% (246,892/271,588) in 1995 and 79.8% (220,836/276,682) in 2004. Target subjects were aged >or=20 years in each year of the study. The prevalence of self-reported fairly poor or poor health was lowest in 1995 and then increased every year until 2001, when it appeared to reach a plateau. The prevalence of having fairly poor or poor health among women aged 35 to 44, 45 to 54, 55 to 64, and 65 to 74 years were as follows in 1995: 9.2%, 11.7%, 15.3%, and 19.8%, respectively. In 2004, the rates were 13.3%, 17.2%, 22.1%, and 31.7%, respectively. By comparison, the prevalence of self-reported fairly poor or poor health was 8.1%, 9.3%, 13.7%, and 17.9% among men aged 35 to 44, 45 to 54, 55 to 64, and 65 to 74 years, respectively, in 1995. In 2004, these rates were 12.8%, 14.8%, 19.0%, and 27.9%, respectively. CONCLUSIONS: In this survey, conducted every 3 years between 1989 and 2004 in Japanese households, older subjects had a greater prevalence of self-reported fairly poor or poor health than did younger subjects. The proportion of respondents who described their health as poor or fairly poor was highest in 1995. Women generally had a greater prevalence of self-reported poor or fairly poor health.

[Evaluation of new saliva collection device for determination of salivary cotinine, cortisol, dehydroepiandrosterone and testosterone concentrations].

OBJECTIVES: This study was conducted to examine the effectiveness of a new salivary collection device (psi10 mm x 25 mm) made of polypropylene and polyethylene polymers (Salisoft((R))). METHOD: Experiment 1; We measured the absorption capacity of the new device by two methods. Next, we examined whether the device affected the pH of 0.1 M NaHCO(3) solution (pH 8.7) and 0.1 M sodium phosphate buffer solution (pH 6.0). Experiment 2; We compared three saliva collection methods: by passive drool, using a cotton device (Salivette((R))), and using the new device. Saliva samples were collected from twelve men (average age, 31.5 +/- 17.1 years). Saliva samples were assayed for cotinine, cortisol, dehydroepiandrosterone, and testosterone concentrations by enzyme immunoassay, and the pH of saliva samples were measured. RESULTS: After this device was put in the mouth for one minute, 1.28 +/- 0.13 mL (mean +/- SD, N = 6) of saliva samples were obtained. The mean pHs of saliva samples collected using Salisoft((R)) and by passive drool did not differ significantly, whereas that of saliva samples collected with Salivette((R)) was significantly low. Saliva samples collected with Salisoft((R)) and those obtained by passive drool did not show significant differences in the concentrations of cotinine, cortisol, dehydroepiandrosterone, and testosterone. Moreover, significant positive correlations were noted between the concentrations in saliva samples collected with Salisoft((R)) and those in saliva samples collected by passive drool. CONCLUSION: This new device was shown to be suitable for saliva collection for the determination of the concentrations of cotinine and some steroids by enzyme immunoassay.

Sleep-disordered breathing and disorders of glucose metabolism.

AIMS: The authors identified the risk of disorders of glucose metabolism (DGM) for sleep-disordered breathing (SDB). METHODS: We conducted a cross-sectional study in 536 men aged 33-84 years. Patients with diabetes medication were excluded for the analysis and DGM were diagnosed by fasting plasma glucose≥100mg/dl and/or 2h plasma glucose ≥140mg/dl. RESULTS: The prevalence of DGM in subjects with and without severe SDB, which was judged by an apnea-hypopnea index (AHI) of 30, were 64.9% and 53.3%, which showed no significant difference. The adjusted odds ratios (ORs) (95% confidence intervals [CIs]) of the logarithmic-transformed AHI and that of C-reactive protein for DGM were 1.3 (0.87-2.0) and 2.3 (1.5-3.6), respectively. When the subjects were categorized by the severity of SDB, the ORs (95% CIs) of subjects with mild, moderate and severe SDB against subjects without SDB were 2.9 (1.8-4.6), 1.2 (0.72-2.1) and 1.5 (0.8-3.0), respectively. CONCLUSION: A significant association was observed between mild SDB and the presence of DGM in male subjects of this study.

Serotonin rebalances cortical tuning and behavior linked to autism symptoms in 15q11-13 CNV mice.

Serotonin is a critical modulator of cortical function, and its metabolism is defective in autism spectrum disorder (ASD) brain. How serotonin metabolism regulates cortical physiology and contributes to the pathological and behavioral symptoms of ASD remains unknown. We show that normal serotonin levels are essential for the maintenance of neocortical excitation/inhibition balance, correct sensory stimulus tuning, and social behavior. Conversely, low serotonin levels in 15q dup mice (a model for ASD with the human 15q11-13 duplication) result in impairment of the same phenotypes. Restoration of normal serotonin levels in 15q dup mice revealed the reversibility of a subset of ASD-related symptoms in the adult. These findings suggest that serotonin may have therapeutic potential for discrete ASD symptoms.

EM703 improves bleomycin-induced pulmonary fibrosis in mice by the inhibition of TGF-beta signaling in lung fibroblasts.

BACKGROUND: Fourteen-membered ring macrolides have been effective in reducing chronic airway inflammation and also preventing lung injury and fibrosis in bleomycin-challenged mice via anti-inflammatory effects. EM703 is a new derivative of erythromycin (EM) without the bactericidal effects. We investigated the anti-inflammatory and antifibrotic effects of EM703 in an experimental model of bleomycin-induced lung injury and subsequent fibrosis in mice. METHODS: Seven-week-old male ICR mice were used. All experiments used eight mice/group, unless otherwise noted in the figure legends. Bleomycin was administered intravenously to the mice on day 0. EM703 was orally administered daily to mice. All groups were examined for cell populations in the bronchoalveolar lavage (BAL) fluid and for induction of messenger RNA (mRNA) of Smad3 and Smad4 in the lung tissues by reverse transcriptase (RT)-polymerase chainreaction (PCR) on day 7. Fibroblastic foci were assessed histologically, and the hydroxyproline content was chemically determined in the lung tissues on day 28. We performed assay of proliferation and soluble collagen production, and examined the induction of mRNA of Smad3 and Smad4 by RT-PCR in murine lung fibroblast cell line MLg2908. We also examined Smad3, Smad4 and phosphorylated Smad2/3 (p-Smad2/3) protein assay by western blotting in MLg2908. RESULTS: Bleomycin-induced lung fibrosis, and the infiltration of macrophages and neutrophils into the airspace were inhibited by EM703. The expression of Smad3 and Smad4 mRNA was clearly attenuated by bleomycin, but was recovered by EM703. EM703 also inhibited fibroblast proliferation and the collagen production in lung fibroblasts induced by Transforming growth factor-beta (TGF-beta). The expression of Smad3 and Smad4 mRNA in murine lung fibroblasts disappeared due to TGF-beta, but was recovered by EM703. EM703 inhibited the expression of p-Smad2/3 and Smad4 protein in murine lung fibroblasts induced by TGF-beta. CONCLUSION: These findings suggest that EM703 improves bleomycin-induced pulmonary fibrosis in mice by actions of anti-inflammation and regulation of TGF-beta signaling in lung fibroblasts.

[The trial of simple gas analysis of tobacco smoke that can be used for medical education].

OBJECTIVE: The purpose of this study was to check a simple sampling and easy gas analysis of tobacco smoke for effective tobacco intervention in medical education. METHODS: The mainstream of tobacco smoke was sampled by a syringe (50 ml) at five, ten and twenty seconds. The extracted mainstream was moved to a commercial PET bottle (2000 ml), and measured with gas detector tubes. The sidestream, which rises from the tip of the cigarette, was collected into a commercial PET bottle for a duration of 30 or 60 seconds. Formaldehyde, acetaldehyde, ammonia, hydrogen cyanide, and nitrogen oxides (NO, NO2) in the tobacco smoke were measured. Then, these gasses in the tobacco smoke of four brands of cigarettes were compared. This trial was conducted in third-year medical students, and the changes in attitudes to smokers and tobacco itself were investigated. RESULTS: The method of sampling 50 ml for 5 seconds produced the highest concentration of each gas in the mainstream. The gas concentration in the sidestream increased as the sampling time increased. The gas concentration in mainstream of "Lucia" was the highest of the used four brands, and the gas concentrations in the sidestream of "Mild Seven Prime" were higher than those of the other brands. Many medical students obtained knowledge about the toxicity of smoking by this experiment study. CONCLUSION: We studied a simple sampling method of tobacco smoke, and gas analysis with gas detector tubes. This method is recommended for tobacco education and intervention in medical education.

Insomnia as a sequela of sarin toxicity several years after exposure in Tokyo subway trains.

More than 5,000 passengers on Tokyo subway trains were injured with toxic chemicals including the nerve gas "sarin" on March 20, 1995. The purpose of this study was to identify the effect of sarin exposure on insomnia in a cross-sectional study. A self-administered questionnaire concerning sleep-related items was distributed to victims of sarin exposure in October and November, 2003. Questionnaires were completed by 161 of the 163 participants (98.8%), who were selected from 1,500 subjects. Among them, the authors selected 75 women 30 to 69 years of age. Control participants were collected from inhabitants living in Maebachi City, Gunma Prefecture, Japan. For the younger exposed group (under 50 yr. of age), percentages of poor sleep, difficulty falling asleep, intermittent awakening, early morning awakening, a feeling of light overnight sleep, and insomnia were significantly higher than those for the control group. In contrast, the older exposed group (ages 50 to 69 years) had significantly higher prevalence of poor sleep, a feeling of light overnight sleep, and early morning awakening for the exposed group when compared with the control group. The high prevalence of insomnia and insomnia-related factors for victims especially under 50 years of age suggests a need for research on sleep quality after sarin exposure. Although posttraumatic stress disorder is assumed to be a psychological effect of exposure to a toxic substance, a cause-and-effect relationship has not been established.

Biological markers, lifestyles and metabolic syndrome in workers.

OBJECTIVE: The association between metabolic syndrome (MetS) and biological inflammatory or anti-coagulant markers were clarified in combination with lifestyle factors. PATIENTS AND METHODS: The target subjects were 5102 working men without metabolic diseases, aged 30-60 years old. The authors measured the serum levels of high-sensitivity C-reactive protein (CRP), uric acid and plasma fibrinogen as potential key biomarkers of MetS. RESULTS: Mean values of uric acid, log-transformed serum CRP and plasma fibrinogen increased significantly as the number of components of MetS increased after adjustment for age. Multivariate analysis revealed significant associations between the presence of MetS and age, habitual exercise, not current smoking, the log-transformed value of serum CRP and serum uric acid, with odds ratios (ORs) of 1.03 (95% confidence interval (CI): 1.02-1.04; p<0.001), 0.77 (95% CI: 0.65-0.90; p<0.01), 0.82 (95% CI: 0.70-0.96; p<0.05), 3.2 (95% CI: 2.6-3.9; p<0.001) and 1.5 (95% CI: 1.4-1.6; p<0.001), respectively for the presence of MetS. CONCLUSION: Elevated serum level of CRP, uric acid, not habitual exercise and current smoking were associated with MetS in this cross-sectional study.

[Determination of the optical purity of N-nitrosofenfluramine found in the Chinese slimming diet].

From 2001 to the summer of 2002, more than 800 cases of liver damage were reported in Japan among people taking Chinese diet aids. The Japanese Ministry of Health, Labor and Welfare has recently announced that N-nitrosofenfluramine was the hepatotoxic compound contained in the diet aids based on animal experiments performed by the National Institute of Health Sciences. Although N-nitrosofenfluramine is a derivative of fenfluramine, a previously used antiobesity drug, neither pharmacologic nor toxicologic properties have been reported for N-nitroso fenfluramine. It should be noted that N-nitrosofenfluramine has two optical isomers, although it is not yet known which isomer damages the liver and other organs. The Japanese Ministry of Health, Labor and Welfare has not commented on this point. Pursuing this question, 10 types of Chinese slimming aid samples including those obtained from patients with fulminating hepatitis were analyzed by NMR, GC/MS, and a newly established HPLC method using a chiral separation column. It was found that the N-nitrosofenfluramine in all of the toxic diet aids was the (S)-isomer form. No (R)-isomer was detected. These results strongly suggest that the nitroso-compound in the diets must be prepared from pharmacologically active (S)-fenfluramine (dexfenfluramine). Thus the pharmacologic and toxicologic properties of each isomer should be investigated.

Substrate specificity of human granzyme 3: analyses of the P3-P2-P1 triplet using fluorescence resonance energy transfer substrate libraries.

Granzyme 3 (Gr3) is known as a tryptase-type member of the granzyme family and exists in the granules of immunocompetent cells. Granule proteases including granzymes, are transported into the cytoplasm of tumor cells or virus-infected cells by perforin function, degrade cytoplasmic or nuclear proteins and subsequently cause the death of the target cells. Recently, although several substrates of Gr3 in vivo have been reported, these hydrolyzed sites were unclear or lacked consistency. Our previous study investigated the optimal amino acid triplet (P3-P2-P1) as a substrate for Gr3 using a limited combination of amino acids at the P2 and P3 positions. In the present study, new fluorescence resonance energy transfer (FRET) substrate libraries to screen P2 and P3 positions were synthesized, respectively. Using these substrate libraries, the optimal amino acid triplet was shown to be Tyr-Phe-Arg as a substrate for human Gr3. Moreover, kinetic analyses also showed that the synthetic substrate FRETS-YFR had the lowest Km value for human Gr3. A substantial number of membrane proteins possessed the triplet Tyr-Phe-Arg and some of them might be in vivo substrates for Gr3. The results might also be a great help for preparing specific inhibitors to manipulate Gr3 activity both in vitro and in vivo.