Biochemical Characterization of Reactive Nitorogen Species by Eosinophil Peroxidase in Tyrosine Nitration

取得学位 : 博士（医学）, 学位授与番号 : 医博甲第1853号, 学位授与年月日 : 平成19年3月22日, 学位授与大学 : 金沢大学, 主査教授 : 中村 裕之, 副査教授 : 山本 博, 中尾 眞

Preparation and Characterization of a Polyclonal Antibody against Brominated Protein

(Di)bromotyrosine is formed by the specific reaction of eosinophil peroxidase and can be used as an eosinophil activation marker. In the present study, an antibody for (di)bromotyrosine in proteins was prepared to investigate the pathogenesis of eosinophil-related diseases such as allergic responses. A rabbit polyclonal antibody was raised against brominated keyhole limpet hemocyanin. The specificity of the antiserum was investigated with an enzyme-linked immunosorbent assay (ELISA). The antiserum recognized brominated bovine serum albumin (BSA) and dibromotyrosine-conjugated BSA. The antiserum also reacted with chlorinated BSA and di-iodotyrosine-conjugated BSA. Moreover, the specificity of the antiserum was investigated using competitive ELISA. Dibromotyrosine and di-iodotyrosine inhibited the recognition of brominated BSA by the antiserum. However, the recognition of brominated BSA by the antiserum was not inhibited by bromotyrosine, chlorotyrosine, iodotyrosine, nitrotyrosine, aminotyrosine, phosphotyrosine, or tyrosine. These results suggested that the epitope of the antiserum is dihalogenated tyrosine. Immunohistochemically, the antiserum stained brominated rat eosinophils but not chlorinated or nitrated eosinophils. In conclusion, an antiserum for dihalogenated protein was prepared. It is expected that the antiserum will be useful for the analysis of the pathogenesis of allergic diseases such as asthma and atopic dermatitis

Association of Dietary Fat and Carbohydrate Consumption and Predicted Ten-year Risk for Developing Coronary Heart Disease in a General Japanese Population

We examined the relationships between dietary carbohydrate, protein, fat, and the ratio of n6/n3 fatty acid intakes with the predicted 10-year coronary heart disease (CHD) risk in a general Japanese population. We used the Framingham risk score to determine the 10-year CHD risk of the subjects, who were employees of 6 companies in a single prefecture in Japan. After excluding the subjects who reported any history of angina pectoris, myocardial infarction, diabetes, or cancer, and those with missing data resulting in the inability of estimation of 10-year CHD risk and food intakes, the final data analysis was carried out for 809 subjects. The logistic regression models revealed a significantly increased odds ratio of 10-year CHD risk in the subjects with the highest tertile of carbohydrate intake (% energy) (odds ratio 3.64, 95% CI, 2.07-6.40);after adjustment for other variables, the odds ratio for the 10-year CHD risk was also higher in the subjects with the highest tertile of carbohydrate intake (odds ratio 1.72, 95% CI, 0.70-4.25). We also found that fat intake and the ratio of n6/n3 fatty acids were inversely associated with the predicted 10-year CHD risk (p for trend＜0.01). The present findings added evidence of a positive association of dietary carbohydrate and inverse associations of total fat and n6/n3 fatty acid ratio with the predicted 10-year CHD risk in a general Japanese population

Biochemical characterization of reactive nitrogen species by eosinophil peroxidase in tyrosine nitration

It is well known that eosinophils are involved in tyrosine nitration. In this study, we evaluated tyrosine nitration by rat eosinophils isolated from peritoneal fl uid and constituent eosinophils in the stomach. Rat peritoneal eosinophils activated with 1 &#956;M phorbol myristate acetate (PMA) and 50 &#956;M NO2 &#65437; showed immunostaining for nitrotyrosine only in smaller cells, despite the fact that eosinophils are capable of producing superoxide (O2·&#65437;). Free tyrosine nitrating capacity after incubation with PMA and NO2 &#65437; was 4-fold higher in eosinophils than in neutrophils. Catalase and &#65400;- and &#65402; -tocopherol inhibited free tyrosine nitration by reactive nitrogen species from eosinophils but not that by peroxynitrite. Superoxide dismutase augmented free tyrosine nitration by activated eosinophils and peroxynitrite. The concentration of nitric oxide released from eosinophils was relatively low (0.32 &#956;M/106 cells/h) and did not contribute to the formation of nitrotyrosine. On the other hand, most constituent eosinophils constituent in the rat stomach stimulated by PMA and NO2 &#65437; showed tyrosine nitration capacity. These results suggest that intact cells other than apoptotic-like eosinophils eluted in the intraperitoneal cavity could not generate reactive species responsible for nitration by a peroxidase-dependent mechanism. In contrast, normal eosinophils in the stomach were capable of nitration, suggesting that the characteristics of eosinophils in gastric mucosa are diff erent from those eluted in the peritoneal cavity.</p

Influences of protein ingestion on glucagon-like peptide (GLP)-1-immunoreactive endocrine cells in the chicken ileum

Influences of a specific dietary nutrient on glucagon-like peptide (GLP)-1-containing cells in the chicken intestine are not yet clear. Significance of dietary protein level on GLP-1-containing cells in the chicken ileum was investigated. Chickens fed control or experimental diets of varying protein levels were examined using immunohistochemical and morphometrical techniques. We show that the protein ingestion had an impact on the activities of GLP-1-immunoreactive cells in the chicken ileum. Weight gains declined with decreasing dietary crude protein (CP) levels, but no significant differences were detected in the daily feed intake and villous height. GLP-1-immunoreactive cells with a round or oval shape were frequently observed in the lower CP level groups (4.5% and 0%). Frequencies of occurrence of GLP-1-immunoreactive cells were 41.1 +/- 4.1, 38.5 +/- 4, 34.8 +/- 3.1 and 34.3 +/- 3.7 (cells/mm(2), mean +/- SD) for dietary CP level of 18%, 9%, 4.5% and 0% groups, respectively and significant differences were recognized between the control and lower CP level groups (P<0.05). Multiple regression analysis indicated a significant correlation between the daily protein intake and frequencies of occurrence of GLP-1-immunoreactive cells. The protein ingestion is one of the signals that influence GLP-1-containing cells in the chicken small intestine.ArticleANIMAL SCIENCE JOURNAL. 85(5):581-587 (2014)journal articl

Disposition of protein-bound 3-nitrotyrosine in rat plasma analysed by a novel protocol for HPLC-ECD

金沢大学医薬保健研究域医学系3-Nitrotyrosine (NTyr) is considered as a biomarker of the generation of reactive nitrogen species (RNS). However, it is still difficult to determine its concentration in biological samples. To develop a reliable and high-throughput method, we optimized the conditions for high performance liquid chromatography and electrochemical detection (HPLC-ECD). The best separation of NTyr was achieved using a highly acidic mobile phase (pH 2.5). The concentration of protein-bound NTyr in plasma protein was 593.6 ± 53.8 fmol/mg in rats treated with lipopolysaccharide (LPS) and 114.4 ± 27.6 fmol/mg in control. After intravenous administration of in vitro-nitrated plasma protein, NTyr concentration decreased; the half-life was 63.4 ± 16.8 h. Consistently, protein-bound NTyr concentration in plasma after LPS treatment declined gradually, but was detectable for 1 week. Our protocol is reproducible and suitable for analysing multiple clinical samples to study RNS production in vivo. © 2007 The Japanese Biochemical Society

Associations of Serum Retinol, alpha-Tocopherol, and gamma-Tocopherol with Biomarkers among Healthy Japanese Men

Retinol, alpha-tocopherol, and gamma-tocopherol are fat-soluble vitamins acting as antioxidants via the prevention of lipid oxidation. Little is known about circulatory levels in healthy individuals. The present cross-sectional study aimed at elucidating the relationship between these antioxidants and clinical biomarkers in 206 male (median age 41 years, range 23-67) employees from companies located in the Okayama Prefecture, Japan. Subjects younger than 40 years (n = 94) showed a positive association of the frequency of alcohol consumption with the circulating retinol (beta = 0.344, p = 0.001) and gamma-tocopherol levels (beta = 0.219, p = 0.041), and an inverse association of fast insulin with serum retinol (beta = -0.301, p = 0.009). In participants older than 40 years (n = 112) we found that an inverse association of HOMA-R with serum retinol (beta = -0.262, p = 0.021), a-tocopherol (beta = -0.236, p = 0.035), and gamma-tocopherol levels (beta = -0.224, p = 0.052); and cigarette smoking was inversely associated with the levels of serum a-tocopherol (beta = -0.286, p = 0.008) and gamma-tocopherol (beta = -0.229, p = 0.040). We further found negative relationships between serum ferritin and the retinol (beta = -0.211, p = 0.032) and a-tocopherol levels (beta = -0.223, p = 0.022) in men over 40 years of age. The present study suggests that the circulatory levels of antioxidant vitamins may modulate the action of insulin and that higher levels of iron might decrease the levels of antioxidant vitamins in the blood

Influences of protein ingestion on glucagon-like peptide (GLP)-1-immunoreactive endocrine cells in the chicken ileum

Influences of a specific dietary nutrient on glucagon-like peptide (GLP)-1-containing cells in the chicken intestine are not yet clear. Significance of dietary protein level on GLP-1-containing cells in the chicken ileum was investigated. Chickens fed control or experimental diets of varying protein levels were examined using immunohistochemical and morphometrical techniques. We show that the protein ingestion had an impact on the activities of GLP-1-immunoreactive cells in the chicken ileum. Weight gains declined with decreasing dietary crude protein (CP) levels, but no significant differences were detected in the daily feed intake and villous height. GLP-1-immunoreactive cells with a round or oval shape were frequently observed in the lower CP level groups (4.5% and 0%). Frequencies of occurrence of GLP-1-immunoreactive cells were 41.1 +/- 4.1, 38.5 +/- 4, 34.8 +/- 3.1 and 34.3 +/- 3.7 (cells/mm(2), mean +/- SD) for dietary CP level of 18%, 9%, 4.5% and 0% groups, respectively and significant differences were recognized between the control and lower CP level groups (P<0.05). Multiple regression analysis indicated a significant correlation between the daily protein intake and frequencies of occurrence of GLP-1-immunoreactive cells. The protein ingestion is one of the signals that influence GLP-1-containing cells in the chicken small intestine

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target