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

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

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

Association study of the KCNJ3 gene as a susceptibility candidate for schizophrenia in the Chinese population

We recently reported the results of a genome-wide association study (GWAS) of schizophrenia in the Japanese population. In that study, a single nucleotide polymorphism (SNP) (rs3106653) in the KCNJ3 (potassium inwardly rectifying channel, subfamily J, member 3) gene located at 2q24.1 showed association with schizophrenia in two independent sample sets. KCNJ3, also termed GIRK1 or Kir3.1, is a member of the G protein-activated inwardly rectifying K+ channel (GIRK) group. GIRKs are widely distributed in the brain and play an important role in regulating neural excitability through the activation of various G protein-coupled receptors. In this study, we set out to examine this association using a different population. We first performed a gene-centric association study of the KCNJ3 gene, by genotyping 38 tagSNPs in the Chinese population. We detected nine SNPs that displayed significant association with schizophrenia (lowest P = 0.0016 for rs3106658, Global significance = 0.036). The initial marker SNP (rs3106653) examined in our prior GWAS in the Japanese population also showed nominally significant association in the Chinese population (P = 0.028). Next, we analyzed transcript levels in the dorsolateral prefrontal cortex of postmortem brains from patients with schizophrenia and bipolar disorder and from healthy controls, using real-time quantitative RT-PCR. We found significantly lower KCNJ3 expression in postmortem brains from schizophrenic and bipolar patients compared with controls. These data suggest that the KCNJ3 gene is genetically associated with schizophrenia in Asian populations and add further evidence to the “channelopathy theory of psychiatric illnesses”