Development and validation of bioanalytical methods for Imidafenacin (KRP-197/ONO-8025) and its metabolites in human plasma by liquid chromatography-tandem mass spectrometry

Imidafenacin (KRP-197/ONO-8025, IM), 4-(2-methyl-1H-imidazol-1-yl)-2,2-diphenylbutanamide, is a new antimuscarinic agent currently under application for the indication of treatment of overactive bladder in Japan. We developed and validated the sensitive and selective bioanalytical methods for the extremely low levels of IM and its metabolite, M-2 (Method 1), M-4 (Method 2) and M-9 (Method 3) in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In each method, plasma sample was extracted by solid phase extraction, separated on a semi-micro high performance liquid chromatography column and detected by tandem mass spectrometer with an atmospheric pressure chemical ionization or ionspray interface. Selected reaction monitoring mode was used for quantification. Each method was found to have acceptable accuracy, precision, stability, selectivity and linearity over the concentration range of 10-500 pg/mL for IM and M-2, 10-1000 pg/mL for M-4 and 50-5000 pg/mL for M-9. Using these analytical methods, concentration profiles of IM and its metabolites in human plasma were successfully determined even in the low pg/mL levels after oral administration of IM at the therapeutic dosage of 0.1 mg.     

Morning-evening preference: sleep pattern spectrum and lifestyle habits among Japanese junior high school pupils

We surveyed the sleep-wake patterns and lifestyle habits in a sample of Japanese first to third year junior high school children (n=638, age 12 to 15 yrs), of whom 29.3% were evening type, 64.1% intermediate type, and 6.6% morning type in preference. The morningness-eveningness (M-E) score was lower (more evening typed), 16.1 vs. 15.4 in first compared to third year students. There were significant gender differences, with girls showing a greater evening preference. Evening preference was associated with longer sleep latency, shortened sleep duration during schooldays and weekends, bad morning feeling, and episodes of daytime sleepiness. In contrast, morning preference was associated with higher sleep drive and better sleep-wake parameters and lifestyle habits. Our results suggest the morning preference should be promoted among junior high school children to increase the likelihood of more regular sleep-wake patterns and lifestyle habits.     

Guidelines for the Management of Helicobacter pylori Infection in Japan: 2009 Revised Edition

Background:  Over the past few years, the profile of Helicobacter pylori infection has changed in Japan. In particular, the relationship between H. pylori and gastric cancer has been demonstrated more clearly. Accordingly, the committee of the Japanese Society for Helicobacter Research has revised the guidelines for diagnosis and treatment of H. pylori infection in Japan.
Materials and Methods:  Four meetings of guidelines preparation committee were held from July 2007 to December 2008. In the new guidelines, recommendations for treatment have been classified into five grades according to the Minds Recommendation Grades, while the level of evidence has been classified into six grades. The Japanese national health insurance system was not taken into consideration when preparing these guidelines.
Results:  Helicobacter pylori eradication therapy achieved a Grade A recommendation, being useful for the treatment of gastric or duodenal ulcer, for the treatment and prevention of H. pylori -associated diseases such as gastric cancer, and for inhibiting the spread of H. pylori infection. Levels of evidence were determined for each disease associated with H. pylori infection. For the diagnosis of H. pylori infection, measurement of H. pylori antigen in the feces was added to the tests not requiring biopsy. One week of proton-pump inhibitor-based triple therapy (including amoxicillin and metronidazole) was recommended as second-line therapy after failure of first-line eradication therapy.
Conclusion:  The revised Japanese guidelines for H. pylori are based on scientific evidence and avoid the administrative restraints that applied to earlier versions .     

Vacuolar H(+)-pyrophosphatase purified from pear fruit

A vacuolar H(+)-translocating inorganic pyrophosphatase was purified from pear fruit through selective detergent treatments, Superose 6 and Mono Q column chromatography. The specific activity of the purified enzyme was 850 mumol h-1 mg protein-1. The Mr of V-PPase was 66 kDa by SDS-PAGE and the polypeptide cross-reacted with the antiserum against V-PPase of mung bean. The purified V-PPase was stimulated by potassium and inhibited by calcium and N, N'-dicyclohexylcarbodiimide.     

Phylogenetic relationships among JC virus strains in Japanese/Koreans and Native Americans speaking Amerind or Na-Dene

Many genetic studies using human mtDNA or the Y chromosome have been conducted to elucidate the relationships among the three Native American groups speaking Amerind, Na-Dene, and Eskimo-Aleut. Human polyomavirus JC (JCV) may also help to gain insights into this issue. JCV isolates are classified into more than 10 geographically distinct genotypes (designated subtypes here), which were generated by splits in the three superclusters, Types A, B, and C. A particular subtype of JCV (named MY) belonging to Type B is spread in both Japanese/Koreans and Native Americans speaking Amerind or Na-Dene. In this study, we evaluated the phylogenetic relationships among MY isolates worldwide, using the whole-genome approach, with which a highly reliable phylogeny of JCV isolates can be reconstructed. Thirty-six complete sequences belonging to MY (10 from Japanese/Koreans, 24 from Native Americans, and 2 from others), together with 54 belonging to other subtypes around the world, were aligned and subjected to phylogenetic analysis using the neighbor-joining and maximum-likelihood methods. In the resultant phylogenetic trees, the MY sequences diverged into two Japanese/Korean and five Native American clades with high bootstrap probabilities. Two of the Native American clades contained isolates mainly from Na-Denes and the others contained isolates mainly from Amerinds. The Na-Dene clades were not clustered together, nor were the Amerind clades. In contrast, the two Japanese/Korean clades were clustered at a high bootstrap probability. We concluded that there is no distinction between Amerinds and Na-Denes in terms of indigenous JCVs, although they are linguistically distinguished from each other.     

Identification of mature plasma cells in early rat yolk sac. A possible origin from the endodermal cell layer: immunohistochemistry and immunoelectron microscopic study

Plasma cells play a pivotal role in the immune system and are responsible for the synthesis and release of immunoglobulins. Numerous in vitro culture experiments on the yolk sac demonstrated the generation of mature cells of the myeloid and lymphoid lineages under appropriate culture conditions. However, there are no reports describing the development of mature lymphoid cells in the yolk sac so far. For this reason, we undertook this study to investigate the development of antibody-containing plasma cells during early yolk sac haematopoiesis. Immunohistochemistry and immunoelectron microscopy were employed in the study. Results of this work demonstrated very weak immune staining for the intracytoplasmic IgA, IgG, and IgM at days 10 and 11 of embryonic life, while dark staining was obtained at 12 days. Positive staining was localized to the endodermal cell layer. Electron microscopic examinations revealed the existence of cells with the typical characteristics of plasma cells inside the endodermal cell layer, which may suggest their endodermal origin. To further verify the nature of these cells, intracytoplasmic immunoglobulins were demonstrated by immunoelectron microscopy. The present study demonstrated emergence of mature functioning plasma cells in early rat yolk sac. In a previous work we hypothesized the possibility of endodermal origin of yolk sac macrophages. This study adds additional evidence to support that hypothesis. The possible role of plasma cells in the yolk sac is discussed.     

Antagonistic interactions between the SA and JA signaling pathways in Arabidopsis modulate expression of defense genes and gene-for-gene resistance to cucumber mosaic virus

Gene-for-gene resistance to a yellow strain of cucumber mosaic virus [CMV(Y)] is conferred by the dominant RESISTANCE to CMV(Y) (RCY1) allele in the Arabidopsis thaliana ecotype C24. RCY1-conferred resistance to CMV(Y) and expression of the Pathogenesis-related 1 (PR-1) and PR-5 genes are partially compromised by the eds5 mutation and the nahG transgene that block accumulation of salicylic acid (SA). In contrast, the RCY1-conferred resistance to CMV(Y) is not affected by the jasmonic acid (JA)-insensitive coi1 and jar1 mutations. Interestingly, we report here that in contrast to the eds5 RCY1 plant, the eds5 coi1 RCY1 double-mutant plant exhibited a higher level of resistance to CMV(Y). Presence of the coi1 mutant allele also restored the CMV(Y)-activated expression of the PR-1 and PR-5 gene in the eds5 coi1 RCY1 plant. In contrast to the PR-1 and PR-5 genes, expression of the JA-dependent PLANT DEFENSIN 1.2 (PDF1.2) and HEVEIN-LIKE PROTEIN (HEL) genes was elevated in the CMV(Y)-inoculated leaves of the eds5 RCY1 plant, but not in the virus-inoculated leaves of the wild-type RCY1 and coi1 RCY1 plants. We propose that antagonistic interactions between the SA and JA signaling mechanisms modulate defense gene expression and the activation of RCY1-conferred gene-for-gene resistance to CMV(Y).     

Molecular cloning and expression of pyruvate kinase from globefish (Fugu rubripes) skeletal muscle

A cDNA clone encoding pyruvate kinase (PK) was isolated from a skeletal muscle cDNA library of globefish (Fugu rubripes), which is a kind of lower vertebrate. The full-length cDNA of globefish skeletal muscle pyruvate kinase (FM-PK) is approximately 2 kb and encodes a protein comprising 530 amino acids. The FM-PK gene is spanning approximately 4.8 kb and consists of 11 exons. FM-PK mRNA was detected in muscle and heart using Northern blots. The recombinant FM-PK (rFM-PK) was expressed in a baculovirus-insect cell system and purified using ion-exchange chromatography. The purified rFM-PK was shown to exist a 230 kDa homotetramer composed of 57 kDa subunits. Gel filtration showed 230000 as the tetramer of the subunit. The apparent K(m) (or S(0.5)) and the Hill coefficient for phosphoenolpyruvate (PEP) and ADP are 0.14 mM, 1.3 and 0.30 mM 0.98 at pH 7.4, respectively, when the enzyme is saturated with the second substrate. The rFM-PK is strongly activated by fructose-1,6-bisphosphate, the apparent K(m) for PEP changes to 0.059 mM and the Hill coefficient to 1.1. ATP, which is the product of the enzyme reaction, inhibits activity. This is the first report to show the full-length cDNA and amino acid sequence of PK for a species of fish.