Treatment with β2‐Adrenoceptor Agonist in Vivo Induces Human Clock Gene,Per1, mRNA Expression in Peripheral Blood

This study examined whether in vivo exposure to a β₂‐adrenoceptor agonist, tulobuterol, induces human Period1 (hPer1) mRNA expression in cells from peripheral whole blood. In one experiment, oral tulobuterol was administered to five healthy volunteers at 22:00 h, while in another, a transdermally tulobuterol patch was applied to the same five subjects at 20:00 h. In each experiment, serum tulobuterol concentrations were measured at four time points, and total RNA was isolated from peripheral blood cells for determinations of hPer1 mRNA expression by real‐time polymerase chain reaction. Both the tulobuterol tablet and the transdermal patch increased hPer1 mRNA expression, suggesting that analyses of human peripheral blood cells could reliably represent peripheral clock gene mRNA expression in vivo.     

Rapid PaperMicrobial Oxygenation of Organo-metallic Bissulfides Leading to Formation of Planar Chirality and C2 Symmetry

1,2-Bis(methylthiomethyl)ferrocene (3) was oxidized by Corynebacterium equi IFO 3730 to give monosulfoxide 4 in two diastereomeric forms with (1S,2R,S_S) and (1S,2R,R_S) configurations in a ratio of 4:1, while 1,1′-bis(methylthiomethyl)ferrocene (5) was oxidized by Penicillium frequentans IFO 5692 to (R)-monosulfoxide 6 and then preferentially to (R,R)-bissulfoxide 7. Thus, the bacterial monooxygenase generated specific planar chirality in the metallocenic monosulfoxide, and the fungal enzyme formed C₂ symmetry in the bissulfoxide.     

miR-146a Polymorphism (rs2910164) Predicts Colorectal Cancer Patients’ Susceptibility to Liver Metastasis

miR-146a plays important roles in cancer as it directly targets NUMB, an inhibitor of Notch signaling. miR-146a is reportedly regulated by a G>C polymorphism (SNP; rs2910164). This polymorphism affects various cancers, including colorectal cancer (CRC). However, the clinical significance of miR-146a polymorphism in CRC remains unclear. A total of 59 patients with CRC were divided into 2 groups: a CC/CG genotype (n = 32) and a GG genotype (n = 27), based on the miR-146a polymorphism. cDNA microarray analysis was performed using 59 clinical samples. Significantly enriched gene sets in each genotype were extracted using GSEA. We also investigated the association between miR-146a polymorphism and miR-146a, NUMB expression or migratory response in CRC cell lines. The CC/CG genotype was associated with significantly more synchronous liver metastasis (p = 0.007). A heat map of the two genotypes showed that the expression profiles were clearly stratified. GSEA indicated that Notch signaling and JAK/STAT3 signaling were significantly associated with the CC/CG genotype (p = 0.004 and p = 0.023, respectively). CRC cell lines with the pre-miR-146a/C revealed significantly higher miR-146a expression (p = 0.034) and higher NUMB expression and chemotactic activity. In CRC, miR-146a polymorphism is involved in liver metastasis. Identification of this polymorphism could be useful to identify patients with a high risk of liver metastasis in CRC.     

Combining mapping of physiological quantitative trait loci and transcriptome for cold tolerance for counteracting male sterility induced by low temperatures during reproductive stage in rice

Male sterility induced by low temperatures (LTs) during the reproductive stage is a major constraint for temperate zone rice. To detect physiological quantitative trait loci (QTLs), we modeled genotypic variation in the physiological processes involved in low temperature spikelet sterility on the basis of anther length (AL), a proxy for microspore and pollen grain number per anther. The model accounted for 83% of the genotypic variation in potential AL at normal temperature and the ability to maintain AL at LT. We tested the model on 208 recombinant inbred lines of cold‐tolerant ‘Tohoku‐PL3’ (PL3) × cold‐sensitive ‘Akihikari’ (AH) for 2 years. QTLs for spikelet fertility (FRT) at LT were detected on chromosomes 5 (QTL for Cold Tolerance at Reproductive stage, qCTR5) and 12 (qCTR12). qCTR12 was annotated with the ability to maintain AL under LTs. qCTR5 was in a region shared with QTLs for culm length and heading date. Genome‐wide expression analysis showed 798 genes differentially expressed in the spikelets between the parents at LTs. Of these, 12 were near qCTR5 and 23 were near qCTR12. Gene expression analysis confirmed two candidate genes for qCTR5 (O‐methyltransferase ZRP4, Os05g0515600; beta‐1,3‐glucanase‐like protein, Os05g0535100) and one for qCTR12 (conserved hypothetical protein, Os12g0550600). Nucleotide polymorphisms (21 deletions, 2 insertions and 10 single nucleotide polymorphisms) in PL3 were found near the candidate conserved hypothetical protein (Os12g0550600) and upstream in PL3, but not in AH. Haplotype analysis revealed that this gene came from ‘Kuchum’. The combination of mapping physiological QTLs with gene expression analysis can be extended to identify other genes for abiotic stress response in cereals.     

Electrochemical screening of recombinant protein solubility in Escherichia coli using scanning electrochemical microscopy (SECM)

A microbial array chip with collagen gel spots entrapping living Escherichia coli (E. coli) DH5alpha was applied for the screening of recombinant protein solubilities. The alpha-fragment of beta-galactosidase (betaGal) was fused to the target protein, namely, maltose-binding protein (MBP), to monitor the solubility of MBP. Scanning electrochemical microscopy (SECM) was used to detect the release of p-aminophenol from E. coli cells catalyzed by intracellular betaGal. Comparison of the SECM-based method with the Western blotting-based method indicated that the current response obtained using SECM increased with an increase in the betaGal activity and therefore, with the soluble fraction of MBP in the host cells.     

Natural variation of ebony gene controlling thoracic pigmentation in Drosophila melanogaster

We identified the causal genetic variation for the difference in the thoracic trident pigmentation intensity between two wild-derived strains of Drosophila melanogaster. It was found to be the difference in expression level of ebony, which codes for an enzyme in the melanin-synthesis pathway and has pleiotropic effects on vision and behavior.     

Double-layered collagen gel hemisphere for cell invasion assay: successful visualization and quantification of cell invasion activity

Although various methods for collagen gel-based cell invasion assays have been described, there continues to be a need for a simpler and more objective assay. Here, we describe an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) system that satisfies these requirements, and we demonstrate the advantages of this new system for visualizing cell movements during invasion. DL-CGH consists of a central core collagen layer surrounded by an outer cover collagen layer. A droplet of collagen I solution (containing cells to be examined) naturally forms a small hemisphere on the bottom of the culture dish. After this central core layer gels, a second droplet is placed atop the first gel, encapsulating it completely. The hemisphere is submerged in the medium and cultured. The invasive activity of cells that infiltrate from the inner to the outer layer can be evaluated optically. Using this in vitro system, we measured the inhibitory effect of E-cadherin expression on cancer cell invasion. DL-CGH also allowed visualization of interactions between invading cancer cells and the stroma. Cancer cells, which lack the proteases required for direct entrance into the three-dimensional collagen matrix, were seen to slip like amoebas through matrix gaps generated by the pericellular proteolytic activity of fibroblasts. [Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resources: Movies 1-3; 4a and b].     

Familial Hyperaldosteronism Type 3 with a Rapidly Growing Adrenal Tumor: An In Situ Aldosterone Imaging Study

Primary aldosteronism is most often caused by aldosterone-producing adenoma (APA) and bi-lateral adrenal hyperplasia. Most APAs are caused by somatic mutations of various ion channels and pumps, the most common being the inward-rectifying potassium channel KCNJ5. Germ line mutations of KCNJ5 cause familial hyperaldosteronism type 3 (FH3), which is associated with severe hyperaldosteronism and hypertension. We present an unusual case of FH3 in a young woman, first diagnosed with primary aldosteronism at the age of 6 years, with bilateral adrenal hyperplasia, who underwent unilateral adrenalectomy (left adrenal) to alleviate hyperaldosteronism. However, her hyperaldosteronism persisted. At the age of 26 years, tomography of the remaining adrenal revealed two different adrenal tumors, one of which grew substantially in 4 months; therefore, the adrenal gland was removed. A comprehensive histological, immunohistochemical, and molecular evaluation of various sections of the adrenal gland and in situ visualization of aldosterone, using matrix-assisted laser desorption/ionization imaging mass spectrometry, was performed. Aldosterone synthase (CYP11B2) immunoreactivity was observed in the tumors and adrenal gland. The larger tumor also harbored a somatic β-catenin activating mutation. Aldosterone visualized in situ was only found in the subcapsular regions of the adrenal and not in the tumors. Collectively, this case of FH3 presented unusual tumor development and histological/molecular findings.