Bioimaging Probes for Reactive Oxygen Species and Reactive Nitrogen Species

Reactive oxygen species (ROS) play key roles in many pathogenic processes, including carcinogenesis, inflammation, ischemia-reperfusion injury and signal transduction. Also, reactive nitrogen species (RNS) cause various biological events such as neurodegenerative disorders. Sensitive and specific detection methods for ROS and RNS in biological samples should be useful for elucidation of biological events both in vitro and in vivo. Fluorescent probes based on small organic molecules have become indispensable tools in modern biology because they provide dynamic information concerning the localization and quantity of biological molecules of interest, without the need of genetic engineering of the sample. In this review, we recount some recent achievements in the field of small molecular fluorescent probes. First, the probes for nitric oxide and peroxynitrite as RNS are introduced and the probes of hydroxyl radical, hydrogen peroxide, hypochlorous and singlet oxygen as ROS are discussed, based on the fluorescence off/on switching mechanisms including photoinduced electron transfer and spirocyclization processes, and with some applications for in vitro and in vivo systems

A series of ENU-induced single-base substitutions in a long-range cis-element altering Sonic hedgehog expression in the developing mouse limb bud

AbstractMammal–fish-conserved-sequence 1 (MFCS1) is a highly conserved sequence that acts as a limb-specific cis-acting regulator of Sonic hedgehog (Shh) expression, residing 1 Mb away from the Shh coding sequence in mouse. Using gene-driven screening of an ENU-mutagenized mouse archive, we obtained mice with three new point mutations in MFCS1: M101116, M101117, and M101192. Phenotype analysis revealed that M101116 mice exhibit preaxial polydactyly and ectopic Shh expression at the anterior margin of the limb buds like a previously identified mutant, M100081. In contrast, M101117 and M101192 show no marked abnormalities in limb morphology. Furthermore, transgenic analysis revealed that the M101116 and M100081 sequences drive ectopic reporter gene expression at the anterior margin of the limb bud, in addition to the normal posterior expression. Such ectopic expression was not observed in the embryos carrying a reporter transgene driven by M101117. These results suggest that M101116 and M100081 affect the negative regulatory activity of MFCS1, which suppresses anterior Shh expression in developing limb buds. Thus, this study shows that gene-driven screening for ENU-induced mutations is an effective approach for exploring the function of conserved, noncoding sequences and potential cis-regulatory elements

Molecular imaging of aberrant crypt foci in the human colon targeting glutathione S-transferase P1-1

Aberrant crypt foci (ACF), the earliest precursor lesion of colorectal cancers (CRCs), are a good surrogate marker for CRC risk stratification and chemoprevention. However, the conventional ACF detection method with dye-spraying by magnifying colonoscopy is labor- and skill-intensive. We sought to identify rat and human ACF using a fluorescent imaging technique that targets a molecule specific for ACF. We found that glutathione S-transferase (GST) P1-1 was overexpressed in ACF tissues in a screening experiment. We then synthesized the fluorogenic probe, DNAT-Me, which is fluorescently quenched but is activated by GSTP1-1. A CRC cell line incubated with DNAT-Me showed strong fluorescence in the cytosol. Fluorescence intensities correlated significantly with GST activities in cancer cell lines. When we sprayed DNAT-Me onto colorectal mucosa excised from azoxymethane-treated rats and surgically resected from CRC patients, ACF with strong fluorescent signals were clearly observed. The ACF number determined by postoperative DNAT-Me imaging was almost identical to that determined by preoperative methylene blue staining. The signal-to-noise ratio for ACF in DNAT-Me images was significantly higher than that in methylene blue staining. Thus, we sensitively visualized ACF on rat and human colorectal mucosa by using a GST-activated fluorogenic probe without dye-spraying and magnifying colonoscopy

Large 3' UTR of sugar beet rps3 is truncated in cytoplasmic male-sterile mitochondria

Genomic alteration near or within mitochondrial gene is often associated with cytoplasmic male sterility (CMS). Its influence on the expression of the mitochondrial gene was proposed as one of the possible causes of CMS. In sugar-beet mitochondrial rps3, whose downstream 1056-bp region contains Norf246, an apparently non-functional open reading frame (ORF), was deleted in CMS mitochondria. In our previous study, normal rps3 (3.8 kb), CMS rps3 (2.7 kb), and Norf246 (3.8 kb and 0.9 kb) were shown to be transcribed. The present study was conducted to determine whether the deletion affected gene expression. Reverse transcription (RT)-PCR analysis revealed the co-transcription of rps3 and Norf246. By circularized RNA (CR) RT-PCR analysis, the 5' and 3' termini of the 3.8-kb and the 0.9-kb transcripts were determined. The results suggested that the 3.8-kb transcripts were the rps3 mRNA bearing an ∼464-base 5' untranslated region (UTR) and ∼1508-base 3' UTR, whereas no functional ORF was observed in the 0.9-kb transcripts. CR-RT-PCR revealed that the 3' UTR of the 2.7-kb transcripts was reduced to ∼460 bases. However, no difference in the accumulation of RPS3 polypeptide and RNA editing was detected by protein gel blot analysis and cDNA sequencing. Although the deleted region encoded the truncated-atp9 that was edited, no influence on the pattern and frequency of RNA editing of genuine atp9 was evident. The results eliminated rps3 as a candidate for the CMS gene, making preSatp6, a unique ORF fused with CMS atp6, the sole CMS-associated region in sugar beet

Is RNA editing implicated in group II intron survival in the angiosperm mitochondrial genome?

Introns may be considered as optional because they are removed from mRNA molecules, but introns are fairly preserved for unknown reasons. Previously, the mitochondrial rps3 gene of sugar beet (Beta vulgaris L., Caryophyllales) was shown to represent a unique example of an intron loss. We have determined the distribution of the rps3 intron in 19 Caryophyllalean species. The intron was absent from the Amaranthaceae and the Achatocarpaceae. In the Caryophyllaceae, Dianthus japonicus rps3 was pseudogenized but the intronic sequence was retained. Intact intron-bearing rps3 copies were cloned from Portulaca grandiflora and Myrtillocactus geometrizans, members of the sister clade of the Amaranthaceae-Achatocarpaceae-Caryophyllaceae clade. Most of the C-to-U RNA-editing sites in Portulaca and Myrtillocactus rps3 transcripts were homologous in the two species as well as in the sugar beet rps3, which, unlike other 12 rps3 transcripts, lacks editing in the exonic regions around the intron. Provided that the loss of editing preceded the loss of rps3 intron, it appears conceivable that a requirement for editing could have prevented the loss of group-II introns retained in angiosperm mitochondrial genomes. This interpretation is an alternative to the conventional one that views the loss of editing as a mere trace of RNA-mediated gene conversion