Fluorescent properties of oligonucleotides doubly modified with an indole-fused cytosine analog and 2-aminopurine

Single- and double-stranded oligodeoxynucleotides (ODNs) incorporating both 2-aminopurine (2AP) and an indole-fused cytosine analog (PPI) were prepared and studied for their fluorescence properties. PPI and 2AP can be excited simultaneously by irradiation at 300 nm, with emission observed at 500 nm for PPI and 370 nm for 2AP. We demonstrated the utility of these properties in the dual fluorescence labeling of ODNs giving well-separated emission peaks. In addition, both of the fluorescence signals of a doubly modified ODN changed independently, reflecting the local duplex formation at the regions containing 2AP or PPI. Potential applications of this strategy for the dual fluorescence labeling of oligonucleotides with 2AP and PPI include monitoring local structure alterations of functional nucleic acids and the multiplex detection of biologically important nucleic acids.     

A new class of aggregation inhibitor of amyloid-β peptide based on an O-acyl isopeptide

Inhibition of amyloid β peptide (Aβ) aggregation is a potential therapeutic approach to treat Alzheimer’s disease. We report that an O-acyl isopeptide of Aβ1–42 (1) containing an ester bond at the Gly²⁵-Ser²⁶ moiety inhibits Aβ1–42 fibril formation at equimolar ratio. Inhibitory activity was retained by an N-Me-β-Ala²⁶ derivative (2), in which the ester of 1 was replaced with N-methyl amide to improve chemical stability at physiological pH. Inhibition was verified by fluorescence anisotropy, Western blot, and atomic force microscopy. This report suggests a new class of Aβ aggregation inhibitor based on modification of Aβ1–42 at Gly²⁵-Ser²⁶.     

Intragastric administration of capsiate, a transient receptor potential channel agonist, triggers thermogenic sympathetic responses

The sympathetic thermoregulatory system controls the magnitude of adaptive thermogenesis in correspondence with the environmental temperature or the state of energy intake and plays a key role in determining the resultant energy storage. However, the nature of the trigger initiating this reflex arc remains to be determined. Here, using capsiate, a digestion-vulnerable capsaicin analog, we examined the involvement of specific activation of transient receptor potential (TRP) channels within the gastrointestinal tract in the thermogenic sympathetic system by measuring the efferent activity of the postganglionic sympathetic nerve innervating brown adipose tissue (BAT) in anesthetized rats. Intragastric administration of capsiate resulted in a time- and dose-dependent increase in integrated BAT sympathetic nerve activity (SNA) over 180 min, which was characterized by an emergence of sporadic high-activity phases composed of low-frequency bursts. This increase in BAT SNA was abolished by blockade of TRP channels as well as of sympathetic ganglionic transmission and was inhibited by ablation of the gastrointestinal vagus nerve. The activation of SNA was delimited to BAT and did not occur in the heart or pancreas. These results point to a neural pathway enabling the selective activation of the central network regulating the BAT SNA in response to a specific stimulation of gastrointestinal TRP channels and offer important implications for understanding the dietary-dependent regulation of energy metabolism and control of obesity.     

Treatment with IL-27 attenuates experimental colitis through the suppression of the development of IL-17-producing T helper cells

Inflammatory bowel disease (IBD) represents a group of chronic inflammatory diseases characterized by inflammation and relapsing gastrointestinal disorders. Recent studies have shown that Th17 cells, which are well known as key mediators of chronic inflammation, have a pivotal role in onset and development of IBD in humans and mice, alike. In recent years, it has been reported that IL-27, which is an IL-12-related heterodimeric cytokine consisting of EBI3 and p28 subunits, act directly on naive T cells to suppress the differentiation of Th17 cells. However, effects of exogenous IL-27 on the IBD are not well elucidated. To clarify the suppressive effect of IL-27 treatment on IBD, we applied the flexible linking method to EBI3 and p28 subunits and generated a single-chain human IL-27 (scIL-27). scIL-27 inhibited xenogenic mouse Th17 cell differentiation in vitro, indicating that scIL-27 also acts in mouse immune systems. In a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mouse acute colitis model, subcutaneous scIL-27 treatment significantly improved the colon length, extent of necrosis, and ulceration and thickened epithelium and several pathological scores in a dose-dependent manner. scIL-27 clearly suppressed several inflammatory cytokines, including IL-17, in inflamed colon, except for anti-inflammatory cytokine IL-10. The mesenteric lymph node cells from scIL-27-treated mice also exhibited a reduced inflammatory response and, furthermore, a lower population of Th17 cells than those of PBS-treated mice. Finally, we showed the therapeutic efficacy of scIL-27 on TNBS-induced colitis even after active colitis was established. These results suggest new possible therapeutic approaches for IBD, including disorders such as Crohn's disease and ulcerative colitis.     

Acoustic overstimulation activates 5'-AMP-activated protein kinase through a temporary decrease in ATP level in the cochlear spiral ligament prior to permanent hearing loss in mice

Inner ear disorders are known to be elicited by mitochondrial dysfunction, which decreases the ATP level in the inner ear. 5′-AMP-activated protein kinase (AMPK) is a serine/threonine kinase activated by metabolic stress and by an increase in the AMP/ATP ratio. To elucidate the involvement of AMPK-derived signals in noise-induced hearing loss, we investigated whether in vivo acoustic overstimulation would activate AMPK in the cochlea of mice. Std-ddY mice were exposed to 8 kHz octave band noise at a 90-, 110- or 120-dB sound pressure level (SPL) for 2 h. Exposure to the noise at 110 or 120 dB SPL produced outer hair cell death in the organ of Corti and permanent hearing loss. Exposure to the noise at 120-dB SPL elevated the level of the phospho-AMPK α-subunit (p-AMPKα), without affecting the protein level of this subunit, immediately and at 12-h post-exposure in the lateral wall structures including the spiral ligament and stria vascularis. In the hair cells and spiral ganglion cells, no marked change in the level of p-AMPKα was observed at any time post-exposure. The level of phospho-c-Jun N-terminal kinase (p-JNK) was increased in the lateral wall structures at 2- to 4-h post-exposure at 120 dB SPL. Noise exposure significantly, but temporarily, decreased the ATP level in the spiral ligament, in an SPL-dependent manner at 110 dB and above. Likewise, elevation of p-AMPKα and p-JNK levels was also observed in the lateral wall structures post-exposure to noise at an SPL of 110 dB and above. Taken together, our data suggest that AMPK and JNK were activated by ATP depletion in the cochlear spiral ligament prior to permanent hearing loss induced by in vivo acoustic overstimulation.     

Polyphenolic compounds in clove and pimento and their antioxidative activities

Two new polyphenolic glucosides, 6'-O-acetylisobiflorin (1) and (2S)-3-(4-hydroxy-3-methoxyphenyl)-propane-1,2-diol 1-O-(6'-O-galloyl)-β-D-glucoside (2), were respectively isolated from the flower buds of Syzygium aromaticum and berries of Pimenta dioica. Each structure was elucidated on the basis of spectral analyses (NMR, MS and [α](D)) and chemical conversion. A total of twenty-seven known compounds from the plants were also characterized. The antioxidative activity of their extracts and the twenty-nine isolates including gallo- and ellagitannins was estimated by oxygen radical absorbance capacity (ORAC) assay, and eugenol (3), which was the most abundant ingredient in each plant extract, showed the most potent antioxidative activity [ORAC value of 39,270 μmol TE (trolox equivalent)/g].     

Screening of substrate peptide sequences for tissue-type transglutaminase (TGase 2) using T7 phage cDNA library

Transglutaminase (TGase) is a family of enzymes that catalyzes cross-linking reaction between glutamine- and lysine residue of substrate proteins in several mammalian biological events. Substrate proteins for TGase and their physiological relevance have been still in research, continuously expanding. In this study, we have established a novel screening system that enables identification of cDNA sequence encoding favorable primary structure as a substrate for tissue-type transglutaminase (TGase 2), a multifunctional and ubiquitously expressing isozyme. By the screening, we identified several T7 phage clones that displayed substrate peptides for TGase 2 as a translated product from human brain cDNA library. Among the selected clones, the C-terminal region of IKAP, IkappaB kinase complex associated protein, appeared as a highly reactive substrate sequence for TGase 2. This system will open possibility of rapid identification of substrate sequences for transglutaminases at a genetic level.