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.     

Nucleosomal arrays self‐assemble into supramolecular globular structures lacking 30‐nm fibers

The existence of a 30‐nm fiber as a basic folding unit for DNA packaging has remained a topic of active discussion. Here, we characterize the supramolecular structures formed by reversible Mg²⁺‐dependent self‐association of linear 12‐mer nucleosomal arrays using microscopy and physicochemical approaches. These reconstituted chromatin structures, which we call “oligomers”, are globular throughout all stages of cooperative assembly and range in size from ~50 nm to a maximum diameter of ~1,000 nm. The nucleosomal arrays were packaged within the oligomers as interdigitated 10‐nm fibers, rather than folded 30‐nm structures. Linker DNA was freely accessible to micrococcal nuclease, although the oligomers remained partially intact after linker DNA digestion. The organization of chromosomal fibers in human nuclei in situ was stabilized by 1 mM MgCl₂, but became disrupted in the absence of MgCl₂, conditions that also dissociated the oligomers in vitro. These results indicate that a 10‐nm array of nucleosomes has the intrinsic ability to self‐assemble into large chromatin globules stabilized by nucleosome–nucleosome interactions, and suggest that the oligomers are a good in vitro model for investigating the structure and organization of interphase chromosomes.     

Protein S-guanylation by the biological signal 8-nitroguanosine 3',5'-cyclic monophosphate

The signaling pathway of nitric oxide (NO) depends mainly on guanosine 3',5'-cyclic monophosphate (cGMP). Here we report the formation and chemical biology of a nitrated derivative of cGMP, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), in NO-mediated signal transduction. Immunocytochemistry demonstrated marked 8-nitro-cGMP production in various cultured cells in an NO-dependent manner. This finding was confirmed by HPLC plus electrochemical detection and tandem mass spectrometry. 8-Nitro-cGMP activated cGMP-dependent protein kinase and showed unique redox-active properties independent of cGMP activity. Formation of protein Cys-cGMP adducts by 8-nitro-cGMP was identified as a new post-translational modification, which we call protein S-guanylation. 8-Nitro-cGMP seems to regulate the redox-sensor signaling protein Keap1, via S-guanylation of the highly nucleophilic cysteine sulfhydryls of Keap1. This study reveals 8-nitro-cGMP to be a second messenger of NO and sheds light on new areas of the physiology and chemical biology of signal transduction by NO.     

Eudistomidin G,a new β-carboline alkaloid from the Okinawan marine tunicate Eudistoma glaucus and structure revision of eudistomidin B

A new β-carboline alkaloid, eudistomidin G (1), has been isolated from the Okinawan marine tunicate Eudistoma glaucus, and the structure was elucidated from spectroscopic data. Furthermore, the structure of eudistomidin B (2), which has been isolated from the same tunicate, was revised from 2a to 2b by detailed analyses of spectroscopic data. Asymmetric synthesis of the revised structure (2b) of eudistomidin B (2) and its (1S,10S)-diastereomer (2c) has been accomplished with the Noyori catalytic asymmetric hydrogen-transfer reaction. The absolute configuration of eudistomidin B (2) was confirmed to be 2b possessing (1R,10S)-configuration, from comparison of the ¹H NMR data, CD spectra, [α]_D values, and HPLC analysis of 2b, 2c, and natural eudistomidin B.     

Electron acquisition system constructed from an NAD-independent D-lactate dehydrogenase and cytochrome c2 in Rhodopseudomonas palustris No. 7

The activities of NAD-independent D- and L-lactate dehydrogenases (D-LDH, L-LDH) were detected in Rhodopseudomonas palustris No. 7 grown photoanaerobically on lactate. One of these enzymes, D-LDH, was purified as an electrophoretically homogeneous protein (M(r), about 235,000; subunit M(r) about 57,000). The pI was 5.0. The optimum pH and temperature of the enzyme were pH 8.5 and 50 degrees C, respectively. The Km of the enzyme for D-lactate was 0.8 mM. The enzyme had narrow substrate specificity (D-lactate and DL-2-hydroxybutyrate). The enzymatic activity was competitively inhibited by oxalate (Ki, 0.12 mM). The enzyme contained a FAD cofactor. Cytochrome c(2) was purified from strain No. 7 as an electrophoretically homogeneous protein. Its pI was 9.4. Cytochrome c(2) was reduced by incubating with D-LDH and D-lactate.     

Highly potent PDE4 inhibitors with therapeutic potential

Based on the hypothesis that the dose-limiting side effects of PDE4 inhibitors could be mediated via the central nervous system (CNS), design and synthesis of a hydrophilic analogue is considered to be one approach to improving the side-effect profile of Ariflo 1. Water-soluble piperidine derivatives were found to possess therapeutic potential.     

Essential structural features of acetogenins: role of hydroxy groups adjacent to the bis-THF rings

The presence of two hydroxy groups adjacent to the THF ring(s) is a common structural feature of natural acetogenins. To elucidate the role of each hydroxy group in the inhibitory action of acetogenins, we synthesized three acetogenin analogues which lack either or both of the hydroxy groups, and investigated their inhibitory activities with bovine heart mitochondrial complex I. Our results indicate that the presence of either of the two hydroxy groups sufficiently sustains a potent inhibitory effect.     

Expression of Fibroblast growth factor 19 (Fgf19) during chicken embryogenesis and eye development, compared with Fgf15 expression in the mouse

The normal development of eyes relies on proper signaling through Fibroblast growth factor (FGF) receptors, but the source and identity of cognate ligands have remained largely unknown. We have found that Fgf19 is expressed in the developing chicken retina. In situ hybridization discloses dynamic expression patterns for Fgf19 in the optic vesicle, lens primordia and retinal horizontal cells. Overall expression pattern of Fgf19 during chicken embryogenesis was also examined: Fgf19 is expressed in the regions associated with cranial placodes induction, boundary regions of rhombomeres, somites, specific groups of neural cells in midbrain, hindbrain, and those derived from epibranchial placodes, and the apical ectodermal ridge of limb buds. Expression pattern of the Fgf19-orthologous gene Fgf15 was further examined in the mouse developing eye. Fgf15 is expressed in the optic vesicle, a subset of progenitor cells of neural retina, and emerging ganglion and amacrine cells during retinogenesis.     

Highly potent PDE4 inhibitors with therapeutic potential

The hypothesis that the dose-limiting side effects of PDE4 inhibitors could be mediated via the central nervous system prompted us to design and synthesize a hydrophilic piperidine analog to improve the side effect profile of Ariflo 1, which is an orally active second-generation PDE4 inhibitor. During evaluation of various water-soluble piperidine analogs, 2a-b, 11b-14b, and 17a showed therapeutic potential in cross-species comparison studies. The following three findings were obtained: (1) The hydroxamic acid group, a well known metal chelator, caused a marked increase of inhibitory activity. (2) Water-soluble piperidine analogs lacked the configurational isomerism of Ariflo 1 without loss of inhibitory activity. (3) Replacement of the 4-methoxy residue with a difluoromethoxy residue led to an increase of in vivo potency. Structure-activity relationships are presented. Single-dose rat pharmacokinetic data for 11b, 12b, and 17a are also presented.     

Design and synthesis of novel hydrophilic spacers for the reduction of nonspecific binding proteins on affinity resins

Tubulin and actin often bind nonspecifically to affinity chromatography resins, complicating research toward identifying the cellular targets. Reduction of nonspecific binding proteins is important for success in finding such targets. We herein disclose the design, synthesis, and effectiveness in reduction of nonspecific binding proteins, of novel hydrophilic spacers (2-5), which were introduced between matrices and a ligand. Among them, tartaric acid derivative (5) exhibited the most effective reduction of nonspecific binding proteins, whilst maintaining binding of the target protein. Introduction of 5 on TOYOPEARL reduced tubulin and actin by almost 65% and 90% compared to that without the hydrophilic spacer, respectively, with effective binding to the target protein, FKBP12.