Coenzyme Q10 as a potent compound that inhibits Cdt1–geminin interaction

A human replication initiation protein Cdt1 is a very central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by directly binding to it. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1–geminin imbalance, for example by geminin silencing with siRNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. In the present study, we first established a high throughput screening system based on modified ELISA (enzyme linked immunosorbent assay) to identify compounds that interfere with human Cdt1–geminin binding. Using this system, we found that coenzyme Q₁₀ (CoQ₁₀) can inhibit Cdt1–geminin interaction in vitro. CoQ compound is an isoprenoid quinine that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. CoQ₁₀, having a longer isoprenoid chain, was the strongest inhibitor of Cdt1–geminin binding in the tested CoQs, with 50% inhibition observed at concentrations of 16.2 μM. Surface plasmon resonance analysis demonstrated that CoQ₁₀ bound selectively to Cdt1, but did not interact with geminin. Moreover, CoQ₁₀ had no influence on the interaction between Cdt1 and mini-chromosome maintenance (MCM)4/6/7 complexes. These results suggested that CoQ₁₀ inhibits Cdt1–geminin complex formation by binding to Cdt1 and thereby could liberate Cdt1 from inhibition by geminin. Using three-dimensional computer modeling analysis, CoQ₁₀ was considered to interact with the geminin interaction interface on Cdt1, and was assumed to make hydrogen bonds with the residue of Arg243 of Cdt1. CoQ₁₀ could prevent the growth of human cancer cells, although only at high concentrations, and it remains unclear whether such an inhibitory effect is associated with the interference with Cdt1–geminin binding. The application of inhibitors for the formation of Cdt1–geminin complex is discussed.     

Directional degradation of β-chitin by chitinase A1 revealed by a novel reducing end labelling technique

A novel procedure for labelling the molecular ends of β-chitin crystals has been established. By introducing a hydrazide derivative of biotin at the reducing end of a chitin chain, followed by a specific interaction between biotin and streptavidin coupled with a colloidal gold particle, the chain directionality of β-chitin microcrystals could be directly visualized by transmission electron microscopy. This method allowed to certify the parallelism of the chitin chains in the β-chitin microcrystals, and also to label the reducing tips of β-chitin microcrystals degraded by Bacillus circulans chitinase A1. With these substrates, the labelling occurred only at their tapered tip, which indicates that the digestion of these crystals proceeded from their reducing end. The generalization of this new labelling method to other polysaccharide crystals is discussed.     

Enzymatic resolution of 2-phenoxy-1-propanols through the enantioselective acylation mediated by Achromobacter sp. lipase

2-(Substituted phenoxy)-1-propanols, e.g. 2-(4-chlorophenoxy)-1-propanol, belonging to primary alcohols with an oxygen atom at the stereocenter, were resolved with moderate to good enantioselectivity, as judged by the value of enantiomeric ratio E (up to 27), through the enantioselective acylation with vinyl butanoate mediated by the little-known lipase from Achromobacter sp. in diisopropyl ether, after the examination of potential factors affecting the reaction such as organic solvents and acyl donors.     

Bacillus licheniformis protease-catalyzed peptide synthesis via the kinetically controlled approach using the carbamoylmethyl ester as an acyl donor in anhydrous acetonitrile

Summary The couplings ofN-protected amino acid esters with amino acid amides proved to be carried out in anhydrous acetonitrile in the presence ofBacillus licheniformis protease (subtilisin Carlsberg) immobilized on Celite. The maximal peptide yields were obtained with the immobilized enzyme prepared through lyophilization from a pH 10.7 buffer solution. A series of dipeptide syntheses and several segment condensations were achieved generally in high yields by the combined use of the immobilized enzyme prepared from this pH and the carbamoylmethyl ester as the acyl donor.     

Biotransformation of phenolic compounds by the cultured cells of Catharanthus roseus

The cultured cells of Catharanthus roseus were able to convert 2-, 3-, and 4-hydroxybenzyl alcohols into their corresponding hydroxybenzyl-β- -glucopyranosides or β- -glucopyranosylbenzyl alcohols, and then convert 2- and 3-hydroxybenzyl-β- -glucopyranosides into primeverosides and vicianosides. Further, the C. roseus cells were capable of hydroxylation of 2-hydroxybenzoic acid to afford 2,5-dihydroxybenzoic acid and then glucosylation of the newly introduced phenolic hydroxyl group.     

IL-22-Producing RORγt-Dependent Innate Lymphoid Cells Play a Novel Protective Role in Murine Acute Hepatitis

Retinoid-related orphan receptor (ROR) γt is known to be related to the development and function of various immunological compartments in the liver, such as Th17 cells, natural killer T (NKT) cells, and innate lymphoid cells (ILCs). We evaluated the roles of RORγt-expressing cells in mouse acute hepatitis model using RORγt deficient (RORγt^−/−) mice and RAG-2 and RORγt double deficient (RAG-2^−/− × RORγt^−/−) mice. Acute hepatitis was induced in mice by injection with carbon tetrachloride (CCl₄), to investigate the regulation of liver inflammation by RORγt-expressing cells. We detected RORC expression in three compartments, CD4⁺ T cells, NKT cells, and lineage marker-negative SCA-1⁺Thy1^high ILCs, of the liver of wild type (WT) mice. CCl₄-treated RORγt^−/− mice developed liver damage in spite of lack of RORγt-dependent cells, but with reduced infiltration of macrophages compared with WT mice. In this regard, ILCs were significantly decreased in RAG-2^−/− × RORγt^−/− mice that lacked T and NKT cells. Surprisingly, RAG-2^−/− × RORγt^−/− mice developed significantly severer CCl₄-induced hepatitis compared with RAG-2^−/− mice, in accordance with the fact that hepatic ILCs failed to produce IL-22. Lastly, anti-Thy1 monoclonal antibody (mAb), but not anti-NK1.1 mAb or anti-asialo GM1 Ab administration exacerbated liver damage in RAG-2^−/− mice with the depletion of liver ILCs. Collectively, hepatic RORγt-dependent ILCs play a part of protective roles in hepatic immune response in mice.     

Phylogenetic analysis of a novel sulfate-reducing magnetic bacterium, RS-1, demonstrates its membership of the δ-Proteobacteria

Abstract Most of the 16S ribosomal RNA gene of a sulfate-reducing magnetic bacterium, RS-1, was sequenced, and phylogenetic analysis was carried out. The results suggest that RS-1 is a member of the δ-Proteobacteria, and it appears to represent a new genus. RS-1 is the first bacterium reported outside the α-Proteobacteria that contains magnetite inclusions. RS-1 therefore disrupts the correlation between the α-Proteobacteria and possession of magnetite inclusions, and that between the δ-Proteobacteria and possession of greigite inclusions. The existence of RS-1 also suggests that intracellular magnetite biomineralization is of multiple evolutionary origins.     

Movement Patterns and Residency of the Critically Endangered Horseshoe Crab Tachypleus tridentatus in a Semi-Enclosed Bay Determined Using Acoustic Telemetry

The horseshoe crab Tachypleus tridentatus is critically endangered in Japan due to rapidly decreasing numbers resulting from the loss of tidal flats and sandy beaches, and the deterioration of coastal environments. We monitored the year-round migratory patterns and residency of this species in a coastal embayment at Tsuyazaki, Japan, using acoustic telemetry. Total 20 adult crabs (15 males and 5 females) were tagged with ultrasonic transmitters and tracked during two periods (2006–2008; n = 10 and 2007–2009; n = 10). Adult crabs were more active during periods of higher water temperatures and their activity peaked in July, during the spawning period. Water temperature appeared to be one of the key factors influencing the movement patterns for the species. Moreover, the crabs tended to be more active at night than in the day. The nocturnal activity pattern was clearly evident before and during the reproductive period (May–August). Tracking data also showed that one pair-bond was maintained for a maximum of 17 days after the pair-bonded female had spawned. Overall, 11 males (73% of 15 individuals) remained in the bay area over winter, whereas three females (60% of 5 individuals) overwintered outside of the bay. Telemetry data showed that over 60% (13 of 20) of tagged crabs overwintered within the bay where there are sandy beaches, mudflats, and scattered seagrass beds. This year-round residence by adult T. tridentatus in the bay area identifies it as a critical habitat for the management of this species, regardless of life-stage. Not only is it a comprehensive management strategy that effectively reflects this species’ habitat use patterns but also its implementation, such as the establishment of a protected area, would contribute to its conservation.     

Calcitonin gene-related peptide and substance P in the pharynx and lung of the bullfrog,Rana catesbeiana

Indirect double immunofluorescence labelling in the pharynx and lung of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of two neuropeptides. In the pharynx, immunoreactive calcitonin gene-related peptide (CGRP) and substance P (SP) were localized in nerve fibers distributed within and just beneath the ciliated epithelium. In the lung, CGRP and SP were localized in nerve fibers in five principal locations: 1) within the smooth muscle layer in the interfaveolar septa; 2) in the luminal thickened edges of the septa; 3) around the pulmonary vasculature; 4) within, and 5) under the ciliated epithelium. Within the smooth muscle layer in the septa, luminal thickened septa, and around blood vessels, almost all fibers showed coexistence of CGRP and SP. Within and just beneath the ciliated epithelium in the thickened septa, all fibers showed coexistence of CGRP and SP. No immunoreactivity for vasoactive intestinal polypeptide, neuropeptide Y, galanin, somatostatin, FMRFamide, and leucine-and methionine-enkephalins was detected in the nerve fibers within the larynx and the lung. Together with our previous data, the present findings suggest that peptidergic mechanisms are involved in the regulation of amphibian respiratory systems throughout their life.