A novel multifunctional cellulolytic enzyme screened from metagenomic resources representing ruminal bacteria

Metagenomic resources representing ruminal bacteria were screened for novel exocellulases using a robotic, high-throughput screening system, the novel CelEx-BR12 gene was identified and the predicted CelEx-BR12 protein was characterized. The CelEx-BR12 gene had an open reading frame (ORF) of 1140 base pairs that encoded a 380-amino-acid-protein with a predicted molecular mass of 41.8 kDa. The amino acid sequence was 83% identical to that of a family 5 glycosyl hydrolase from Prevotella ruminicola 23. Codon-optimized CelEx-BR12 was overexpressed in Escherichia coli and purified using Ni–NTA affinity chromatography. The Michaelis–Menten constant (K_m value) and maximal reaction velocity (V_max values) for exocellulase activity were 12.92 μM and 1.55 × 10⁻⁴ μmol min⁻¹, respectively, and the enzyme was optimally active at pH 5.0 and 37 °C. Multifunctional activities were observed against fluorogenic and natural glycosides, such as 4-methylumbelliferyl-β-d-cellobioside (0.3 U mg⁻¹), CMC (105.9 U mg⁻¹), birch wood xylan (132.3 U mg⁻¹), oat spelt xylan (67.9 U mg⁻¹), and 2-hydroxyethyl-cellulose (26.3 U mg⁻¹). Based on these findings, we believe that CelEx-BR12 is an efficient multifunctional enzyme as endocellulase/exocellulase/xylanase activities that may prove useful for biotechnological applications.     

Maintenance of cellular ATP level by caloric restriction correlates chronological survival of budding yeast

The free radical theory of aging emphasizes cumulative oxidative damage in the genome and intracellular proteins due to reactive oxygen species (ROS), which is a major cause for aging. Caloric restriction (CR) has been known as a representative treatment that prevents aging; however, its mechanism of action remains elusive. Here, we show that CR extends the chronological lifespan (CLS) of budding yeast by maintaining cellular energy levels. CR reduced the generation of total ROS and mitochondrial superoxide; however, CR did not reduce the oxidative damage in proteins and DNA. Subsequently, calorie-restricted yeast had higher mitochondrial membrane potential (MMP), and it sustained consistent ATP levels during the process of chronological aging. Our results suggest that CR extends the survival of the chronologically aged cells by improving the efficiency of energy metabolism for the maintenance of the ATP level rather than reducing the global oxidative damage of proteins and DNA.     

Marine algal fucoxanthin inhibits the metastatic potential of cancer cells

Metastasis is major cause of malignant cancer-associated mortality. Fucoxanthin has effect on various pharmacological activities including anti-cancer activity. However, the inhibitory effect of fucoxanthin on cancer metastasis remains unclear. Here, we show that fucoxanthin isolated from brown alga Saccharina japonica has anti-metastatic activity. To check anti-metastatic properties of fucoxanthin, in vitro models including assays for invasion, migration, actin fiber organization and cancer cell–endothelial cell interaction were used. Fucoxanthin inhibited the expression and secretion of MMP-9 which plays a critical role in tumor invasion and migration, and also suppressed invasion of highly metastatic B16-F10 melanoma cells as evidenced by transwell invasion assay. In addition, fucoxanthin diminished the expressions of the cell surface glycoprotein CD44 and CXC chemokine receptor-4 (CXCR4) which play roles in migration, invasion and cancer–endothelial cell adhesion. Fucoxanthin markedly suppressed cell migration in wound healing assay and inhibited actin fiber formation. The adhesion of B16-F10 melanoma cells to the endothelial cells was significantly inhibited by fucoxanthin. Moreover, in experimental lung metastasis in vivo assay, fucoxanthin resulted in significant reduction of tumor nodules. Taken together, we demonstrate, for the first time, that fucoxanthin suppresses metastasis of highly metastatic B16-F10 melanoma cells in vitro and in vivo.     

Isolation of (-)-olivil-9′-O-β-d-glucopyranoside from Sambucus williamsii and its antifungal effects with membrane-disruptive action

In this study, we isolated (-)-olivil-9′-O-β-d-glucopyranoside (OLI9G), a phytochemical from the stem bark of Sambucus williamsii, and investigated the antifungal mechanism of OLI9G against Candida albicans. First of all, the antifungal susceptibility testing and hemolysis assay showed that OLI9G exerted a potent activity without hemolysis compared to the activity of amphotericin B. To investigate the mechanism of action of OLI9G, we first examined membrane depolarization using cyanine dye, 3,3′-dipropylthiacarbocyanine iodide (diSC₃5). The results showed that OLI9G significantly changed the fungal membrane potential. To further understand this activity on the membrane, we did the propidium iodide (PI) influx assay. From the results, OLI9G caused membrane permeabilization in the fungal membrane, and the three dimensional (3D) flow cytometric contour plot from the PI influx assay further showed that the cells had shrunk due to the membrane damage. Finally, the membrane-active mechanism of OLI9G was confirmed by synthesizing a model membrane, calcein-encapsulating large unilamellar vesicles (LUVs). The calcein leakage showed the membrane-disruptive effects caused by direct action of OLI9G. In conclusion, the current study suggests that OLI9G exerts its antifungal activity through a membrane-disruptive action.     

Synthesis and dual biological effects of hydroxycinnamoyl phenylalanyl/prolyl hydroxamic acid derivatives as tyrosinase inhibitor and antioxidant

We previously reported that caffeoyl-amino acidyl-hydroxamic acid (CA-Xaa-NHOH) acted as both a good antioxidant and tyrosinase inhibitor, in particular when caffeic acid was conjugated with proline or amino acids having aromatic ring like phenylalanine. Here, various hydroxycinnamic acid (HCA) derivatives were further conjugated with phenylalanyl hydroxamic acid and prolyl hydroxamic acid (HCA-Phe-NHOH and HCA-Pro-NHOH) to study the structure and activity relationship as both antioxidants and tyrosinase inhibitors. When their biological activities were evaluated, all HCA-Phe-NHOH and HCA-Pro-NHOH exhibited enhanced antioxidant activity compared to HCA alone. Moreover, derivatives of caffeic acid, ferulic acid, and sinapic acid inhibited lipid peroxidation more efficiently than vitamin E analogue (Trolox). In addition, derivatives of caffeic acid and sinapic acid efficiently inhibited tyrosinase activity and reduced melanin content in melanocytes Mel-Ab cell.     

Synthesis and biological evaluation of novel 3,4-diaryl lactam derivatives as triple reuptake inhibitors

A series of 3,4-diarylpyrrolidin-2-one was designed, prepared and evaluated as triple reuptake inhibitors for antidepressant. Most compounds exhibited comparable in vitro efficacy as norepinephrine and dopamine transporter reuptake inhibitors. Especially, 2i showed better potency than GBR-12909 (IC₅₀ = 14 nM) which was used as reference compound for dopamine transporter. In addition, 2a and 2b showed inhibition (5.17 μM–85.6 nM) for three transporters.     

Inhibitory effect on NO production of triterpenes from the fruiting bodies of Ganoderma lucidum

Four new lanostane triterpenes, butyl lucidenate P (1), butyl lucidenate D₂ (2), butyl lucidenate E₂ (3) and butyl lucidenate Q (4) along with 11 known compounds (5–15) were isolated from the fruiting bodies of Ganoderma lucidum. Their chemical structures were established mainly by 1D and 2D NMR techniques and mass spectrometry. Their anti-inflammatory activity was evaluated against LPS-induced NO production in macrophage RAW 264.7 cells. Compounds 1, 3, 4, 9, 10 and 15 showed inhibitory potency with IC₅₀ values of 7.4, 6.4, 4.3, 9.4, 9.2 and 4.5 μM, respectively. Compounds 1, 3 and 15 dose-dependently reduced the LPS-induced iNOS expressions. Preincubation of cell with 1, 3 and 15 significantly suppressed LPS-induced expression of COX-2 protein.     

Phylogenetic analyses of anatomical and nuclear SSU rDNA sequence data indicate that the Dasyaceae and Delesseriaceae (Ceramiales,Rhodophyta) are polyphyletic

The aim of the current investigation was to test the general convention that the Dasyaceae, Delesseriaceae and Rhodomelaceae are all monophyletic families of the red algal order Ceramiales. Phylogenetic relationships among 45 ceramialean taxa were determined, including eight ceramiacean, 18 dasyacean, nine delesseriacean and eight rhodomelacean species, plus two of uncertain ceramialean affinities, based on 34 anatomical characters and nuclear small subunit (SSU) rDNA sequences. Results from our ‘total-evidence’ approach were consistent with the notion that the Dasyaceae, Delesseriaceae and Rhodomelaceae have evolved from a common ancestor within a paraphyletic Ceramiaceae. Our data indicate, however, that the Rhodomelaceae alone is monophyletic at the family level, with both the Dasyaceae and Delesseriaceae polyphyletic. In particular we resolved two independent and well-supported lineages for the included Dasyaceae, viz., a Dasya group and a Heterosiphonia group, which were as distinct from one another as they were from the Rhodomelaceae and the various lineages of Delesseriaceae. The molecular data alone were equivocal on the issue of monophyly of the Dasyaceae. We therefore advocate conservative taxonomic revisions as an interim step towards eventual resolution of familial-level taxonomy in the Ceramiales. Based on our results, the Heterosiphonioideae H.-G. Choi, Kraft, I.K. Lee et G.W. Saunders subfam. nov. is proposed for Heterosiphonia and five closely related genera, and the Dasyoideae Schmitz et Falkenberg is emended for the remaining taxa. Although the Dasyoideae is a natural group, it is in need of a thorough systematic reinvestigation at the generic level. Our analyses indicate that the genus Dasya is polyphyletic or paraphyletic in excluding Dasysiphonia, Eupogodon and Rhodoptilum and that Heterosiphonia japonica also has affinities to this group, taxonomic issues that will be addressed in detail elsewhere.