Characterization of a novel ginsenoside-hydrolyzing α-l-arabinofuranosidase, AbfA, from Rhodanobacter ginsenosidimutans Gsoil 3054T

The gene encoding an α-l-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-[α-l-arabinofuranosyl-(1–6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054^T, and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K _m values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V _max values of 27.1 ± 1.7 and 49.6 ± 4.1 μmol min⁻¹ mg⁻¹ of protein for p-nitrophenyl-α-l-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar groups. These results are the first report of a glycoside hydrolase family 51 α-l-arabinofuranosidase that can transform ginsenoside Rc to Rd.     

Elevated lipopolysaccharide in the colon evokes intestinal inflammation, aggravated in immune modulator-impaired mice

Frequency of gram-negative bacteria is markedly enhanced in inflamed gut, leading to augmented LPS in the intestine. Although LPS in the intestine is considered harmless and, rather, provides protective effects against epithelial injury, it has been suggested that LPS causes intestinal inflammation, such as necrotizing enterocolitis. Therefore, direct effects of LPS in the intestine remain to be studied. In this study, we examine the effect of LPS in the colon of mice instilled with LPS by rectal enema. We found that augmented LPS on the luminal side of the colon elicited inflammation in the small intestine remotely, not in the colon; this inflammation was characterized by body weight loss, increased fluid secretion, enhanced inflammatory cytokine production, and epithelial damage. In contrast to the inflamed small intestine induced by colonic LPS, the colonic epithelium did not exhibit histological tissue damage or inflammatory lesions, although intracolonic LPS treatment elicited inflammatory cytokine gene expression in the colon tissues. Moreover, we found that intracolonic LPS treatment substantially decreased the frequency of immune-suppressive regulatory T cells (CD4(+)/CD25(+) and CD4(+)/Foxp3(+)). We were intrigued to find that LPS-promoted intestinal inflammation is exacerbated in immune modulator-impaired IL-10(-/-) and Rag-1(-/-) mice. In conclusion, our results provide evidence that elevated LPS in the colon is able to cause intestinal inflammation and, therefore, suggest a physiological explanation for the importance of maintaining the balance between gram-negative and gram-positive bacteria in the intestine to maintain homeostasis in the gut.     

Evaluation of novel antioxidant triterpenoid saponins from the halophyte Salicornia herbacea

As a part of an ongoing search for novel antioxidants from the salt marsh plants, bioactivity-isolation and structure determination of constituents from Salicornia herbacea were performed. One new triterpenoid saponin (4), along with three known saponins (1-3), has been isolated from n-BuOH fraction of S. herbacea. On the basis of the spectroscopic methods, the structure of the new saponin 4 was elucidated as 3β-hydroxy-23-oxo-30-noroleana-12, 20(29)-diene-28-oic acid 3-O-β-D-glucuronopyranosyl-28-O-β-d-glucopyranoside. Scavenging effects of saponins 1-4 were examined on 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical and peroxynitrite. Particularly, saponin 3 exerted significant antioxidant activity on both authentic peroxynitrite and peroxynitrite generated from morpholinosydnonimine (SIN-1).     

Proteomics-based strategy to delineate the molecular mechanisms of RhoGDI2-induced metastasis and drug resistance in gastric cancer

Rho GDP dissociation inhibitor 2 (RhoGDI2) was initially identified as a regulator of the Rho family of GTPases. Our recent works suggest that RhoGDI2 promotes tumor growth and malignant progression, as well as enhances chemoresistance in gastric cancer. Here, we delineate the mechanism by which RhoGDI2 promotes gastric cancer cell invasion and chemoresistance using two-dimensional gel electrophoresis (2-DE) on proteins derived from a RhoGDI2-overexpressing SNU-484 human gastric cancer cell line and control cells. Differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 47 differential protein spots were identified; 33 were upregulated, and 14 were downregulated by RhoGDI2 overexpression. Upregulation of SAE1, Cathepsin D, Cofilin1, CIAPIN1, and PAK2 proteins was validated by Western blot analysis. Loss-of-function analysis using small interference RNA (siRNA) directed against candidate genes reveals the need for CIAPIN1 and PAK2 in RhoGDI2-induced cancer cell invasion and Cathepsin D and PAK2 in RhoGDI2-mediated chemoresistance in gastric cancer cells. These data extend our understanding of the genes that act downstream of RhoGDI2 during the progression of gastric cancer and the acquisition of chemoresistance.     

Phenotypic characterization of Hanwoo (native Korean cattle) cloned from somatic cells of a single adult

We investigated phenotypic differences in Hanwoo cattle cloned from somatic cells of a single adult. Ten genetically identical Hanwoo were generated by somatic cell nuclear transfer from a single adult. Weights at birth, growing pattern, horn and noseprint patterns were characterized to investigate phenotypic differences. The weights of clones at 6 and 12 months were slightly heavier than that of the donor. A horn pattern analysis revealed that seven clones had exactly the same horn pattern as the donor cow, whereas three were different. Although similarities such as general appearance can often be used to identify individual cloned animals, no study has characterized noseprint patterns for this end. A noseprint pattern analysis of all surviving clones showed that all eight animals had distinct noseprints. Four were similar to the donor, and the remaining four had more secondary-like characteristics.     

Ramlibacter ginsenosidimutans sp. nov., with ginsenoside-converting activity

A novel beta-proteobacterium, designated BXN5-27(T), was isolated from soil of a ginseng field of Baekdu Mountain in China, and was characterized using a polyphasic approach. The strain was Gram-staining-negative, aerobic, motile, non-spore-forming, and rod shaped. Strain BXN5-27(T) exhibited beta-glucosidase activity that was responsible for its ability to transform ginsenoside Rb? (one of the dominant active components of ginseng) to compound Rd. Phylogenetic analysis based on 16S rRNA gene sequences showed that this strain belonged to the family Comamonadaceae; it was most closely related to Ramlibacter henchirensis TMB834(T) and Ramlibacter tataouinensis TTB310(T) (96.4% and 96.3% similarity, respectively). The G+C content of the genomic DNA was 68.1%. The major menaquinone was Q-8. The major fatty acids were C??:?, summed feature 4 (comprising C??:? omega7c and/or iso-C??:? 2OH), and C??:? cyclo. Genomic and chemotaxonomic data supported the affiliation of strain BXN5-27(T) to the genus Ramlibacter. However, physiological and biochemical tests differentiated it phenotypically from the other established species of Ramlibacter. Therefore, the isolate represents a novel species, for which the name Ramlibacter ginsenosidimutans sp. nov. is proposed, with the type strain being BXN5-27(T) (= DSM 23480(T) = LMG 24525(T) = KCTC 22276(T)).