Induction of IL-10-producing CD4+CD25+ T cells in animal model of collagen-induced arthritis by oral administration of type II collagen

Induction of oral tolerance has long been considered a promising approach to the treatment of chronic autoimmune diseases, including rheumatoid arthritis (RA). Oral administration of type II collagen (CII) has been proven to improve signs and symptoms in RA patients without troublesome toxicity. To investigate the mechanism of immune suppression mediated by orally administered antigen, we examined changes in serum IgG subtypes and T-cell proliferative responses to CII, and generation of IL-10-producing CD4+CD25+ T-cell subsets in an animal model of collagen-induced arthritis (CIA). We found that joint inflammation in CIA mice peaked at 5 weeks after primary immunization with CII, which was significantly less in mice tolerized by repeated oral feeding of CII before CIA induction. Mice that had been fed with CII also exhibited increased serum IgG1 and decreased serum IgG2a as compared with nontolerized CIA animals. The T-cell proliferative response to CII was suppressed in lymph nodes of tolerized mice also. Production of IL-10 and of transforming growth factor-beta from mononuclear lymphocytes was increased in the tolerized animals, and CD4+ T cells isolated from tolerized mice did not respond with induction of IFN-gamma when stimulated in vitro with CII. We also observed greater induction of IL-10-producing CD4+CD25+ subsets among CII-stimulated splenic T cells from tolerized mice. These data suggest that when these IL-10-producing CD4+CD25+ T cells encounter CII antigen in affected joints they become activated to exert an anti-inflammatory effect.     

Inhibitory effects of glycoprotein-120 (G-120) from Ulmus davidiana Nakai on cell growth and activation of matrix metalloproteinases

Excessive breakdown of extracellular matrix by metalloproteinases (MMPs) occurs in many pathological conditions. Consequently, methods for inhibiting MMP activity have therapeutic potential. In this study, we investigated the effect of G-120, a 120 kDa glycoprotein purified from the Oriental herbal plant, Ulmus davidiana Nakai (UDN), on the activity and production of several MMPs by evaluating its growth inhibitory effect on NIH 3T3 cells. Tritium uptake assays showed that proliferation of NIH 3T3 cells was strongly suppressed, and G-120-mediated inhibition of DNA synthesis proved to involve a cytostatic, rather than a cytotoxic, effect, as shown by cytotoxicity and apoptosis assays. More importantly, G-120 strongly reduced the gelatinolytic and collagenase activities of MMP proteins, as well as expression of MMP-2 and MMP-9. Electrophoretic mobility shift assays revealed that it suppressed the DNA binding activity of NF-kappaB. Collectively, our observations show that G-120 strongly inhibits the activation of MMPs and NF-kappaB.     

Expression of thermostable alkaline protease gene from Thermoactinomyces sp. E79 in E. coli and heat activation of the gene product

The expression of Thermoactinomyces sp. E79 protease gene cloned into E. coli was highly host-dependent and the levels of protease expression was most stable in E. coli RR1 and E. coli HB101. Heating the intracellular extract at 70°C for 15 min converted the inactive recombinant E79 protease to its active mature form and also resulted in purification of the enzyme in a single step. Addition of 10 mM CaCl2 to the E79 protease decreased its autolysis and increased its thermal stability. © Rapid Science Ltd. 1998     

Ruegeria meonggei sp. nov., an alphaproteobacterium isolated from ascidian Halocynthia roretzi

A Gram-negative, strictly aerobic, non-flagellated and rod-shaped bacterial strain, designated MA-E2-3^T, was isolated from an ascidian (Halocynthia roretzi) collected from the South Sea, South Korea. Strain MA-E2-3^T was found to grow optimally at 30 °C, at pH 7.0–8.0 and in the presence of 2.0–3.0 % (w/v) NaCl. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain MA-E2-3^T fell within the clade comprising Ruegeria species, clustering consistently with the type strain of Ruegeria halocynthiae, with which it exhibited 98.2 % sequence similarity. Sequence similarities to the type strains of the other recognized Ruegeria species were 94.7–97.7 %. Strain MA-E2-3^T was found to contain Q-10 as the predominant ubiquinone and C_18:1 ω7c as the predominant fatty acid. The major polar lipids of strain MA-E2-3^T were identified as phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid. The DNA G+C content of strain MA-E2-3^T was determined to be 58.0 mol%. Mean DNA–DNA relatedness values between strain MA-E2-3^T and the type strains of four phylogenetically closely related Ruegeria species were in the range of 13–23 %. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain MA-E2-3^T is separated from other Ruegeria species. On the basis of the data presented, strain MA-E2-3^T (=KCTC 32450^T = CECT 8411^T) represents a novel species of the genus Ruegeria, for which the name Ruegeria meonggei sp. nov. is proposed.     

β-propeller phytase hydrolyzes insoluble Ca(2+)-phytate salts and completely abrogates the ability of phytate to chelate metal ions

Phytate is an antinutritional factor that influences the bioavailability of essential minerals by forming complexes with them and converting them into insoluble salts. To further our understanding of the chemistry of phytate's binding interactions with biologically important metal cations, we determined the stoichiometry, affinity, and thermodynamics of these interactions by isothermal titration calorimetry. The results suggest that phytate has multiple Ca(2+)-binding sites and forms insoluble tricalcium- or tetracalcium-phytate salts over a wide pH range (pH 3.0-9.0). We overexpressed the β-propeller phytase from Hahella chejuensis (HcBPP) that hydrolyzes insoluble Ca(2+)-phytate salts. Structure-based sequence alignments indicated that the active site of HcBPP may contain multiple calcium-binding sites that provide a favorable electrostatic environment for the binding of Ca(2+)-phytate salts. Biochemical and kinetic studies further confirmed that HcBPP preferentially recognizes its substrate and selectively hydrolyzes insoluble Ca(2+)-phytate salts at three phosphate group sites, yielding the final product, myo-inositol 2,4,6-trisphosphate. More importantly, ITC analysis of this final product with several cations revealed that HcBPP efficiently eliminates the ability of phytate to chelate several divalent cations strongly and thereby provides free minerals and phosphate ions as nutrients for the growth of bacteria. Collectively, our results provide significant new insights into the potential application of HcBPP in enhancing the bioavailability and absorption of divalent cations.     

<Emphasis Type="Italic">Shewanella upenei</Emphasis> sp. nov., a lipolytic bacterium isolated from bensasi goatfish <Emphasis Type="Italic">Upeneus bensasi</Emphasis>

A Gram-staining-negative, motile, non-spore-forming and rod-shaped bacterial strain, 20-23R^T, was isolated from intestine of bensasi goatfish, Upeneus bensasi, and its taxonomic position was investigated by using a polyphasic study. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain 20-23R^T belonged to the genus Shewanella. Strain 20-23R^T exhibited 16S rRNA gene sequence similarity values of 99.5, 99.2, and 97.5% to Shewanella algae ATCC 51192^T, Shewanella haliotis DW01^T, and Shewanella chilikensis JC5^T, respectively. Strain 20-23R^T exhibited 93.1–96.0% 16S rRNA gene sequence similarity to the other Shewanella species. It also exhibited 98.3–98.4% gyrB sequence similarity to the type strains of S. algae and S. haliotis. Strain 20-23R^T contained simultaneously both menaquinones and ubiquinones; the predominant menaquinone was MK-7 and the predominant ubiquinones were Q-8 and Q-7. The fatty acid profiles of strain 20–23R^T, S. algae KCTC 22552^T and S. haliotis KCTC 12896^T were similar; major components were iso-C_15:0, C_16:0, C_16:1 ω7c and/or iso-C_15:0 2-OH and C_17:1 ω8c. The DNA G+C content of strain 20-23R^T was 53.9 mol%. Differential phenotypic properties and genetic distinctiveness of strain 20–23R^T, together with the phylogenetic distinctiveness, revealed that this strain is distinguishable from recognized Shewanella species. On the basis of the data presented, strain 20-23R^T represents a novel species of the genus Shewanella, for which the name Shewanella upenei sp. nov. is proposed. The type strain is 20–23R^T (=KCTC 22806^T =CCUG 58400^T).     

Streptococcus mutans GS-5 antigen I/II stimulates cell survival in serum deprived-cultures through PI3K/Akt pathways

The antigen I/II (AgI/II) protein is a major surface protein that mediates the attachment of Streptococcus mutans (S. mutans) to the saliva-coated pellicle. Numerous studies have investigated not only the mechanisms by which AgI/II signaling is transduced within cells, but have also attempted to use AgI/II-specific antibodies to treat dental caries and host immune responses. However, little information is available about the effects of AgI/II on basic cellular events in bone cells. In this study, we examined the effects of the His-tagged recombinant N-terminal half of the AgI/II protein (rAgI/II-N) generated from S. mutans GS-5 on the viability, proliferation, and cell cycle progression of primary calvarial osteoblasts. We also investigated the mechanisms involved in the rAgI/II-N-mediated survival of serum-starved osteoblasts. We found that rAgI/II treatment attenuated the serum deprivation-induced decrease in cell viability and proliferation of osteoblasts. rAgI/II-N also prevented the loss of mitochondrial membrane potential (MMP), alterations in levels of two key mitochondrial Bcl-2 family proteins, and the accumulation of numerous cells into the sub-G(1) phase that were observed in serum-starved osteoblasts. Pharmacological inhibitors of phosphoinositide 3-kinase (PI3K), but not of extracellular signal-regulated kinase or Ras, blocked the rAgI/II-N-mediated protection against serum deprivation-induced cell death. Additional experiments revealed that the integrin α5β1-mediated PI3K pathway is required for rAgI/II-N-mediated Akt phosphorylation in osteoblasts. Collectively, these results suggest that rAgI/II-N induces survival signals in serum-starved osteoblasts through integrin-induced PI3K/Akt signaling pathways.     

Ornithinibacillus scapharcae sp. nov., isolated from a dead ark clam

A novel Gram-positive, aerobic, motile, hemolytic, endospore-forming and rod-shaped bacterium TW25^T was isolated from a dead ark clam during a mass mortality event on the South coast of Korea. The strain grew optimally at 30°C, at pH 8–9, and with 1% (w/v) NaCl. The 16S rRNA gene sequence analysis indicated that strain TW25^T was associated with the genus Ornithinibacillus and that it was most closely related to the type strain of Ornithinibacillus californiensis (98.5% similarity). The dominant cellular fatty acids were iso-C15:0, anteiso-C15:0 and C16:0. The peptidoglycan amino acid type was A4β, containing l-ornithine and d-aspartic acid. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, four unidentified phospholipids, two unidentified aminolipids and two unidentified lipids. The major respiratory quinone was menaquinone-7 (MK-7). The G + C content of genomic DNA was 36.7 mol%. DNA–DNA hybridization experiments with related strains revealed lower than 11 ± 3% relatedness. Based on this polyphasic taxonomic study, strain TW25^T represents a novel species in the genus Ornithinibacillus, for which the name Ornithinibacillus scapharcae sp. nov. is proposed. The type strain is TW25^T (=KACC 15116^T = JCM 17314^T).     

Molecular characterization of a tandem-repeat galectin-9 (RuGlec9) from Korean rose bitterling (Rhodeus uyekii)

Galectin-9 is a b-galactoside-binding lectin that regulates many cellular functions, ranging from cell adhesion to pathogen recognition. We isolated and characterized the cDNA of tandem-repeat galectin-9 (RuGlec9) from the Korean rose bitterling (Rhodeus uyekii), an endemic Korean fish belonging to the Acheilognathinae subfamily of the Cyprinidae family. RuGlec9 cDNA is 1486 bp long and encodes a polypeptide of 323 amino acids containing two carbohydrate-recognition domains connected by a linker peptide. The deduced amino acid sequence of RuGlec9 shows 45-84% amino acid sequence identity to other galectin-9 sequences, including those from mammals and fish. RuGlec9 appeared in a large cluster with other galectin-9 sequences from fish and is more closely related to galectin-9 from Danio rerio than to those of other fish and mammals. RuGlec9 mRNA was expressed highly in the testis, spleen, intestine, stomach, and liver, and moderately in the brain, kidney, ovary, and gills of normal Korean rose bitterling. RuGlec9 mRNA expression in the spleen was increased by lipopolysaccharide. These results suggest that RuGlec9 plays a role in innate immunity in Korean rose bitterling.