Oral administration of an immunostimulatory DNA sequence from Bifidobacterium longum improves Th1/Th2 balance in a murine model

We have reported the antiallergic activities of the immunostimulatory oligodeoxynucleotide (ODN) BL07S, identified from genomic DNA of Bifidobacterium longum BB536 from in vitro and in vivo studies. The present study evaluated the efficiency of ODN BL07S in preventing allergic responses by oral administration. Oral administration of BL07S suppressed serum ovalbumin (OVA)-specific immunoglobulin (Ig) E levels and improved the OVA-specific IgG2a/IgG1 ratio. ODN BL07S increased Th1 cytokine and decreased Th2 cytokine production in splenocytes. These results suggest that immunostimulatory ODNs are potentially associated with the antiallergic effects of probiotics.     

Improved physical and biochemical features of a collagen membrane by conjugating with soluble egg shell membrane protein

Soluble egg shell membrane protein (SEP) prepared from the egg shell membrane by performic acid oxidation and pepsin digestion was applied to prepare a membrane conjugated with pepsin-solubilized collagen (PSC) by cross-linking with water-soluble carbodiimide. SEP enabled a conjugated membrane to be constructed with a well-developed network structure having thicker collagen fibrils and higher stability than the PSC membrane alone, as indicated by reduced solubility and collagenase-digestion, increased denaturation temperature, and superior flexibility. The SEP-PSC membrane (SEP:PSC of 1:1, w/w) had lower sweatiness than the PSC membrane. Fibroblasts and keratinocytes adhered to the SEP-PSC-coated wells (SEP:PSC of 1:10, w/w) as a fundamental model of an artificial skin similar to that to the PSC-coated wells due to similar expression level of several cell-adhesion proteins. An SEP-PSC coating accelerated the cell growth of fibroblasts, whereas the cell growth of keratinocytes did not change, or decreased slightly due to more rapid differentiation indicated by a more substantial expression of differentiation marker proteins than a PSC-coating without SEP.     

Autoantigen Ro52 is an E3 ubiquitin ligase

Anti-Ro/SSA antibodies are classic autoantibodies commonly found in patients with Sj?gren's syndrome, a chronic autoimmune disease characterized by dryness of the eyes and mouth. The autoantibodies recognize a RING-finger protein, Ro52, whose function is still unknown. Since many RING-finger proteins have been identified as E3 ubiquitin ligases, this study was designed to determine whether Ro52 functions as an E3 ubiquitin ligase. For this purpose, recombinant Ro52 was purified from bacterial lysate and used to investigate its activity of E3 ubiquitin ligase in vitro. Its enzymatic activity was also tested in HEK293T cells using wild-type Ro52 and its RING-finger mutant. Our results indicated that Ro52 ubiquitinates itself in cooperation with E2 ubiquitin-conjugating enzyme UbcH5B, thereby validating that Ro52 is a RING-finger-type E3 ubiquitin ligase. Importantly, this ubiquitin modification is predominantly monoubiquitination, which does not target Ro52 to the proteasome for degradation.     

Solution structure and dynamics of Ufm1, a ubiquitin-fold modifier 1

The ubiquitin-fold modifier 1 (Ufm1) is one of various ubiquitin-like modifiers and conjugates to target proteins in cells through Uba5 (E1) and Ufc1 (E2). The Ufm1-system is conserved in metazoa and plants, suggesting its potential roles in various multicellular organisms. Herein, we analyzed the solution structure and dynamics of human Ufm1 (hsUfm1) by nuclear magnetic resonance spectroscopy. Although the global fold of hsUfm1 is similar to those of ubiquitin (Ub) and NEDD8, the cluster of acidic residues conserved in Ub and NEDD8 does not exist on the Ufm1 surface. 15N spin relaxation data revealed that the amino acid residues of hsUfm1 exhibiting conformational fluctuations form a cluster at the C-terminal segment and its spatial proximity, which correspond to the versatile ligand-binding sites of Ub and other ubiquitin-like proteins (Ubls). We suggest that Ub and other Ubl-modifiers share a common feature of potential conformational multiplicity, which might be associated with the broad ligand specificities of these proteins.     

Involvement of the ATR- and ATM-dependent checkpoint responses in cell cycle arrest evoked by pierisin-1

Pierisin-1 identified from the cabbage butterfly, Pieris rapae, is a novel mono-ADP-ribosylating toxin that transfers the ADP-ribose moiety of NAD at N(2) of dG in DNA. Resulting mono-ADP-ribosylated DNA adducts cause mutations and the induction of apoptosis. However, little is known about checkpoint responses elicited in mammalian cells by the formation of such bulky DNA adducts. In the present study, it was shown that DNA polymerases were blocked at the specific site of mono-ADP-ribosylated dG, which might lead to the replication stress. Pierisin-1 treatment of HeLa cells was found to induce an intra-S-phase arrest through both ataxia telangiectasia mutated (ATM) and Rad3-related (ATR) and ATM pathways, and ATR pathway also contributes to a G(2)-M-phase delay. In the colony survival assays, Rad17(-/-) DT40 cells showed greater sensitivity to pierisin-1-induced cytotoxicity than wild-type and ATM(-/-) DT40 cells, possibly due to defects of checkpoint responses, such as the Chk1 activation. Furthermore, apoptotic 50-kb DNA fragmentation was observed in the HeLa cells, which was well correlated with occurrence of phosphorylation of Chk2. These results thus suggest that pierisin-1 treatment primarily activates ATR pathway and eventually activates ATM pathway as a result of the induction of apoptosis. From these findings, it is suggested that mono-ADP-ribosylation of DNA causes a specific type of fork blockage that induces checkpoint activation and signaling.     

Functional analysis of spoT, relA and dksA genes on quinolone tolerance in Pseudomonas aeruginosa under nongrowing condition

To assess the contribution of ppGpp in antibiotic tolerance to quinolone in Pseudomonas aeruginosa, knockout mutants of the genes involved or linked with the stringent response, such as relA, spoT and dksA, were constructed and investigated for their antibiotic susceptibility to quinolones. The survival of the dksA and spoT mutants in the presence of 8 microg/ml of ofloxacin and 1 microg/ml of ciprofloxacin were shown to be approximately 20-180 and 10-40 times respectively, higher than the same for the wild type strain. The intracellular levels of ppGpp determined with high performance liquid chromatography (HPLC) demonstrated that spoT and dksA mutants possess higher basal levels of ppGpp. The data suggest that elevated basal levels of ppGpp may be responsible for rendering these mutants tolerant to quinolones and expand the importance of ppGpp as an antimicrobial target in P. aeruginosa.     

Solo/Trio8, a membrane-associated short isoform of Trio, modulates endosome dynamics and neurite elongation

With DNA microarrays, we identified a gene, termed Solo, that is downregulated in the cerebellum of Purkinje cell degeneration mutant mice. Solo is a mouse homologue of rat Trio8-one of multiple Trio isoforms recently identified in rat brain. Solo/Trio8 contains N-terminal sec14-like and spectrin-like repeat domains followed by a single guanine nucleotide exchange factor 1 (GEF1) domain, but it lacks the C-terminal GEF2, immunoglobulin-like, and kinase domains that are typical of Trio. Solo/Trio8 is predominantly expressed in Purkinje neurons of the mouse brain, and expression begins following birth and increases during Purkinje neuron maturation. We identified a novel C-terminal membrane-anchoring domain in Solo/Trio8 that is required for enhanced green fluorescent protein-Solo/Trio8 localization to early endosomes (positive for both early-endosome antigen 1 [EEA1] and Rab5) in COS-7 cells and primary cultured neurons. Solo/Trio8 overexpression in COS-7 cells augmented the EEA1-positive early-endosome pool, and this effect was abolished via mutation and inactivation of the GEF domain or deletion of the C-terminal membrane-anchoring domain. Moreover, primary cultured neurons transfected with Solo/Trio8 showed increased neurite elongation that was dependent on these domains. These results suggest that Solo/Trio8 acts as an early-endosome-specific upstream activator of Rho family GTPases for neurite elongation of developing Purkinje neurons.     

Effect of interleukin-18 on metastasis of mouse osteosarcoma cells

The effect of interleukin-18 (IL-18) on metastasis of highly metastatic LM8 mouse osteosarcoma cells was investigated using nude mice treated with anti-asialo GM1 serum to exclude anti-tumor actions of IL-18 through activation of T and natural killer cells. Injection of LM8 cells which do not express IL-18 receptor β into a tail vain resulted in the formation of pulmonary and hepatic metastatic foci. Daily injection of mice with IL-18 starting the fifth day from the cell injection had no significant effect on the number of metastatic foci, while five daily injections of IL-18 before and after the cell injection resulted in marked decreases. Culture of LM8 cells with IL-18 for 5 days before the injection into mice produced no significant effect on the number of pulmonary and hepatic metastatic foci. In contrast, pretreatment of mice with IL-18 for 5 days before the cell injection markedly decreased metastatic foci. The retention of LM8 cells in the lung 24 h after their injection was also reduced by the pretreatment of mice with IL-18. Serum obtained from mice pretreated with IL-18 for 5 days suppressed mobility of LM8 cells but IL-18 itself did not. These results suggest that IL-18 inhibits metastasis of LM8 cells partly by inducing a factor(s) in the host which suppresses cell mobility.