T cell hyporesponsiveness induced by oral administration of ovalbumin is associated with impaired NFAT nuclear translocation and p27kip1 degradation

Oral tolerance is an important physiological component of the immune system whereby the organism avoids dangerous reactions such as hypersensitivity to ingested food proteins and other luminal Ags which may cause tissue damage and inflammation. In addition, it has been shown in animal models and in humans that oral tolerance can be applied to controlling undesired immune responses, including autoimmune diseases, allergies, and organ transplant rejections. However, the molecular mechanisms of oral tolerance have been poorly defined. In this study, we investigated the molecular basis underlying the hyporesponsiveness of orally tolerant CD4 T cells using a TCR transgenic mouse system in which oral tolerance was induced by long-term feeding with high dose Ag. We demonstrate that the hyporesponsive state of the CD4 T cells was maintained by a selective impairment in the TCR-induced calcium/NFAT signaling pathway and in the IL-2R-induced degradation of p27(kip1) and cell cycle progression. Thus, physiological mucosal tolerance is revealed to be associated with a unique type of T cell hyporesponsiveness which differs from previously described anergic T cells.     

Roles for the two-hybrid system in exploration of the yeast protein interactome

Comprehensive analysis of protein-protein interactions is a challenging endeavor of functional proteomics and has been best explored in the budding yeast. The yeast protein interactome analysis was achieved first by using the yeast two-hybrid system in a proteome-wide scale and next by large-scale mass spectrometric analysis of affinity-purified protein complexes. While these interaction data have led to a number of novel findings and the emergence of a single huge network containing thousands of proteins, they suffer many false signals and fall short of grasping the entire interactome. Thus, continuous efforts are necessary in both bioinformatics and experimentation to fully exploit these data and to proceed another step forward to the goal. Computational tools to integrate existing biological knowledge buried in literature and various functional genomic data with the interactome data are required for biological interpretation of the huge protein interaction network. Novel experimental methods have to be developed to detect weak, transient interactions involving low abundance proteins as well as to obtain clues to the biological role for each interaction. Since the yeast two-hybrid system can be used for the mapping of the interaction domains and the isolation of interaction-defective mutants, it would serve as a technical basis for the latter purpose, thereby playing another important role in the next phase of protein interactome research.     

The 70-kDa heat shock protein response in two intertidal sculpins,Oligocottus maculosus and O. snyderi: relationship of hsp70 and thermal tolerance

The role that hsp70 plays in influencing thermal tolerance of a whole animal is not clearly understood. We explored this question by examining liver hsp70 response in the tidepool sculpin (Oligocottus maculosus) and fluffy sculpin (O. snyderi), which have distinct distribution patterns in the intertidal zone. The tidepool sculpin is in upper and lower tidepools, while the fluffy sculpin is exclusively in lower tidepools during a low tide. We conducted experiments in order to investigate: (1) habitat water temperatures; (2) upper thermal tolerance limits; (3) the cellular hsp70 response to changes in water temperature in nature; (4) induction temperatures for hepatic hsp70 and hsp70 mRNA; and (5) effects of long-term heat stress on liver hsp70 levels, in these sculpins. Accordingly, we found: (1) the tidepool sculpin was exposed to a wider temperature range in nature; (2) the tidepool sculpin had higher lethal and induction temperatures for hsp70; (3) the liver hsp70 level of the tidepool sculpin was less sensitive to changes in water temperatures; and (4) the tidepool sculpin had higher constitutive hsp70 levels in nature, compared with the fluffy sculpin. From these results, we proposed that the less thermally sensitive tidepool sculpin may enhance its thermal tolerance by having a large pool of cellular hsp70, thus allowing it to inhabit the upper intertidal zone with relatively large and unpredictable fluctuations in environmental variables.     

Leukemia inhibitory factor relaxes arteries through endothelium-dependent mechanism

Leukemia inhibitory factor (LIF) is a cytokine, which inhibits angiogenesis and decreases endothelial cell proliferation and migration, suggesting that LIF may modulate vascular tone. In this study, we examined the effects of LIF on the tone of rat arteries. The isometric tension of ring preparations from rat superior mesenteric arteries was continuously measured. LIF relaxed the mesenteric arteries in a dose-dependent manner, when the arterial rings were precontracted with phenylephrine. The relaxation was totally inhibited by mechanical removal of endothelium. N(G)-nitro-L-arginine methyl ester did not affect the relaxation by LIF. Ca(2+)-dependent K channel (KCa) blockers, apamin with charybdotoxin, inhibited the relaxation by LIF. Catalase, an enzyme which scavenges hydrogen peroxide, also inhibited the relaxation by LIF. Endothelium-derived hyperpolarizing factor relaxes smooth muscle cells and the effect is blocked by KCa and catalase. Our results suggest that LIF regulates vascular tone through the effect of this factor.     

Tyr-199 and charged residues of pharaonis Phoborhodopsin are important for the interaction with its transducer

pharaonis Phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a retinal protein in Natronobacterium pharaonis and is a receptor of negative phototaxis. It forms a complex with its transducer, pHtrII, in membranes and transmits light signals by protein-protein interaction. Tyr-199 is conserved completely in phoborhodopsins among a variety of archaea, but it is replaced by Val (for bacteriorhodopsin) and Phe (for sensory rhodopsin I). Previously, we (Sudo, Y., M. Iwamoto, K. Shimono, and N. Kamo, submitted for publication) showed that analysis of flash-photolysis data of a complex between D75N and the truncated pHtrII (t-Htr) give a good estimate of the dissociation constant K(D) in the dark. To investigate the importance of Tyr-199, K(D) of double mutants of D75N/Y199F or D75N/Y199V with t-Htr was estimated by flash-photolysis and was approximately 10-fold larger than that of D75N, showing the significant contribution of Tyr-199 to binding. The K(D) of the D75N/t-Htr complex increased with decreasing pH, and the data fitted well with the Henderson-Hasselbach equation with a single pK(a) of 3.86 +/- 0.02. This suggests that certain deprotonated carboxyls at the surface of the transducer (possibly Asp-102, Asp-104, and Asp-106) are needed for the binding.     

Probing the proton channel and the retinal binding site of Natronobacterium pharaonis sensory rhodopsin II

The sensory rhodopsin II from Natronobacterium pharaonis (NpSRII) was mutated to try to create functional properties characteristic of bacteriorhodopsin (BR), the proton pump from Halobacterium salinarum. Key residues from the cytoplasmic and extracellular proton transfer channel of BR as well as from the retinal binding site were chosen. The single site mutants L40T, F86D, P183E, and T204A did not display altered function as determined by the kinetics of their photocycles. However, the photocycle of each of the subsequent multisite mutations L40T/F86D, L40T/F86D/P183E, and L40T/F86D/P183E/T204A was quite different from that of the wild-type protein. The reprotonation of the Schiff base could be accelerated approximately 300- to 400-fold, to approximately two to three times faster than the corresponding reaction in BR. The greatest effect is observed for the quadruple mutant in which Thr-204 is replaced by Ala. This result indicates that mutations affecting conformational changes of the protein might be of decisive importance for the creation of BR-like functional properties.     

Muscle-specific RING finger-1 interacts with titin to regulate sarcomeric M-line and thick filament structure and may have nuclear functions via its interaction with glucocorticoid modulatory element binding protein-1

Signaling from receptor tyrosine kinases (RTKs)* requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1-Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. Here, we demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1-Grb2 (S/G) or a Sos-1-E3b1-Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1-Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell.     

Simple non-ion-paired high-performance liquid chromatographic method for simultaneous quantitation of carboxylate and lactone forms of 14 new camptothecin derivatives

SN-38 (7-ethyl-10-hydroxycamptothecin) is an active metabolite derived from the semi-synthetic compound camptothecin (CPT) named Irinotecan (CPT-11). The antitumor activity of SN-38 is 1000-fold more potent than the parent CPT-11. Fourteen new derivatives of camptothecin have recently been developed by Yakult Honsha (Tokyo, Japan). Here we describe a simple and cost-effective high-performance liquid chromatography (HPLC) method without an ion-pairing agent, which allows the simultaneous determination of both lactone and carboxylate forms of SN-38 and other camptothecin derivatives. A weak linear relationship between the HPLC retention factors (ln k') and the cellular concentrations of these compounds was observed. These results suggest that low-polarity compounds easily accumulate in cancer cells and may circumvent drug resistance. The HPLC analysis herein described is expected to greatly assist in derivative synthesis and chemical modification of camptothecin-based antitumor drugs.