Proteinases participating in the processing and activation of prolegumain in primary cultured rat macrophages

The mammalian legumain is a recently identified lysosomal cysteine proteinase belonging to the clan CD and homologous to plant legumain. This enzyme has the characteristic of specifically hydrolyzing peptide bonds after asparagine residues. As in the case of papain-type cysteine proteinases, legumain is synthesized as an inactive zymogen, and processed into a mature form localized in lysosomes. However, the mechanism of its activation remains unclear. In this study, we analyze which types of proteinases may participate in the processing of legumain in rat primary cultured macrophages using various proteinase inhibitors after 24 h treatment with Bafilomycin A1, a vacuolar ATPase inhibitor. The processing of legumain in macrophages was accomplished by papain-type cysteine proteinases other than cathepsin B.     

Estrogenic activity of an environmental pollutant, 2-nitrofluorene,after metabolic activation by rat liver microsomes

In this study, the metabolic activation of 2-nitrofluorene (NF) to estrogenic compounds was examined. NF was negative in estrogen reporter assays using estrogen-responsive yeast and human breast cancer cell line MCF-7. However, the compound exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH. Minor estrogenic activity was observed when liver microsomes of untreated or phenobarbital-treated rats were used instead of those from 3-methylcholanthrene-treated rats. When the compound was incubated with the liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH, 7-hydroxy-2-nitrofluorene (7-OH-NF) was formed as a major metabolite. However, little of the metabolite was formed by liver microsomes of untreated or phenobarbital-treated rats. Rat recombinant cytochrome P450 1A1 exhibited a significant oxidase activity toward NF, affording 7-OH-NF. Liver microsomes of phenobarbital-treated rats also enhanced oxidase activity toward NF. In this case, 9-hydroxy-2-nitrofluorene was formed. 7-OH-NF exhibited a significant estrogenic activity, while the activity of 9-hydroxy-2-nitrofluorene was much lower. These results suggest that the estrogenic activity of NF was due to formation of the 7-hydroxylated metabolite by liver microsomes.     

Effects of different beta73 amino acids on formation of 14-stranded fibers of Hb S versus double-stranded crystals of Hb C-Harlem

Hb S (alpha(2)beta(2)(6Glu-->Val)) forms polymers, while Hb C-Harlem (alpha(2)beta(2)(6Glu-->Val,73Asp-->Asn)) forms crystals upon oversaturation. Since the only difference between the two is the beta73 amino acid, it follows that this site is a critical determinant in promoting either polymerization or crystallization. Beta73 Asp in Hb S forms a hydrogen bond with beta4 Thr, while beta73 Asn in Hb C-Harlem may inhibit this interaction as well as increase the hydrophobicity at the EF helix beta6 Val acceptor sites. Two new beta73 Hb S variants (beta73 His and Leu) were constructed and analyzed to define other amino acids facilitating formation of Hb S-like polymers versus Hb C-Harlem-like crystals. The two variants that were chosen were expected to either (1) enhance formation of the beta73-beta4 hydrogen bond (beta73 His) or (2) inhibit it and increase the hydrophobicity of the EF helix beta6 Val acceptor sites (beta73 Leu). beta73 His Hb S formed fibers but at a lower concentration than Hb S, while beta73 Leu Hb S formed crystals but at a higher concentration than Hb C-Harlem. The solubility of beta73 His Hb S was (1)/(7) of that of Hb S, while the solubility of beta73 Leu Hb S was similar to that of Hb C-Harlem. The delay time prior to polymer or crystal formation depended on Hb concentration. The delay time for beta73 His Hb S was 10(5)-fold shorter than that for Hb S, while that for beta73 Leu Hb S was 10(5)-fold longer in 1.0 M phosphate buffer. NMR results indicate beta73 amino acid changes induce alteration in the beta-chain heme pocket region, while CD results indicate no change in the helical content of the variants. These results suggest that enhancing the beta73-beta4 hydrogen bond and/or induced changes in the heme pocket by the beta73 Asp to His change facilitate formation of Hb S-like fibers. Our results also suggest that removal of the beta73-beta4 hydrogen bond and enhancing the hydrophobicity of the EF helix beta6 Val acceptor sites by the beta73 Asp to Leu or Asn changes delay nuclei formation and facilitate formation of Hb C-Harlem-like crystals.     

Inhibitory effect of curcumin on the invasion of rat ascites hepatoma cells in vitro and ex vivo

Curcumin, a yellow pigment in turmeric, is a food factor withantioxidative activity. The effect of curcumin on the proliferation and invasion of the rat ascites hepatoma AH109Acells was studied in vitro and ex vivo assay systems. Especially, a co-culture system of the hepatoma cellswith mesothelial cells derived from rat mesentery was employed to investigate the invasive motility. Curcumin suppressed thehepatoma slipping motility in a dose-dependent manner up to 5 M and thereafter maintained the effect up to 20 M, whereas this substance exerted little influence on the proliferation of the hepatoma cells at the same concentrations. Sera obtained from rats orally given curcumin also inhibited the AH109A cellular invasive movement when added to the culturemedium. Hepatoma cells previously cultured with hypoxanthineand xanthine oxidase showed a highly invasive activity. Curcumin and curcumin-loaded rat sera suppressed this reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with hypoxanthine, xanthine oxidase and either of curcumin samples. These resultssuggest that the antioxidative property of curcumin may beinvolved in its anti-invasive action.     

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.     

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.