Inhibitory effect of porphyran, prepared from dried "Nori", on contact hypersensitivity in mice

Porphyran is a major component of the red algae, Porphyra tenera and P. yezoensis, which are processed into a sheet type of dried food, "Nori". Porphyran has been reported to activate murine macrophages by in vitro and i.p. injection studies. The contact hypersensitivity (CHS) reaction in mice is commonly used as a model to evaluate the anti-allergic activity of food and food components. We therefore studied the effect of porphyran on the CHS reaction in Balb/c mice to evaluate anti-allergic activity of porphyran. We found that an oral administration of porphyran (2% in drinking water) suppressed the CHS reaction (ear edema) induced by 2,4,6-trinitrochlorobenzene. We also found that porphyran suppressed the serum level of IgE and the production of interferon-gamma (IFN-gamma) in the challenged ear lobe. We conclude from these results that the CHS reaction was suppressed by oral porphyran due to the decreased serum level of IgE and the production of IFN-gamma in the challenged ear lobe.     

Combined effects of ATP on the therapeutic efficacy of antimicrobial drug regimens against Mycobacterium avium complex infection in mice and roles of cytosolic phospholipase A2-dependent mechanisms in the ATP-mediated potentiation of antimycobacterial host resistance

ATP, which serves as a mediator of intramacrophage signaling pathways through purinoceptors, is known to potentiate macrophage antimycobacterial activity. In this study we examined the effects of ATP in potentiating host resistance to Mycobacterium avium complex (MAC) infection in mice undergoing treatment with a drug regimen using clarithromycin and rifamycin and obtained the following findings. First, the administration of ATP in combination with the clarithromycin and rifamycin regimen accelerated bacterial elimination in MAC-infected mice without causing changes in the histopathological features or the mRNA expression of pro- or anti-inflammatory cytokines from those in the mice not given ATP. Second, ATP potentiated the anti-MAC bactericidal activity of macrophages cultivated in the presence of clarithromycin and rifamycin. This effect of ATP was closely related to intracellular Ca2+ mobilization and was specifically blocked by a cytosolic phospholipase A2 (cPLA2) inhibitor, arachidonyl trifluoromethylketone. Third, intramacrophage translocation of membranous arachidonic acid molecules to MAC-containing phagosomes was also specifically blocked by arachidonyl trifluoromethylketone. In the confocal microscopic observation of MAC-infected macrophages, ATP enhanced the intracellular translocation of cPLA2 into MAC-containing phagosomes. These findings suggest that ATP increases the host anti-MAC resistance by potentiating the antimycobacterial activity of host macrophages and that the cPLA2-dependent generation of arachidonic acid from the phagosomal membrane is essential for such a phenomenon.     

Cdt1 phosphorylation by cyclin A-dependent kinases negatively regulates its function without affecting geminin binding

The current concept regarding cell cycle regulation of DNA replication is that Cdt1, together with origin recognition complex and CDC6 proteins, constitutes the machinery that loads the minichromosome maintenance complex, a candidate replicative helicase, onto chromatin during the G(1) phase. The actions of origin recognition complex and CDC6 are suppressed through phosphorylation by cyclin-dependent kinases (Cdks) after S phase to prohibit rereplication. It has been suggested in metazoan cells that the function of Cdt1 is blocked through binding to an inhibitor protein, geminin. However, the functional relationship between the Cdt1-geminin system and Cdks remains to be clarified. In this report, we demonstrate that human Cdt1 is phosphorylated by cyclin A-dependent kinases dependent on its cyclin-binding motif. Cdk phosphorylation resulted in the binding of Cdt1 to the F-box protein Skp2 and subsequent degradation. In contrast, in vitro DNA binding activity of Cdt1 was inhibited by the phosphorylation. However, geminin binding to Cdt1 was not affected by the phosphorylation. Finally we provide evidence that inactivation of Cdk1 results in Cdt1 dephosphorylation and rebinding to chromatin in murine FT210 cells synchronized around the G(2)/M phase. Taken together, these findings suggest that Cdt1 function is also negatively regulated by the Cdk phosphorylation independent of geminin binding.     

Energetic Frustration of Apomyoglobin Folding: Role of the B Helix

Apomyoglobin folds by a sequential mechanism in which the A, G, and H helix regions undergo rapid collapse to form a compact intermediate onto which the central portion of the B helix subsequently docks. To investigate the factors that frustrate folding, we have made mutations in the N-terminus of the B helix to stabilize helical structure (in the mutant G23A/G25A) and to promote native-like hydrophobic packing interactions with helix G (in the mutant H24L/H119F). The kinetic and equilibrium intermediates of G23A/G25A and H24L/H119F were studied by hydrogen exchange pulse labeling and interrupted hydrogen/deuterium exchange combined with NMR. For both mutants, stabilization of helical structure in the N-terminal region of the B helix is confirmed by increased exchange protection in the equilibrium molten globule states near pH 4. Increased protection is also observed in the GH turn region in the G23A/G25A mutant, suggesting that stabilization of the B helix facilitates native-like interactions with the C-terminal region of helix G. These interactions are further enhanced in H24L/H119F. The kinetic burst phase intermediates of both mutants show increased protection, relative to wild-type protein, of amides in the N-terminus of the B helix and in part of the E helix. Stabilization of the E helix in the intermediate is attributed to direct interactions between E helix residues and the newly stabilized N-terminus of helix B. Stabilization of native packing between the B and G helices in H24L/H119F also favors formation of native-like interactions in the GH turn and between the G and H helices in the ensemble of burst phase intermediates. We conclude that instability at the N-terminus of the B helix of apomyoglobin contributes to the energetic frustration of folding by preventing docking and stabilization of the E helix.     

Pulmonary Collectins Protect Macrophages against Pore-forming Activity of Legionella pneumophila and Suppress Its Intracellular Growth

Pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), play important roles in innate immunity of the lung. Legionella pneumophila is a bacterial respiratory pathogen that can replicate within macrophages and causes opportunistic infections. L. pneumophila possesses cytolytic activity, resulting from insertion of pores in the macrophage membrane upon contact. We examined whether pulmonary collectins play protective roles against L. pneumophila infection. SP-A and SP-D bound to L. pneumophila and its lipopolysaccharide (LPS) and inhibited the bacterial growth in a Ca²⁺-dependent manner. The addition of LPS in the culture blocked the inhibitory effects on L. pneumophila growth by the collectins, indicating the importance of LPS-collectin interaction. When differentiated THP-1 cells were infected with L. pneumophila in the presence of SP-A and SP-D, the number of permeable cells was significantly decreased, indicating that pulmonary collectins inhibit pore-forming activity of L. pneumophila. The number of live bacteria within the macrophages on days 1–4 after infection was significantly decreased when infection was performed in the presence of pulmonary collectins. The phagocytosis experiments with the pH-sensitive dye-labeled bacteria revealed that pulmonary collectins promoted bacterial localization to an acidic compartment. In addition, SP-A and SP-D significantly increased the number of L. pneumophila co-localized with LAMP-1. These results indicate that pulmonary collectins protect macrophages against contact-dependent cytolytic activity of L. pneumophila and suppress intracellular growth of the phagocytosed bacteria. The promotion of lysosomal fusion with Legionella-containing phagosomes constitutes a likely mechanism of L. pneumophila growth suppression by the collectins.     

Effects of chlorogenic acid and its metabolites on spontaneous locomotor activity in mice

Chlorogenic acid possessed a weak caffeine-like psychostimulant property when assessed for its effect on spontaneous locomotor activity in mice. In the evaluation of the effects for the major metabolites of chlorogenic acid which were detected upon incubation with rat feces and/or excreted in urine after oral administration to rats, caffeic and m-coumaric acids were found to be the principal active metabolites, while the others contributed little to this caffeine-like psychostimulant activity.     

A principal role for AtXTH18 in Arabidopsis thaliana root growth: a functional analysis using RNAi plants

Rearrangement of cellulose microfibrils within cell-wall matrices is considered one of the most critical steps in the regulation of both the orientation and extent of cell expansion in plants. Xyloglucan endotransglucosylase/hydrolases (XTHs) are a family of enzymes that mediate the construction and restructuring of load-bearing cross links among cellulose microfibrils. The Arabidopsis thaliana XTH genes AtXTH17, 18, 19, and 20 are phylogenetically closely related to one another and are preferentially expressed in the roots. However, they exhibit different expression profiles within the root and respond to hormonal signals differently. To investigate their functions in root growth, we examined phenotypes of loss-of-function mutants for these genes using T-DNA insertion lines and RNAi plants. These functional analyses disclosed a principal role for the AtXTH18 gene in primary root elongation. Of the four XTH genes, AtXTH18 exhibits the highest level of mRNA expression. We also determined auxin-signaling pathways for these genes using a mutant with a defect in the AXR2/IAA7 gene and found that the expression of AtXTH19 in the elongation/maturation region of the root is under the control of the AXR2/IAA7 signaling pathway.