Helical twists of collagen model peptides

Average helical twists were calculated by the method of Sugeta and Miyazawa (Biopolymers 1967, 5, 673-679) for all of the collagen model peptides analyzed to date. Calculation of the helical twists of all triplets in each peptide strand provided novel insights for several model peptides. In the (Pro-Pro-Gly)n (n = 9 and 10), the helical twists showed cyclic fluctuations between 40 and 65 degrees with a 20 A period, suggesting that their molecular conformations were close enough to the ideal 7/2-helix to show the helical repeat of 20 A. Rather small helical twists in the guest regions of IBP in complex and T3-785 were attributed to the interaction with Integrin I domain and a relaxed conformation caused by three consecutive triplets lacking imino acid residues, respectively. Although most of the triplets used in this study were imino acid-rich triplets, helical twists were scattered in a wide range from 30 to 70 degrees with an overall average of 52.6 degrees . This distribution of helical twists indicated a strong preference for the 7/2-helical conformation (51.4 degrees ) rather than the 10/3-helical model (36 degrees ).     

Rac1 negatively regulates lipopolysaccharide-induced IL-23 p19 expression in human macrophages and dendritic cells and NF-kappaB p65 trans activation plays a novel role

IL-23 is a heterodimeric cytokine composed of a unique p19 subunit and of a p40 subunit that is also common to IL-12. We defined the distinct signaling mechanisms that regulate the LPS-mediated induction of IL-23 p19 and p40 in human macrophages and dendritic cells. We found that the overexpression of dominant-negative Rac1 (N17Rac1) enhanced LPS-induced IL-23 p19 expression but did not alter p40 expression or IL-12 p70 production in PMA-treated THP-1 macrophages and in human monocyte-derived dendritic cells. Although the inhibition of either p38 MAPK or JNK enhanced LPS-induced p19 expression, N17Rac1 did not influence either p38 MAPK or JNK activation. By contrast, N17Rac1 augmented both NF-kappaB gene expression and p65 trans activation stimulated by LPS without affecting the degradation of IkappaB-alpha or DNA binding to NF-kappaB. Furthermore, small interference RNA of NF-kappaB p65 attenuated cellular amounts of p65 and suppressed LPS-induced p19 expression but did not affect p40 expression. Our findings indicate that Rac1 negatively controls LPS-induced IL-23 p19 expression through an NF-kappaB p65 trans activation-dependent, IkappaB-independent pathway and that NF-kappaB p65 regulates LPS-induced IL-23 p19, but not p40, expression, which causes differences in the control of IL-23 p19 and p40 expression by Rac1.     

Phosphorylation of human INO80 is involved in DNA damage tolerance

Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in the DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells.     

Root–shoot communication of the seedlings of Japanese larch and a hybrid species grown in different soil-temperature regimes

We studied the effects of soil temperature (7, 15, and 25°C) on the growth and photosynthesis of seedlings of the Japanese larch (Larix kaempferi) and its hybrid larch (L. gmelinii × L. kaempferi) to simulate early stages of regeneration after disturbance. At a soil temperature of 7°C, the root length per unit root biomass, chlorophyll concentration, and photosynthetic nitrogen-use efficiency (PNUE) were markedly lower in the Japanese larch than in the hybrid larch, which may indicate that the hybrid larch is better at acquiring water and nutrients. At ambient temperatures of 17–25°C, the light-saturated photosynthesis rate (P _sat) of both seedlings grown at a soil temperature of 7°C was lower than at 15 or 25°C. By the 16th week, the needle area, root area, and biomass in seedlings of both types were lower at a soil temperature of 7°C than at soil temperatures of 15 or 25°C. At a soil temperature of 25°C, P _sat and nitrogen uptake were lower in both larch species than at 15°C. The growth of the Japanese larch declined sharply from 15 to 25°C; however, the growth of the hybrid larch decreased only slightly from 15 to 25°C. We conclude that an increased soil temperature may retard larch growth in cold regions, especially in the case of the Japanese larch.     

Skin atrophy in cytoplasmic SOD-deficient mice and its complete recovery using a vitamin C derivative

Intrinsic skin ageing is characterized by atrophy and loss of elasticity. Although the skin hypertrophy induced by photoageing has been studied, the molecular mechanisms of skin atrophy during ageing remain unclear. Here, we report that copper/zinc superoxide dismutase (CuZn-SOD)-deficient mice show atrophic morphology in their skin. This atrophy is accompanied by the degeneration of collagen and elastic fibers, and skin hydroxyproline is also significantly reduced in deficient mice. These imply that the dysfunction of collagen and elastin biosynthesis are involved in the progression of skin thinning. Furthermore, transdermal administration of a vitamin C derivative which can permeate through the membrane, completely reversed the skin thinning and deterioration of collagen and elastin in the mutant mice. These indicate that the vitamin C derivative is a powerful agent for alleviating skin ageing through regeneration of collagen and elastin. The CuZn-SOD-deficient mice might be applicable to evaluation of therapeutic medicines against skin ageing.     

Hormonal regulation of tissue-specific ectopic expression of an Arabidopsis endoplasmic reticulum-type ω-3 fatty acid desaturase (FAD3) gene

A common feature of the membrane lipids of higher plants is a large content of polyunsaturated fatty acids, which typically consist of dienoic and trienoic fatty acids. Two types of omega-3 fatty acid desaturase. which are present in the plastids and in the endoplasmic reticulum (ER), respectively, are responsible for the conversion of dienoic to trienoic fatty acids. To establish a system for investigating the tissue-specific, and hor-mone-regulated expression of the ER-type desaturase gene (FAD3), transgenic plants of Arabidopsis thaliana (L.) Heynh. containing the firefly luciferase gene (LUC) fused to the FAD3 promoter (FAD3::LUC) were constructed. At different times during plant development, FAD3::LUC was actively expressed at two major sites, the vegetative shoot meristem and the floral organs. Transgenic plants with LUC fused to the promoter of FAD7 (FAD7::LUC) which encodes plastid-type desaturase, were also constructed. FAD3::LUC and FAD7::LUC were expressed in the same organs during reproductive growth, but not during vegetative growth. In plants exposed to both auxin and cytokinin, FAD3::LUC expression was ectopically induced in the root tissues. However, this induction by auxin and cytokinin was inhibited when abscisic acid was also present. FAD3::LUC expression could be induced in the roots by auxin and cytokinin if the hormones were applied during vegetative growth, but not if they were applied during germination or reproductive growth. Analysis of the fatty acid composition in the roots of Arabidopsis fad mutant and wild-type plants confirmed that the response of FAD3::LUC expression to various hormones reflected the response of endogenous FAD3 gene expression. These results suggest that the expression of ER-type desaturase is regulated through synergistic and antagonistic hormonal interactions, and that such hormonal regulation and the tissue specificity of the expression of this gene are further modified in accordance with the growth phase in plant development.     

Resolvin E1 dampens airway inflammation and hyperresponsiveness in a murine model of asthma

Resolvin E1 (RvE1; 5S, 12R, 18R-trihydroxyeicosapentaenoic acid) is an anti-inflammatory lipid mediator derived from the omega-3 fatty acid eicosapentaenoic acid (EPA). It has been recently shown that RvE1 is involved in the resolution of inflammation. However, it is not known whether RvE1 is involved in the resolution of asthmatic inflammation. To investigate the anti-inflammatory effect of RvE1 in asthma, a murine model of asthma was studied. After RvE1 was administered to mice intraperitoneally, there were decreases in: airway eosinophil and lymphocyte recruitment, specific Th2 cytokine, IL-13, ovalbumin-specific IgE, and airway hyperresponsiveness (AHR) to inhaled methacholine. Moreover, RvE1-treated mice had significantly lower mucus scores compared to vehicle-treated mice based on the number of goblet cells stained with periodic acid-schiff (PAS). These findings provide evidence that RvE1 is a pivotal counterregulatory signal in allergic inflammation and offer novel multi-pronged therapeutic approaches for human asthma.     

Hepatoblast-like cells enriched from mouse embryonic stem cells in medium without glucose, pyruvate, arginine, and tyrosine

In order to enrich hepatocytes differentiated from embryonic stem cells, we developed a novel medium. Since only hepatocytes have the activity of ornithine transcarbamylase, phenylalanine hydroxylase, galactokinase, and glycerol kinase, we expected that hepatocytes would be enriched in a medium without arginine, tyrosine, glucose, and pyruvate, but supplemented with ornithine, phenylanaline, galactose, and glycerol (hepatocyte-selection medium, HSM). Embryoid bodies were transferred onto dishes coated with gelatin in HSM after 4 days of culture. At 18 days after embryoid body formation, a single type of polygonal cell survived with an enlarged intercellular space and micorvilli. These cells were positive for indocyanine green uptake and for mRNAs of albumin, transthyretin, and alpha-feto protein, but negative for mRNAs of tyrosine aminotransferase, alpha1-antitrypsin, glucose-6-phosphatase, and phosphoenol pyruvate carboxykinase. Since cells in HSM were positive for cytokeratin (CK)8 and CK18 (hepatocyte markers) and for CK19 (a marker of bile duct epithelial cells), we concluded that they were hepatoblasts. They showed weaker expression of CCAAT/enhancer-binding protein (C/EBP)alpha than fetal liver (18.5 days of gestation) and expression of C/EBPbeta at a similar level to that of fetal liver. These data support our conclusion that HSM allows the selection of hepatoblast-like cells.