Occurrence of the primary cell wall polysaccharide rhamnogalacturonan II in pteridophytes, lycophytes, and bryophytes. Implications for the evolution of vascular plants

Borate ester cross-linking of the cell wall pectic polysaccharide rhamnogalacturonan II (RG-II) is required for the growth and development of angiosperms and gymnosperms. Here, we report that the amounts of borate cross-linked RG-II present in the sporophyte primary walls of members of the most primitive extant vascular plant groups (Lycopsida, Filicopsida, Equisetopsida, and Psilopsida) are comparable with the amounts of RG-II in the primary walls of angiosperms. By contrast, the gametophyte generation of members of the avascular bryophytes (Bryopsida, Hepaticopsida, and Anthocerotopsida) have primary walls that contain small amounts (approximately 1% of the amounts of RG-II present in angiosperm walls) of an RG-II-like polysaccharide. The glycosyl sequence of RG-II is conserved in vascular plants, but these RG-IIs are not identical because the non-reducing L-rhamnosyl residue present on the aceric acid-containing side chain of RG-II of all previously studied plants is replaced by a 3-O-methyl rhamnosyl residue in the RG-IIs isolated from Lycopodium tristachyum, Ceratopteris thalictroides, Platycerium bifurcatum, and Psilotum nudum. Our data indicate that the amount of RG-II incorporated into the walls of plants increased during the evolution of vascular plants from their bryophyte-like ancestors. Thus, the acquisition of a boron-dependent growth habit may be correlated with the ability of vascular plants to maintain upright growth and to form lignified secondary walls. The conserved structures of pteridophyte, lycophyte, and angiosperm RG-IIs suggests that the genes and proteins responsible for the biosynthesis of this polysaccharide appeared early in land plant evolution and that RG-II has a fundamental role in wall structure.     

Two morphological types of cell appendages on a strongly adhesive bacterium, Acinetobacter sp. strain Tol 5

Two morphological types of appendages, an anchor-like appendage and a peritrichate fibril-type appendage, have been observed on cells of an adhesive bacterium, Acinetobacter sp. strain Tol 5, by use of recently developed electron microscopic techniques. The anchor extends straight to the substratum without branching and tethers the cell body at its end at distances of several hundred nanometers, whereas the peritrichate fibril attaches to the substratum in multiple places, fixing the cell at much shorter distances.     

Cathepsin L is crucial for a Th1-type immune response during Leishmania major infection

Prior to the activation of CD4+ T cells, exogenous proteins are digested by endo/lysosomal enzymes in antigen-presenting cells (APCs) to produce antigenic peptides that are presented on MHC class II molecules. In the studies described here, the functional significance of cathepsin L for antigen processing and Th1/Th2 differentiation in experimental leishmaniasis was investigated. We first demonstrated that cathepsin L is one of the candidates for endo/lysosomal enzymes in the processing of soluble Leishmania antigen (SLA) by using CLIK148, a specific inhibitor of cathepsin L. Treatment of BALB/c or DBA/2 mice with CLIK148 exacerbated the disease by enhancing an SLA-specific Th2-type response such as IL-4 production. CLIK148 did not exert any direct influence on Leishmania major promastigotes themselves or on the course of L. major infection in SCID mice. Taken together, these findings suggest that treatment of host mice with CLIK148 affects the processing of SLA in APCs, resulting in the potentiation of Th2-type immune responses and thus leading to exacerbation of the disease. Furthermore, endo/lysosomal cathepsin L was found to be functionally distinct from previously described cathepsins B and D.     

Potent and orally active ET(A) selective antagonists with 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acid structures

The synthesis and structure-activity relationships of a series of 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids are described. Our efforts have been focused on modification of the aryl ring at the 5-position and the alkyl substituent at the 2-position of the bottom 4-methoxyphenyl ring in an effort to develop orally available ET(A) selective antagonists with safer profiles in terms of the P-450 enzyme inhibitory activity. Incorporation of a hydroxymethyl group as an alkyl substituent in methylenedioxyphenyl and 6-dihydrobenzofuran derivatives led to the identification of orally bioavailable ET(A) selective antagonists 1f and 7f. These compounds also showed not only excellent binding affinity (IC(50) < 0.10nM, more than 800-fold selectivity for the ET(A) receptor over the ET(B) receptor) but also sufficient oral bioavailability, 48% and 56%, respectively, in rats. Furthermore, these compounds did not exhibit either competitive or mechanism-based inhibition of human cytochrome P450 enzymes.     

Contribution of IL-1 beta and TNF-alpha to the initiation of the peripheral lung response to atmospheric particulates (PM10)

Alveolar macrophages (AM) play a key role in clearing atmospheric particulates from the lung surface and stimulating epithelial cells to produce proinflammatory mediators. The present study examines the role of "acute response" cytokines TNF-alpha and IL-1 beta released by AM exposed to ambient particulate matter with a diameter of <10 microm (PM(10)) in amplifying the proinflammatory mediator expression by A549 cells and human bronchial epithelial cells (HBEC). The results showed that supernatants from human AM incubated 24 h with PM(10) (100 microg/ml) contained more TNF-alpha, IL-1 beta, granulocyte-macrophage colony stimulating factor, IL-6, and IL-8 than nonexposed AM supernatants. The 3-h treatment of A549 cells with PM(10)-exposed AM supernatants increased TNF-alpha, IL-1 beta, IL-8, regulated on activation normal T-cells expressed and secreted (RANTES), and leukemia inhibitory factor mRNA compared with the treatment with nonexposed AM supernatants and, compared with untreated A549 cells, additionally increased ICAM-1 and monocyte chemotactic protein-1 mRNA. Preincubating PM(10)-exposed AM supernatants with anti-IL-1 beta antibodies reduced all the above mediators as well as VEGF mRNA expression (P < 0.05), while anti-TNF-alpha antibodies were less effective (P > 0.05), and the combination of the two antibodies most effective. When HBEC were treated similarly, anti-TNF-alpha antibodies had the greatest effect. In A549 cells PM(10)-exposed AM supernatants increased NF-kappa B, activator protein (AP)-1 and specificity protein 1 binding, while anti-TNF-alpha and anti-IL-1 beta antibodies reduced NF-kappa B and AP-1 binding. We conclude that AM-derived TNF-alpha and IL-1 beta provide a major stimulus for the production of proinflammatory mediators by lung epithelial cells and that their relative importance may depend on the type of epithelial cell target.     

Exposure to ambient particles accelerates monocyte release from bone marrow in atherosclerotic rabbits

Exposure to air pollution [particulate matter, particles <10 microm (PM(10))] causes a systemic inflammatory response that includes stimulation of the bone marrow (BM) and progression of atherosclerosis. Monocytes are known to play a key role in atherogenesis by migration into subendothelial lesions where they appear as foam cells. The present study was designed to quantify the BM monocyte response in Watanabe heritable hyperlipidemic (WHHL) rabbits after PM(10) exposure. WHHL rabbits were given twice weekly intrapharyngeal instillations of 5 mg of PM(10) for 4 wk to a total of 40 mg and compared with control WHHL or New Zealand White (NZW) rabbits. The thymidine analog 5'-bromo-2'-deoxyuridine was used to label dividing cells in the BM and a monoclonal antibody to identify monocytes in peripheral blood. The transit time of monocytes through the BM was faster in WHHL than in NZW rabbits (30.4 +/- 1.9 h vs. 35.2 +/- 0.9 h, WHHL vs. NZW; P < 0.05). PM(10) instillation exposure increased circulating band cell counts, caused rapid release of monocytes from the BM, and further shortened their transit time through the BM to 23.2 +/- 1.6 h (P < 0.05). The percentage of alveolar macrophages containing particles in the lung correlated with the BM transit time of monocytes (r(2) = 0.45, P <0.05). We conclude that atherosclerosis increases the release of monocytes from the BM, and PM(10) exposure accelerates this process in relation to the amount of particles phagocytosed by alveolar macrophages.     

Up-regulation of cysteinyl leukotriene 1 receptor by IL-13 enables human lung fibroblasts to respond to leukotriene C4 and produce eotaxin

Cysteinyl leukotrienes (CysLTs) play an important role in eosinophilic airway inflammation. In addition to their direct chemotactic effects on eosinophils, indirect effects have been reported. Eotaxin is a potent eosinophil-specific chemotactic factor produced mainly by fibroblasts. We investigated whether CysLTs augment eosinophilic inflammation via eotaxin production by fibroblasts. Leukotriene (LT)C(4) alone had no effect on eotaxin production by human fetal lung fibroblasts (HFL-1). However, LTC(4) stimulated eotaxin production by IL-13-treated fibroblasts, thereby indirectly inducing eosinophil sequestration. Unstimulated fibroblasts did not respond to LTC(4), but coincubation or preincubation of fibroblasts with IL-13 altered the response to LTC(4). To examine the mechanism(s) involved, the expression of CysLT1R in HFL-1 was investigated by quantitative real-time PCR and flow cytometry. Only low levels of CysLT1R mRNA and no CysLT1R protein were expressed in unstimulated HFL-1. In contrast, stimulation with IL-13 at a concentration of 10 ng/ml for 24 h significantly up-regulated both CysLT1R mRNA and protein expression in HFL-1. The synergistic effect of LTC(4) and IL-13 on eotaxin production was abolished by CysLT1R antagonists pranlukast and montelukast. These findings suggest that IL-13 up-regulates CysLT1R expression, which may contribute to the synergistic effect of LTC(4) and IL-13 on eotaxin production by lung fibroblasts. In the Th2 cytokine-rich milieu, such as that in bronchial asthma, CysLT1R expression on fibroblasts might be up-regulated, thereby allowing CysLTs to act effectively and increase eosinophilic inflammation.     

A palmitoylated RING finger ubiquitin ligase and its homologue in the brain membranes

Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).