The endothelial cell-produced antiangiogenic cytokine vascular endothelial growth inhibitor induces dendritic cell maturation

Angiogenesis is an essential component of chronic inflammation that is linked to carcinogenesis. In this study, we report that human vascular endothelial growth inhibitor (VEGI, TNF superfamily 15), an endothelial cell-produced antiangiogenic cytokine, induces mouse dendritic cell (DC) maturation, a critical event in inflammation-initiated immunity. VEGI-stimulated bone marrow-derived immature DCs display early activation of maturation signaling molecules NF-kappaB, STAT3, p38, and JNK, and cytoskeleton reorganization and dendrite formation. The activation signals are partially inhibited by using a neutralizing Ab against death domain-containing receptor-3 (DR3) or a truncated form of DR3 consisting of the extracellular domain, indicating an involvement of DR3 in the transmission of VEGI activity. A VEGI isoform, TL1A, does not induce similar activities under otherwise identical experimental conditions. Additionally, the cells reveal significantly enhanced expression of mature DC-specific marker CD83, secondary lymphoid tissue-directing chemokine receptor CCR7, the MHC class-II protein (MHC-II), and costimulatory molecules CD40, CD80, and CD86. Functionally, the cells exhibit decreased Ag endocytosis, increased cell surface distribution of MHC-II, and increased secretion of IL-12 and TNF. Moreover, VEGI-stimulated DCs are able to facilitate the differentiation of CD4+ naive T cells in cocultures. These findings suggest that the anticancer activity of VEGI arises from coupling the inhibition of endothelial cell growth with the promotion of the adaptive immune mechanisms through the stimulation of DC maturation.     

Structural basis for cyclodextrin recognition by Thermoactinomyces vulgaris cyclo/maltodextrin-binding protein

The crystal structure of a Thermoactinomyces vulgaris cyclo/maltodextrin-binding protein (TvuCMBP) complexed with gamma-cyclodextrin has been determined. Like Escherichia coli maltodextrin-binding protein (EcoMBP) and other bacterial sugar-binding proteins, TvuCMBP consists of two domains, an N- and a C-domain, both of which are composed of a central beta-sheet surrounded by alpha-helices; the domains are joined by a hinge region containing three segments. gamma-Cyclodextrin is located at a cleft formed by the two domains. A common functional conformational change has been reported in this protein family, which involves switching from an open form to a sugar-transporter bindable form, designated a closed form. The TvuCMBP-gamma-cyclodextrin complex structurally resembles the closed form of EcoMBP, indicating that TvuCMBP complexed with gamma-cyclodextrin adopts the closed form. The fluorescence measurements also showed that the affinities of TvuCMBP for cyclodextrins were almost equal to those for maltooligosaccharides. Despite having similar folds, the sugar-binding site of the N-domain part of TvuCMBP and other bacterial sugar-binding proteins are strikingly different. In TvuCMBP, the side-chain of Leu59 protrudes from the N-domain part into the sugar-binding cleft and orients toward the central cavity of gamma-cyclodextrin, thus Leu59 appears to play the key role in binding. The cleft of the sugar-binding site of TvuCMBP is also wider than that of EcoMBP. These findings suggest that the sugar-binding site of the N-domain part and the wide cleft are critical in determining the specificity of TvuCMBP for gamma-cyclodextrin.     

Nuclear envelope precursor vesicle targeting to chromatin is stimulated by protein phosphatase 1 in Xenopus egg extracts

The mechanism underlying targeting of the nuclear membrane to chromatin at the end of mitosis was studied using an in vitro cell-free system comprising Xenopus egg membrane and cytosol fractions, and sperm chromatin. The mitotic phase membrane, which was separated from a mitotic phase extract of Xenopus eggs and could not bind to chromatin, became able to bind to chromatin on pretreatment with a synthetic phase cytosol fraction of Xenopus eggs. When the cytosol fraction was depleted of protein phosphatase 1 (PP1) with anti-Xenopus PP1γ1 antibodies, this ability was lost. The addition of recombinant xPP1γ1 to the PP1-depleted cytosol fraction restored the ability. These and other results suggested that dephosphorylation of mitotic phosphorylation sites on membranes by PP1 in the synthetic phase cytosol fraction promoted targeting of the membranes to chromatin. On the other hand, a fragment containing the chromatin-binding domain of lamin B receptor (LBR) but not emerin inhibited targeting of membrane vesicles. It was also shown that PP1 dephosphorylates a phosphate group(s) responsible for regulation of the binding of LBR to chromatin. A possible mechanism involving PP1 and LBR for the regulation of nuclear membrane targeting to chromatin was discussed.     

Early diagenesis of organic matter in lacustrine sediments in terms of methane fermentation

The amounts of organic compounds such as amino acids (proteinaceous substances), fatty acids, and sterols in lacustrine sediments were found to decrease greatly from the mud‐water interface to depths of 20 cm and less at greater core depths. The amounts of methane produced by microbial activity in subsamples incubated for 40 days at 27°C were also found to decrease with core depth, apparently limited by the exhaustion of organic substrates subject to methane fermentation. Decomposition rates of certain organic compounds added to sediment samples were determined by incubation experiments at different temperatures for 40 to 450 days. Experimental results with mixed cultures of microorganisms in lacustrine sediments indicated the following: (a) The marked decomposition of organic compounds such as amino acids, fatty acids, and sterols in the lacustrine sediments resulted mainly in the formation of methane, (b) Unsaturated compounds were more readily decomposed with the ultimate formation of methane than saturated compounds, (c) The susceptibility of saturated fatty acids to methane formation decreased in proportion to their molecular weights.     

Identification of Ypk1 as a Novel Selective Substrate for Nitrogen Starvation-triggered Proteolysis Requiring Autophagy System and Endosomal Sorting Complex Required for Transport (ESCRT) Machinery Components

Nitrogen starvation-mediated reduction of Ypk1 is suggested to suppress translational initiation, possibly in parallel with the target of rapamycin complex 1 (TORC1) signaling. However, the molecular mechanism that regulates Ypk1 in nitrogen-starved cells is poorly understood. Here we report that Ypk1 is a novel selective substrate for nitrogen starvation-triggered proteolysis requiring autophagy system. Among various nutrient starvation methods used to elicit autophagy, rapid Ypk1 degradation was specific to nitrogen starvation. In screening genes required for such nitrogen starvation-specific vacuolar proteolysis, we found that autophagy-related degradation of Ypk1 depended on the endosomal sorting complex required for transport (ESCRT) machinery, which is conventionally thought to function in endosomal trafficking. In microscopic analyses, the disruption of ESCRT subunits resulted in the accumulation of both Ypk1 and autophagosomal Atg8 at a perivacuolar site that was distinct from conventional endosomes. ESCRT machinery was not involved in autophagic flux induced by the TORC1 inhibitor rapamycin, thus suggesting that ESCRT represents an exclusive mechanism of nitrogen starvation-specific proteolysis of Ypk1. Overall, we propose a novel regulation of Ypk1 that is specific to nitrogen limitation.     

X-ray structures of the microglia/macrophage-specific protein Iba1 from human and mouse demonstrate novel molecular conformation change induced by calcium binding

The ionized calcium-binding adaptor molecule 1 (Iba1) with 147 amino acid residues has been identified as a calcium-binding protein, expressed specifically in microglia/macrophages, and is expected to be a key factor in membrane ruffling, which is a typical feature of activated microglia. We have determined the crystal structure of human Iba1 in a Ca(2+)-free form and mouse Iba1 in a Ca(2+)-bound form, to a resolution of 1.9 A and 2.1 A, respectively. X-ray structures of Iba1 revealed a compact, single-domain protein with two EF-hand motifs, showing similarity in overall topology to partial structures of the classical EF-hand proteins troponin C and calmodulin. In mouse Iba1, the second EF-hand contains a bound Ca(2+), but the first EF-hand does not, which is often the case in S100 proteins, suggesting that Iba1 has S100 protein-like EF-hands. The molecular conformational change induced by Ca(2+)-binding of Iba1 is different from that found in the classical EF-hand proteins and/or S100 proteins, which demonstrates that Iba1 has an unique molecular switching mechanism dependent on Ca(2+)-binding, to interact with target molecules.     

Typing of bfpA genes of enteropathogenic Escherichia coli isolated in Thailand and Japan by heteroduplex mobility assay

We developed a rapid genetic approach for screening bfpA variants of enteropathogenic E. coli(EPEC) using a heteroduplex mobility assay (HMA). A total of 204 human EPEC strains were isolated in Thailand and Japan. Of 34 bfpA-positive EPEC strains, bfpA variants were classified into 5 HMA-types. Different HMA-types were found in EPEC of the same serotypes. The results suggest that HMA is a simple and easy method to analyze polymorphism of bfpA gene, and can be used in laboratories without large apparatus such as sequencers.     

Swarming of Pseudomonas aeruginosa PAO1 without differentiation into elongated hyperflagellates on hard agar minimal medium

Polar flagellated Pseudomonas aeruginosa PAO1 demonstrated extensive spreading growth in 2 days on 1.5% agar medium. Such spreading growth of P. aeruginosa PAO1 strains was absent on Luria-Bertani 1.5% agar medium, but remarkable on Davis minimal synthetic agar medium (especially that containing 0.8% sodium citrate and 1.5% Eiken agar) under aerobic 37 degrees C conditions. Analyses using isogenic mutants and complementation transformants showed that bacterial flagella and rhamnolipid contributed to the surface-spreading behavior. On the other hand, a type IV pilus-deficient pilA mutant did not lose the spreading growth activity. Flagella staining of PAO1 T cells from the frontal edge of a spreading colony showed unipolar and normal-sized rods with one or two flagella. Thus, the polar flagellate P. aeruginosa PAO1 T appears to swarm on high-agar medium by producing biosurfactant rhamnolipid and without differentiation into an elongated peritrichous hyperflagellate.     

Stat6 signaling suppresses VLA-4 expression by CD8+ T cells and limits their ability to infiltrate tumor lesions in vivo

VLA-4 plays a critical role in T cell trafficking into inflammatory sites. Our recent studies have suggested that VLA-4 expression on CD8+ T cells is negatively controlled by IL-4 and serves as a functionally distinguishing variable for why Type-1, but not Type-2, CD8+ T cells are able to traffic into tumors. In this study, using in vitro culture of murine CD8+ T cells under Type-1 and Type-2 cytokine conditions, we show that IL-4-mediated down-regulation of VLA-4 expression is completely abrogated in Stat6-deficient CD8+ T cells. Conversely, CD8+ T cells expressing a constitutively active mutant form Stat6 (Stat6VT) failed to express VLA-4 even in the absence of IL-4-stimulation. Notably, Type-2 CD8+ T cells developed from Stat6-/- but not wild-type mice were competent to migrate into tumor lesions in vivo. These results suggest that Stat6-signaling is necessary and sufficient to restrict CD8+ T cell expression of VLA-4 (by IL-4), thereby serving as a regulator for CD8+ T cell infiltration into tumors.     

Solution structure of the state 1 conformer of GTP-bound H-Ras protein and distinct dynamic properties between the state 1 and state 2 conformers

Ras small GTPases undergo dynamic equilibrium of two interconverting conformations, state 1 and state 2, in the GTP-bound forms, where state 2 is recognized by effectors, whereas physiological functions of state 1 have been unknown. Limited information, such as static crystal structures and (31)P NMR spectra, was available for the study of the conformational dynamics. Here we determine the solution structure and dynamics of state 1 by multidimensional heteronuclear NMR analysis of an H-RasT35S mutant in complex with guanosine 5'-(β, γ-imido)triphosphate (GppNHp). The state 1 structure shows that the switch I loop fluctuates extensively compared with that in state 2 or H-Ras-GDP. Also, backbone (1)H,(15)N signals for state 2 are identified, and their dynamics are studied by utilizing a complex with c-Raf-1. Furthermore, the signals for almost all the residues of H-Ras·GppNHp are identified by measurement at low temperature, and the signals for multiple residues are found split into two peaks corresponding to the signals for state 1 and state 2. Intriguingly, these residues are located not only in the switch regions and their neighbors but also in the rigidly structured regions, suggesting that global structural rearrangements occur during the state interconversion. The backbone dynamics of each state show that the switch loops in state 1 are dynamically mobile on the picosecond to nanosecond time scale, and these mobilities are significantly reduced in state 2. These results suggest that multiconformations existing in state 1 are mostly deselected upon the transition toward state 2 induced by the effector binding.