Caspase cleavage releases a nuclear protein fragment that stimulates phospholipid scrambling at the plasma membrane

1. Download : Download high-res image (162KB)
2. Download : Download full-size image

     

Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 degrees C, but stopped growing at the non-permissive temperature of 39 degrees C. In the presence of receptor activator of NFkappaB ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 degrees C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.     

Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding

Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to d-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2′-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.     

The Role of Matrix Metalloproteinase in the Intimal Sarcoma-Like Cells Derived from Endarterectomized Tissues from a Chronic Thromboembolic Pulmonary Hypertension Patient

Sarcoma-like cells (SCLs) were derived from endarterectomized tissue of a single chronic thromboembolic pulmonary hypertension (CTEPH) patient during incubation of those thrombi at second passage as described at our previous report. These cells had malignant potential, with an increased expression of matrix metalloproteinase-14 (MMP-14), leading to tumor emboli within pulmonary arteries in in vivo studies. The purpose of this study was to perform a more detailed evaluation of the characteristics of SCLs, and to elucidate the role of the increased expression of MMP-14 expression in the growth and death of these cells. In order to elucidate the characteristics of SCLs and to confirm the protein expression of MMP-14, three-dimentional culture, invasion assays, a Western blot analysis and immunohistochemical studies were performed. To examine the role of MMP-14 in tumorigenesis, the metalloproteinase inhibitor, batimastat, was administered to SCID mice which were subcutaneously injected with SCLs. Those mice were sacrificed on day 14 and the tumor volume was evaluated. A Western blot analysis showed the increased expression of MMP-14 in comparison to the expression in lung adenocarcinoma cells (A549). Immunohistochemistry showed that SCLs were positive for vimentin, MMP-14, MMP-2 and CD44. However, endothelial markers, such as CD31 and von Willebrand factor (vWF), were negative. The in vivo studies demonstrated that batimastat could suppress the growth of the subcutaneous tumors formed by the SCLs. This study suggested that MMPs had critical roles on the pathological activities of SCLs and that batimastat might have anti-proliferative and anti-invasive effects on these cells.     

Competitive abilities and distribution ranges of four asexual Daphnia pulex lineages and Daphnia mitsukuri in Eurasian continental islands

Daphnia pulex and D. mitsukuri are morphologically similar and distributed in small lowland Japanese lakes. Daphnia pulex in Japan reproduces by obligate parthenogenesis and is composed of four different lineages (JPN1–JPN4) with North American origins. Although this species is found throughout Japan, JPN1 has the largest distribution range, followed by JPN2. Daphnia mitsukuri is a putatively endemic species that is now rarely found in Japan. Since Daphnia share the same algal food, the difference in distribution ranges among the four asexual D. pulex lineages and D. mitsukuri may be caused by exploitative competition for algal food. To examine this possibility, we measured the threshold food concentration (TFC) at zero-net growth rate and the food concentration required for 50% of the individuals to survive (FC50). In addition, we examined the effects of temperature on the somatic growth rate (SGR) and the mortality rates of four asexual D. pulex lineages and D. mitsukuri. The experiments showed that TFC was lowest in D. pulex JPN1 and highest in D. mitsukuri, supporting the idea that the largest distribution range of JPN1 is due to its superiority in inter-lineage competition, and that the distribution area of D. mitsukuri is reduced through exploitative competition with D. pulex. However, although JPN2 was found in relatively large areas, it had higher TFC than JPN3 and JPN4, which appear in very limited areas. These results clearly showed that the difference in distribution ranges among D. pulex lineages are not necessarily determined by their competitive superiority alone.     

Benzimidazole tethered pyrrolo[3,4-b]quinoline with broad-spectrum activity against fungal pathogens

Fungal infections caused by Candida and Cryptococcus are particularly dangerous for immunocompromised individuals. In this study, we identified that benzimidazole fused pyrrolo[3,4-b]quinoline compounds have potent antifungal activity against several clinical isolates of pathogenic fungal strains. Specifically, the compound 6a did not show cytotoxicity against mammalian cells at a concentration that inhibits the growth of fungal strains. In addition, the compound 6a also significantly reduced the metabolic activity of fungal cells in the Candida albicans biofilms. Collectively, our results indicate that benzimidazole fused quinoline compounds have a potential to develop as an antifungal agents.     

Deficiency of Chemokine Receptor CCR1 Causes Osteopenia Due to Impaired Functions of Osteoclasts and Osteoblasts

Chemokines are characterized by the homing activity of leukocytes to targeted inflammation sites. Recent research indicates that chemokines play more divergent roles in various phases of pathogenesis as well as immune reactions. The chemokine receptor, CCR1, and its ligands are thought to be involved in inflammatory bone destruction, but their physiological roles in the bone metabolism in vivo have not yet been elucidated. In the present study, we investigated the roles of CCR1 in bone metabolism using CCR1-deficient mice. Ccr1^−/− mice have fewer and thinner trabecular bones and low mineral bone density in cancellous bones. The lack of CCR1 affects the differentiation and function of osteoblasts. Runx2, Atf4, Osteopontin, and Osteonectin were significantly up-regulated in Ccr1^−/− mice despite sustained expression of Osterix and reduced expression of Osteocalcin, suggesting a lower potential for differentiation into mature osteoblasts. In addition, mineralized nodule formation was markedly disrupted in cultured osteoblastic cells isolated from Ccr1^−/− mice. Osteoclastogenesis induced from cultured Ccr1^−/− bone marrow cells yielded fewer and smaller osteoclasts due to the abrogated cell-fusion. Ccr1^−/− osteoclasts exerted no osteolytic activity concomitant with reduced expressions of Rank and its downstream targets, implying that the defective osteoclastogenesis is involved in the bone phenotype in Ccr1^−/− mice. The co-culture of wild-type osteoclast precursors with Ccr1^−/− osteoblasts failed to facilitate osteoclastogenesis. This finding is most likely due to a reduction in Rankl expression. These observations suggest that the axis of CCR1 and its ligands are likely to be involved in cross-talk between osteoclasts and osteoblasts by modulating the RANK-RANKL-mediated interaction.     

Crystal Structure of the ATPPase Subunit and Its Substrate-Dependent Association with the GATase Subunit: A Novel Regulatory Mechanism for a Two-Subunit-Type GMP Synthetase from Pyrococcus horikoshii OT3

Guanosine 5′-monophosphate synthetase(s) (GMPS) catalyzes the final step of the de novo synthetic pathway of purine nucleotides. GMPS consists of two functional units that are present as domains or subunits: glutamine amidotransferase (GATase) and ATP pyrophosphatase (ATPPase). GATase hydrolyzes glutamine to yield glutamate and ammonia, while ATPPase utilizes ammonia to convert adenyl xanthosine 5′-monophosphate (adenyl-XMP) into guanosine 5′-monophosphate. Here we report the crystal structure of PH-ATPPase (the ATPPase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon Pyrococcus horikoshii OT3). PH-ATPPase consists of two domains (N-domain and C-domain) and exists as a homodimer in the crystal and in solution. The N-domain contains an ATP-binding platform called P-loop, whereas the C-domain contains the xanthosine 5'-monophosphate (XMP)-binding site and also contributes to homodimerization. We have also demonstrated that PH-GATase (the glutamine amidotransferase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon P. horikoshii OT3) alone is inactive, and that all substrates of PH-ATPPase except for ammonia (Mg²⁺, ATP and XMP) are required to stabilize the active complex of PH-ATPPase and PH-GATase subunits.     

Identification of the rat IgA Fc receptor encoded in the leukocyte receptor complex

Human FcRI (CD89) is a myeloid-specific IgA Fc receptor encoded in the leukocyte receptor complex. Thus far, no gene coding for FcRI has been identified in mice. Here, we show that, unlike mice, rats have the gene (Fcar) coding for FcRI. The rat Fcar gene has an exon-intron structure essentially identical to that of the human counterpart and is encoded in the leukocyte receptor complex on Chromosome 1. Southern blot analysis using the rat Fcar as a probe revealed hybridizing bands in Chinese and Syrian hamsters and gerbils, but not in mice, indicating that Fcar was lost in the lineage leading to mice after the divergence of rats and mice. Identification of FcRI in rats should facilitate the elucidation of the in vivo role of this receptor.The sequence data reported in this paper have been submitted to the DDBJ/EMBL/GenBank databases under accession numbers: AB109766, AB109767, and AB109768     

Forced expression of murine IL-17E induces growth retardation, jaundice, a Th2-biased response, and multiorgan inflammation in mice.

IL-17 is a proinflammatory cytokine, and its in vivo expression induces neutrophilia in mice. IL-17E is a recently described member of an emerging family of IL-17-related cytokines. IL-17E has been shown to bind IL-17Rh1, a protein distantly related to the IL-17R, suggesting that IL-17E probably possesses unique biological functions. In this study, we have identified the murine ortholog of IL-17E and developed transgenic mice to characterize its actions in vivo. Biological consequences of overexpression of murine (m)IL-17E, both unique to IL-17E and similar to IL-17, were revealed. Exposure to mIL-17E resulted in a Th2-biased response, characterized by eosinophilia, increased serum IgE and IgG1, and a Th2 cytokine profile including elevated serum levels of IL-13 and IL-5 and elevated gene expression of IL-4, IL-5, IL-10, and IL-13 was observed in many tissues. Increased gene expression of IFN-gamma in several tissues and elevated serum TNF-alpha were also noted. In addition, IL-17E induces G-CSF production in vitro and mIL-17E-transgenic mice had increased serum G-CSF and exhibit neutrophilia, a property shared by IL-17. Moreover, exposure to mIL-17E elicited pathological changes in multiple tissues, particularly liver, heart, and lungs, characterized by mixed inflammatory cell infiltration, epithelial hyperplasia, and hypertrophy. Taken together, these findings suggest that IL-17E is a unique pleiotropic cytokine and may be an important mediator of inflammatory and immune responses.