Abstract
The J-PARC E16 experiment measures mass spectra of low-mass vector mesons,
ρ
,
ω
, and
ϕ
, in nuclei via
e
+
e
-
decays. Vector mesons are produced in
p
A reactions. A spectrometer with ...a large acceptance and a high-intensity beam up to 1
× 10
10
protons per ∼2-sec duration pulse are utilized to collect a sufficient number
of vector mesons. We have developed a spectrometer, which has two types of electron
identification detectors: a hadron blind detector (HBD) and a lead-glass calorimeter.
Commissioning runs have been performed at the J-PARC high-momentum beam line. The HBD
successfully observed on average 11 ± 1 photoelectrons for an incident electron. We achieved
the pion rejection of 0.9 ± 0.2% with the electron detection efficiency of 61 ± 4% for
the HBD, which was consistent of the expected performance.
Vesicular trafficking plays an important role in a virulence mechanism of the enteric protozoan parasite Entamoeba histolytica as secreted and lysosomal cysteine protease (CP) contributes to both ...cytolysis of tissues and degradation of internalized host cells. Despite the primary importance of intracellular sorting in pathogenesis, the molecular mechanism of CP trafficking remains largely unknown. In this report we demonstrate that transport of CP is regulated through a specific interaction of Rab7A small GTPase (EhRab7A) with the retromerlike complex. The amoebic retromerlike complex composed of Vps26, Vps29, and Vps35 was identified as EhRab7A-binding proteins. The amoebic retromerlike complex specifically bound to GTP-EhRab7A, but not GDP-EhRab7A through the direct binding via the carboxy terminus of EhVps26. In erythrophagocytosis the retromerlike complex was recruited to prephagosomal vacuoles, the unique preparatory vacuole of digestive enzymes, and later to phagosomes. This dynamism was indistinguishable from that of EhRab7A, and consistent with the premise that the retromerlike complex is involved in the retrograde transport of putative hydrolase receptor(s) from preparatory vacuoles and phagosomes to the Golgi apparatus. EhRab7A overexpression caused enlargement of lysosomes and decrease of the cellular CP activity. The reduced CP activity was restored by the coexpression of EhVps26, implying that the EhRab7A-mediated transport of CP to phagosomes is regulated by the retromerlike complex.
In mammals, Rab5 and Rab7 play a specific and coordinated role in a sequential process during phagosome maturation. Here,
we report that Rab5 and Rab7 in the enteric protozoan parasite Entamoeba ...histolytica , Eh Rab5 and Eh Rab7A, are involved in steps that are distinct from those known for mammals. Eh Rab5 and Eh Rab7A were localized to independent small vesicular structures at steady state. Priming with red blood cells induced the formation
of large vacuoles associated with both Eh Rab5 and Eh Rab7A (â p re p hagosomal v acuoles (PPV)â) in the amoeba within an incubation period of 5â10 min. PPV emerged de novo physically and distinct from phagosomes. PPV were gradually acidified and matured by fusion with lysosomes containing a digestive
hydrolase, cysteine proteinase, and a membrane-permeabilizing peptide amoebapore. After Eh Rab5 dissociated from PPV, 5â10 min later, the Eh Rab7A-PPV fused with phagosomes, and Eh Rab7A finally dissociated from the phagosomes. Immunoelectron and light micrographs showed that PPV contained small vesicle-like
structures containing fluid-phase markers and amoebapores, which were not evenly distributed within PPV, suggesting that the
mechanism was similar to multivesicular body formation in PPV generation. In contrast to Rab5 from other organisms, Eh Rab5 was involved exclusively in phagocytosis, but not in endocytosis. Overexpression of wild-type Eh Rab5 enhanced phagocytosis and the transport of amoebapore to phagosomes. Conversely, expression of an Eh Rab5Q67L GTP form mutant impaired the formation of PPV and phagocytosis. Altogether, we propose that the amoebic Rab5 plays
an important role in the formation of unique vacuoles, which is essential for engulfment of erythrocytes and important for
packaging of lysosomal hydrolases, prior to the targeting to phagosomes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The rapid and efficient phagocytosis of apoptotic cells plays a critical role in preventing secondary necrosis, inflammation as well as in tissue remodeling and regulating immune responses. However, ...the molecular details of engulfment are just beginning to be elucidated. Among the Rho family GTPases, previous studies have implicated a role for Rac and Cdc42 in the uptake of apoptotic cells by phagocytes, yet the role of Rho has remained unclear. Here, we present evidence that Rho-GTP levels decrease during engulfment. RhoA seems to negatively affect basal engulfment, such that inhibition of Rho-mediated signaling in phagocytes enhanced the uptake of apoptotic targets. Activation of endogenous Rho or overexpression of constitutively active forms of Rho also inhibited engulfment. By testing mutants of RhoA that selectively activate downstream effectors, the Rho-kinase seemed to be primarily responsible for this inhibitory effect. Taken together, these data suggest that inhibition of Rho- and Rho-kinase-mediated signaling might be important during engulfment, which could have important implications for several clinical trials involving inhibition of the Rho kinase.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The prompt clearance of cells undergoing apoptosis is critical during embryonic development, normal tissue turnover, as well as inflammation and autoimmunity. The molecular details of the engulfment ...of apoptotic cells are not fully understood. ced-6 and its human homologue gulp, encode an adapter protein, whose function in engulfment is highly evolutionarily conserved; however, the upstream and downstream components of CED-6 mediated signaling are not known. Recently, ced-1 has been shown to encode a transmembrane protein on phagocytic cells, with two functional sequence motifs in its cytoplasmic tail that are important for engulfment. In this study, using a combination of biochemical approaches and yeast two-hybrid analysis, we present evidence for a physical interaction between GULP/CED-6 and one of the two motifs (NP XY motif) in the cytoplasmic tail of CED-1. The phosphotyrosine binding domain of GULP was necessary and sufficient for this interaction. Since the precise mammalian homologue of CED-1 is not known, we undertook a database search for human proteins that contain the motifs shown to be important for CED-1 function and identified CD91/LRP (low density lipoprotein receptor-related protein) as one candidate. Interestingly, recent studies have also identified CD91/LRP as a receptor involved in the phagocytosis of apoptotic cells in mammals. The GULP phosphotyrosine binding domain was able to specifically interact with one specific NP XY motif in the CD91 cytoplasmic tail. During these studies we have also identified the mouse GULP sequence. These studies suggest a physical link between CED-1 or CD91/LRP and the adapter protein CED-6/GULP during engulfment of apoptotic cells and further elucidate the pathway suggested by the genetic studies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Using human myeloperoxidase cDNA as a probe, a chromosomal gene related to myeloperoxidase was isolated from a human gene library. Comparison of the amino acid sequence deduced from the nucleotide ...sequence of the cloned gene with that of human eosinophil peroxidase purified from buffy coats has indicated that the isolated gene is the chromosomal gene for human eosinophil peroxidase. Like human myeloperoxidase gene, human eosinophil peroxidase gene consists of 12 exons and 11 introns spanning about 12 kilobases. The gene can code for a protein of 715 amino acids with a calculated Mr of 81,036. The heavy chain and the light chain of eosinophil peroxidase were located on the COOH and NH2 terminus of the protein, respectively. The coding sequences of eosinophil peroxidase and myeloperoxidase show homologies of 72.4% at the nucleotide and 69.8% at the amino acid level, while little homology was found in the 5′-flanking region. Northern hybridization and S1 mapping analysis of RNA from human leukemic cells have indicated that the eosinophil peroxidase gene is expressed in the eosinophilic subline of human HL-60 cells but not in the neutrophilic subline or in parental HL-60 cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Although the cellular and molecular mechanisms underlying the delayed-type hypersensitivity (DTH) reaction have been investigated, the functions of infiltrating leukocytes and skin resident cells in ...the elicitation phase of the DTH reaction are not completely understood. To gain more insight into the role of these cells in the DTH reaction, we identified about 250 cDNA fragments showing elevated expression during the DNCB-induced guinea pig skin DTH reaction by differential display analysis. Characterization of 50 of them led to the identification of 28 genes whose expression was elevated in the DNCB-induced DTH reactive tissue. Sequencing of the 28 cDNA fragments and homology search analysis demonstrated that 10 of them represented known genes, some of which, in particular elafin (an elastase inhibitor) and ferritin, are considered to play roles in the DTH reaction. The other 18 fragments are probably derived from unknown genes. Cloning of the cDNAs of one of these genes indicated that it is that for guinea pig tryptophanyl-tRNA synthetase (WRS), a protein found to be induced by interferon-γ and upregulated during the late stages of mononuclear phagocyte maturation in vitro. Strong induction of the WRS gene during the DTH reaction suggests its involvement in the in vivo immune response.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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