Membrane potential changes associated with pinocytosis of serum lipoproteins in L cells

L cells exhibit spontaneous oscillations of membrane potential in accord with fluctuations of the cytoplasmic Ca²⁺ concentration. Upon addition of low-density lipoprotein (LDL), L cells show a prolonged hyperpolarization which is followed by an increase in the frequency of membrane potential oscillations. These membrane potential changes induced by LDL were inhibited by Ca²⁺-channel blockers. LDL-induced membrane potential changes were accompanied by a vigorous pinocytosis which was coupled with the formation of ring-like ridge structures on the cell surface. These electrical and morphological changes were also induced by high-density lipoprotein (HDL) but not by very-low-density lipoprotein (VLDL). These results suggest that the application of LDL or HDL to the membrane surface elicits a rapid influx of Ca²⁺ into the cytosol, resulting in membrane hyperpolarization. A rise in cytoplasmic Ca²⁺ may be implicated in the primary factor for the pinocytic process.     

PCR and Culture Identification of Pathogenic Leptospira spp. from Coastal Soil in Leyte,Philippines, after a Storm Surge during Super Typhoon Haiyan (Yolanda)

Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. Most of the outbreaks of leptospirosis occur after floods caused by heavy rain in countries where Leptospira spp. are endemic. It has been believed that the overflow of seawater rarely causes outbreaks of leptospirosis because the leptospires are killed by salt water. On 8 November 2013, a storm surge caused by Super Typhoon Haiyan (Yolanda) inundated the entire coastal areas of Tacloban and Palo in Leyte, Philippines. The present study was carried out in order to determine whether the environmental leptospires in soil were able to survive after the storm surge in the affected areas. We collected 23 wet soil samples along the coastal areas of Tacloban and Palo 2 months after the storm surge. The samples were suspended in HEPES buffer, and the supernatants were cultured in liquid or semisolid Korthof''s medium supplemented with five antimicrobial agents to inhibit the growth of contaminants. Leptospires were isolated from primary cultures of 22 out of 23 samples. The DNA of pathogenic Leptospira species was detected in 11 samples (47.8%) by analysis of flaB by nested PCR. Eventually, two pathogenic Leptospira strains were isolated and showed the highest 16S rRNA gene sequence similarity to Leptospira kmetyi. When these isolates were experimentally mixed with soil, they were found to survive in seawater for 4 days. These results show the possibility that leptospires living in soil survived after the storm surge. Our findings may serve as a warning that when seawater inundates the land during a storm surge or a tsunami, an outbreak of leptospirosis could occur in the disaster-stricken area.     

Highly stereoselective synthesis of (2'R)-[2'-2H]-deoxyribonucleosides and synthesis of their oligonucleotides.

Highly stereoselective synthesis of (2'R)-[2'-2H]-2'-deoxyribonucleosides (2'R:2'S = > 99:1) were accomplished by treating 2'-bromo-3',5'-O-TPDS-2'-deoxyribonucleosides with tributyltin deuteride at lower temperatures such as -60 degrees C in the presence of triethylborane. Moreover, synthesis of some oligodeoxyribonucleosides involving them will be described.     

Enterohemorrhagic Escherichia coli effector EspL2 induces actin microfilament aggregation through annexin 2 activation

Enterohemorrhagic Escherichia coli (EHEC) delivers virulence factors into host cells through the type III secretion system (T3SS) to exert the bacterial pathogenicity. EHEC encodes more than 20 type III secretion system-delivered families of effectors that have different functions at different infectious stages and enable a successful infection. One of them, EspL2, is encoded on the SpLE3 phage-like element in EHEC O157:H7 Sakai and is well conserved among various EHEC strains. Here we show that, after delivery into host cells, EspL2 accumulated under adherent bacteria, as did polymerized F-actin. EspL2-expressing EHEC formed three-dimensional, condensed microcolonies, into which the host cell extended plasma membrane protrusions on an F-actin-rich cytoskeleton. EspL2 bound F-actin-aggregating annexin 2 directly, increasing its activity. In addition, annexin 2 depletion abolished the EspL2-dependent formation of condensed microcolonies and F-actin aggregation. The EspL2-induced pseudopod-like protrusion of the host plasma membrane interacted with and supported colonization by the bacteria, independent of Tir-mediated actin polymerization. Thus, EspL2 supports efficient colonization by increasing annexin 2's ability to aggregate Tir-induced F-actin and by modifying the morphology of the host cell membrane.     

Beraprost sodium enhances neovascularization in ischemic myocardium by mobilizing bone marrow cells in rats

Beraprost sodium, an orally active prostacyclin analogue, has vasoprotective effects such as vasodilation and antiplatelet activities. We investigated the therapeutic potential of beraprost for myocardial ischemia. Immediately after coronary ligation of Sprague-Dawley rats, beraprost (200 microg/kg/day) or saline was subcutaneously administered for 28 days. Four weeks after coronary ligation, administration of beraprost increased capillary density in ischemic myocardium, decreased infarct size, and improved cardiac function in rats with myocardial infarction. Beraprost markedly increased the number of CD34-positive cells and c-kit-positive cells in plasma. Also, four weeks after coronary ligation of chimeric rats with GFP-expressing bone marrow, bone marrow-derived cells were incorporated into the infarcted region and its border zone. Treatment with beraprost increased the number of GFP/von Willebrand factor-double-positive cells in the ischemic myocardium. These results suggest that beraprost has beneficial effects on ischemic myocardium partly by its ability to enhance neovascularization in ischemic myocardium by mobilizing bone marrow cells.     

Three-dimensional structure of rat-liver acyl-CoA oxidase in complex with a fatty acid: insights into substrate-recognition and reactivity toward molecular oxygen

The three-dimensional structure of rat-liver acyl-CoA oxidase-II (ACO-II) in a complex with a C12-fatty acid was solved by the molecular replacement method based on the uncomplexed ACO-II structure. The crystalline form of the complex was obtained by cocrystallization of ACO-II with dodecanoyl-CoA. The crystalline complex possessed, in the active-site crevice, only the fatty acid moiety that had been formed through hydrolysis of the thioester bond. The overall dimeric structure and the folding pattern of each subunit are essentially superimposable on those of uncomplexed ACO-II. The active site including the flavin ring of FAD, the crevice embracing the fatty acyl moiety, and adjacent amino acid side chains are superimposably conserved with the exception of Glu421, whose carboxylate group is tilted away to accommodate the fatty acid. One of the carboxyl oxygens of the bound fatty acid is hydrogen-bonded to the amide hydrogen of Glu421, the presumed catalytic base, and to the ribityl 2'-hydroxyl group of FAD. This hydrogen-bonding network correlates well with the substrate recognition/activation in acyl-CoA dehydrogenase. The binding mode of C12-fatty acid suggests that the active site does not close upon substrate binding, but remains spacious during the entire catalytic process, the oxygen accessibility in the oxidative half-reaction thereby being maintained.     

Crystal structures of nonoxidative zinc-dependent 2,6-dihydroxybenzoate (gamma-resorcylate) decarboxylase from Rhizobium sp. strain MTP-10005

Reversible 2,6-dihydroxybenzoate decarboxylase from Rhizobium sp. strain MTP-10005 belongs to a nonoxidative decarboxylase family. We have determined the structures of the following three forms of the enzyme: the native form, the complex with the true substrate (2,6-dihydroxybenzoate), and the complex with 2,3-dihydroxybenzaldehyde at 1.7-, 1.9-, and 1.7-A resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of one (alphabeta)8 triose-phosphate isomerase-barrel domain with three functional linkers and one C-terminal tail. The native enzyme possesses one Zn2+ ion liganded by Glu8, His10, His164, Asp287, and a water molecule at the active site center, although the enzyme has been reported to require no cofactor for its catalysis. The substrate carboxylate takes the place of the water molecule and is coordinated to the Zn2+ ion. The 2-hydroxy group of the substrate is hydrogen-bonded to Asp287, which forms a triad together with His218 and Glu221 and is assumed to be the catalytic base. On the basis of the geometrical consideration, substrate specificity is uncovered, and the catalytic mechanism is proposed for the novel Zn2+-dependent decarboxylation.     

Lipocortin-like 33 kDa protein of guinea pig neutrophil. Its distribution and stimulation-dependent translocation detected by monoclonal anti-33 kDa protein antibody

33 kDa protein of neutrophil is a lipocortin-like protein, as proposed from its biochemical properties, amino acid composition, and the homology of its amino acid sequence to human lipocortin I. The localization and translocation of 33 kDa protein (p33) in blood cells of guinea pig were studied by immunoblotting (Western blotting) and immunocytochemical fluorescence methods using polyclonal and monoclonal mouse anti-p33 antibodies. The protein was determined to be present only in the cytoplasm of neutrophils, but not in cells such as the monocyte, lymphocyte, platelet, and other bone marrow cells. The translocation of the protein from cytoplasm to cell membrane was coupled with the increase in intracellular calcium ion and in superoxide generation induced by a chemotactic factor. These findings suggest that p33 may have an important role not only in the regulatory mechanism of phospholipase A2 (PLA2) activity but also in other transmembrane signaling.