Pleurocidin, a novel antimicrobial peptide, induces human mast cell activation through the FPRL1 receptor.

Pleurocidins are a novel family of α-helical cationic antimicrobial peptides (CAPs) that are structurally and functionally similar to cathelicidins, one of the major CAP families. As cathelicidins stimulate mast cell chemotaxis and mediator release, we postulated that pleurocidins similarly activate mast cells. A screen of 20 pleurocidin peptides revealed that some were capable of degranulating the human mast cell line LAD2 (Laboratory of Allergic Diseases 2). Pleurocidin NRC-04 caused LAD2 to adhere, migrate, degranulate, and release cysteinyl leukotrienes and prostaglandin D2. Moreover, pleurocidin increased intracellular Ca(2+) mobilization in mast cells and induced the production of proinflammatory chemokines such as monocyte chemotactic protein-1/C-C motif chemokine ligand 2 (CCL2) and macrophage inflammatory protein-1ß/CCL4. Our evaluation of possible cellular mechanisms suggested that G proteins, phosphoinositol-3 kinase (PI3K), phospholipase C (PLC), and phosphokinase C (PKC) were involved in pleurocidin-induced mast cell activation as evidenced by the inhibitory effects of pertussis toxin (G protein inhibitor), wortmanin (PI3K inhibitor), U-73122 (PLC inhibitor), and Ro-31-8220 (PKC inhibitor), respectively. We also found that human mast cells expressed the N-formyl-peptide receptor 1 (FPRL1) receptor and FPRL1-specific inhibitor affected pleurocidin-mediated activation of mast cell. Our finding that the novel CAP pleurocidin activated human mast cell through G protein-coupled receptor signaling suggests that this peptide might have immunomodulatory functions.

Ceratomyxa drepanopsettae in the gallbladder of Atlantic halibut, Hippoglossus hippoglossus, from the northwest Atlantic Ocean.

Trophozoites of Ceratomyxa drepanopsettae Averintsev, 1907 (Myxosporea: Ceratomyxidae) containing prominent refractile granules were found in the gallbladders of all but one of eight halibut, the exception being a single juvenile. They ranged in shape and size from globular forms 5-10 micron in diameter, to rounded structures with pseudopodia and one or more processes that were up to 500 micron in length and packed with refractile granules. Some trophozoites were free in the bile, while others were attached to the epithelium of the gallbladder wall by pseudopodia which extended between the microvilli. Many free trophozoites were attached to each other by septate junctions between their pseudopodia. There were small cylindrical papillae on the surface of the trophozoites, and the rounded portions contained two vegetative nuclei, generative cells (some attached by junctions) and, in many cases, feeding vacuoles. During sporogony, a binucleate sporoplasmic cell and the capsulogenic cells of some sporoblasts were engulfed by valvogenic cells before they began to differentiate; whereas other sporoblasts consisted of six cells attached to each other, two being capsulogenic cells containing external tubes, two sporoplasmic cells and two valvogenic cells. There was a septate junction around the opening of the rounded polar capsule of the spore, between the capsulogenic and valvogenic cell. Sporoplasmosomes appeared to form in smooth membraned vesicles, possibly part of the Golgi apparatus. Spores had thin, delicate membrane, and elongate shell-valves, most of which were asymmetric, and bent or folded. A sporoplasm extended on either side of the distinct, straight suture line, but did not penetrate into the valves.

Evidence that kinetochore microtubules in crane-fly spermatocytes disassemble during anaphase primarily at the poleward end.

Anaphase chromosome motion involves the disassembly of kinetochore microtubules. We wished to determine the site of kinetochore microtubule disassembly during anaphase in crane-fly spermatocytes. In crane-fly spermatocyte spindles, monoclonal antibody 6-11B-1 to acetylated alpha-tubulin labels kinetochore microtubules almost exclusively, with an area immediately adjacent to the kinetochore being weakly or not labelled. This 'gap' in acetylation at the kinetochore serves as a natural marker of kinetochore microtubules in the kinetochore fibre. We measured the length of the gap on kinetochore fibres in metaphase and anaphase in order to deduce the fate of the gap during anaphase; we used this information to determine where kinetochore microtubules disassemble in anaphase. Gap lengths were measured from confocal microscope images of fixed spermatocytes dual labelled with 6-11B-1 to acetylated alpha-tubulin and YL1/2 to tyrosinated alpha-tubulin, the latter being used to determine the positions of kinetochores. In metaphase the average gap length was 1.7 microns. In anaphase, the gap appeared to decrease in length abruptly by about 0.4 microns, after which it decreased in length by about 0.2 microns for every 1 microns that the chromosome moved poleward. PacMan models of chromosome movement predict that this 'gap' in staining should disappear in anaphase at a rate equal to that of chromosome movement. Thus, our results do not support theories of chromosome motion that require disassembly solely at the kinetochore; rather, in crane-fly spermatocytes kinetochore microtubule disassembly in anaphase seems to take place primarily at the poles.

Electron-microscopic and immunochemical analysis of kinetochore microtubules after ultraviolet microbeam irradiation of kinetochores.

We used an ultraviolet microbeam to irradiate kinetochores of chromosomes in crane-fly spermatocytes. We used one of two doses, low (0.106 erg microns-2) or high (0.301 erg microns-2), and then studied the microtubules in those spindles using electron microscopy or immunofluorescence microscopy. After irradiation with low doses microtubules are present as usual, with normal fluorescence and in normal numbers. After irradiation with high doses microtubules are no longer associated with the irradiated kinetochore. After irradiation with either dose, non-kinetochore microtubules are in smaller numbers in the irradiated half-spindle than in the non-irradiated half-spindle or in non-irradiated cells. Since irradiation with low doses alters interchromosomal 'signals', but microtubules remain attached to the kinetochore, we argue that low doses of ultraviolet light damage a signal-related function of kinetochores without altering the ability of the kinetochores to bind microtubules.

Ultraviolet microbeam irradiation of microtubules in vitro. The action spectrum for local depolymerization of marginal band microtubules in vitro matches that for reducing birefringence of chromosomal spindle fibres in vivo.

Marginal bands were isolated from newt red blood cells and, using monochromatic light from an ultraviolet microbeam, the marginal band microtubules were irradiated in vitro to produce areas of reduced birefringence (ARBs). The ARBs neither moved nor changed shape after they were formed, though the marginal bands sometimes changed shape during the irradiation. Marginal band ARBs were regions in which the microtubules were locally depolymerized, as determined by electron microscopy and immunofluorescence. The action spectrum for producing ARBs on marginal band microtubules in vitro matches very closely the action spectrum for producing ARBs on crane-fly spermatocyte chromosomal spindle fibres in vivo, which indicates that ARBs in vivo are produced by the ultraviolet light acting directly on the microtubules (as opposed to an intermediate component), and confirms, without complications inherent in the fixation of living cells, that ARBs on spindle fibres in vivo are regions in which microtubules are locally depolymerized.