Terry M. Preston

Purification and characterization of a galactose-specific agglutinin from the haemolymph of the larval stages of the insect Calliphora vomitoria

A lectin was isolated from the haemolymph of the blowfly larva Calliphora vomitoria. It agglutinated a variety of mammalian erythrocytes with varying specificities and was strongly inhibited by D-galactose and fetuin. The activity was also sensitive to chelators of metal ions, heating above 50 degrees C and proteolytic digestion. SDS-PAGE identified a glycoprotein with an Mr of 32,000 under reducing and nonreducing conditions which resolved to a band at pH 5.4 using isoelectric focusing. Using FPLC gel filtration the activity was isolated in a fraction with an Mr of 130,000. It is suggested that the native form of the molecule is a noncovalently associated tetramer.

Functional studies on Calliphora vomitoria haemocyte subpopulations defined by lectin staining and density centrifugation

Haemocyte subpopulations of Calliphora vomitoria have been categorized by their surface staining properties using fluorescently labelled lectins, and their mobilities in Percoll density gradients. These methods of identification were exploited to determine the roles of these cell types in cellular defence reactions. Soybean agglutinin clearly defined the cell subpopulation involved in phagocytosis, while purified thrombocytoid fragments proved to be the main haemocyte population involved in encapsulation and nodule formation.

Evidence for the expression of actomyosin in the infective stage of the sporozoan protist Eimeria

A high-speed supernatant extract was obtained from infective oocysts of Eimeria tenella homogenised in a sucrose-low ionic strength buffer. Immunoblotting showed this soluble, micropore-filtered preparation (designated E1) to be rich in actin. E1 underwent superprecipitation on addition of ATP but not its non-hydrolysable analogue AMP.PMP--behaviour typical of an actomyosin solution. The superprecipitate fluoresced strongly in the presence of rhodamine-phalloidin (indicative of the presence of F-actin) and electron microscopy of negatively-stained preparations of this flocculent matter confirmed the abundance of filamentous material within it. This is the first demonstration of a functional actomyosin isolated from a member of the economically important phylum Apicomplexa.

A prominent microtubule cytoskeleton in Acanthamoeba

A method for preparing by detergent extraction the cytoskeletons of substrate-attached, motile Acanthamoeba castellanii is described. A monoclonal antibody to yeast alpha tubulin has been used to demonstrate the presence of abundant microtubules in the cytoskeleton of this amoeba by fluorescence and whole-mount electron microscopy. Individual microtubules, often more than 10 micron long, interweave to form a well-developed 3-D network pervading the cytoplasm and embracing the nucleus. In some cases immunofluorescent staining reveals distinct nodes in the perinuclear region of this microtubular network.

The cell surface in amoeboid locomotion — studies on the role of cell-substrate adhesion

Abstract The relative strength of attachment to the walls of a glass capillary displayed by Naegleria gruberi amoebae during locomotion under different electrolyte conditions was measured. The proportion of initially adherent cells remaining attached to the capillary after 10 ml of defined solution which had previously been shown to modulate the speed of locomotion and cell-substrate gap distance, was then determined. It was found that an inverse relationship existed between the strength of cell-substrate adhesion and the cell-substrate separation distance. When locomotion speeds were considered it was found that an optimal level of cell-substrate adhesion occurred for maximal cell motility (e.g. in 10 mM NaCl). However locomotion rates of about 30% of this value were found in media producing low adhesivity (i.e. deionized H 2 O) or very high adhesivity (i.e. polylysine solutions).

Biological characteristics of the Calliphora vomitoria agglutinin

The galactose specific agglutinin from Calliphora vomitoria was found to be expressed in the haemolymph of all the larval instars, but could not be detected at any other time during the life cycle. The haemagglutinating activity was insensitive to wounding of the tegument or injection of saline; however, a significant increase in haemagglutinating titre could be induced upon inoculation of the haemocoel with biotic or abiotic particulate material. The agglutinin also actively agglutinated several bacterial species and appeared capable of playing a role in particle--haemocyte interaction. The presence of the purified agglutinin significantly increased the attachment of fetuin-derivatized beads to haemocytes in vitro, and this activity could be specifically reduced by the addition of galactose, suggesting that the agglutinin may act as an opsonin.

Cell-substrate interactions in amoeboid locomotion - a matched reflexion interference and transmission electron microscopy study.

Cell-substrate separation distance were measured on Naegleria gruberi amoebae moving in deionized H2O on an untreated glass substratum (weakly adhesive) and on a polylysine treated glass surface (strongly adhesive). The values obtained by transmission electron microscopy on fixed cells and reflexion interference microscopy on live cells were in broad agreement.

Characterization of p80, a Novel Nuclear and Cytoplasmic Protein in Dinoflagellates

The presence of myosin in dinoflagellates was tested using an anti-Acanthamoeba castellanii myosin II polyclonal antibody on the heterotrophic dinoflagellate Crypthecodinium cohnii Seligo. Western blots revealed the presence of a unique band of 80 kDa in total protein extracts and after immunoprecipitation. Expression of this 80 kDa protein appeared constant during the different phases of the cell cycle. In protein extracts from various other dinoflagellates, this 80 kDa protein was detected only in the autotrophic species Prorocentrum micans Ehr. Screening of a C. cohnii cDNA expression library with this antibody revealed a cDNA coding for an amino acid sequence without homology in the databases. However, particular regions were detected: - a polyglutamine repeat domain in the N-terminal part of the protein, - four peptide sequences associated with GTP-binding sites, - a sequence with slight homology to the rod tail of Caenorhabditis elegans myosin II, -a sequence with homology to a human kinesin motor domain. Immunocytolocalization performed on C. cohnii thin sections with a polyclonal antibody raised against the recombinant protein showed p80 to be present both within the nucleus and in the cytoplasm. Labelling was widespread in the nucleoplasm and more concentrated at the periphery of the permanently condensed chromosomes. In the cytoplasm, labelling appeared in a punctate region close to the nucleus and in the flagellum. Potential functions of this novel protein are discussed.

P75, a novel protein in Dinoflagellates

FVll is a 54 kI>a plasma serine protease zymogen glycoprotein which may be cleaved to produce FVIIa, which binds tightly to cell-exposed tissue factor (TF). The complex is responsible for the initiation of blood coagulation (Stenflo J., Suttie J. (19771 Annu. Rev. Biochern. 46, 157-172). Although FVIIa has been cocrystallised with its cofactor TF (Banner D. et al 0996) Nature 380,41-46), no structure exists for the protease. In certain clinical situations the 56kDa plasma serine protease inhibitor (Serpin) antithrombin III (ATIII) may directly inhibit FVlIa by the formation of a FVIIa/ATIII complex (Lawson J. et al. (1993) I. &of. Chem. 268,767.770).

Molecules involved in the mitosis of dinoflagellate protists

‘URA 671 CNRS, Station Zuologique. Observatoire OcCanologique. 06230 VillefiancheMer We reported earlier that phospholipase C (PLC) and phosphomositide kinase (PI kinase) involved in the turnover of polyphosphoinositides (PPI) may control mitosis in sea urchin eggs (Ciapa et al. (1994), Nature 368, 875-878). Besides PLC second messengers, Pi 3-kinase generates 3phosphorylated inositol lipids which have been proposed to affect a wide variety of cellular processes in other cellular systems (Carpenter, C. L.. Cantley, L. C. (1996), Current Opinion in Cell Biology 8, 153158). We have investigated whether PI 3-kinase could play a role in regulating the sea urchin early embryonic development. We have immunoprecipited from egg extracts a protein of YSkDa w~tb an anti-PI 3 kinase antibody. We found that proteins immunoprecipited with anti-phosphotyrosine antibodies displayed a PI kinase activity sensitive to wortmannih, a specific inhibitor of PI 3kmase. These results suggest that Pi 3-kinase is present in sea urchin eggs. We observed that treatment of eggs with wortmannin did not affect fertilization but led to arrest -of the cell cycle after chromosome condensation and nuclear envelope breakdown bave occured. Although protein and DNA synthesis were not affected, Maturation Promoting.Factor (MPF) activation was inhibited. We found that fertilization induced a transient stimulation of a mitogen-activated protein (MAP) kinase activity at the time of mitosis and during the succeeding mitdtic cycles of sea urchin eggs: We observed that wortmannin delayed MAP kinase stimulation and inhibited its inactivation normally observed when the eggsdivide. It is accepted that high levels of MAP kinase activity are responsible for metaphase arrest of oocytes of various species inch&ins Xenonus and mouse (Sag&a, N. (1996),Tr&ds in Cell B&logy-6, 22-25). Th& fits~with our observation that MAP kinase activity increased when eggs entered mitosis and was maintained at a-high level in eggs arrested by-wottmannin. How MAP kinase and MPF interact during mitosisis still unclear. Our data show that, in wortmannin treated egns, MAP kinase can be activated and remained stimulated without a full activation of MPF, suggesting that MAP kinase may be controlled during the cell cycle bv comuonents other than MPF. Bv regulating MAP kit&e activity, PI 3-k&se &ght be an important element in the control of metaphase arrest in oocytes. MAPK, cychne B et p34”“’ during the acquisition of meiotic maturation competence in goat ooqtes. Thierrv DEDIEU, Isabelle HUE, Emilie LEDAN, Laurence GALL, INRA Unite Biologie de la Fecondation 78352 Jouy-en-Josas cMex