Growth and development in relation to the cell cycle inPhysarum polycephalum

Summary In strain CL ofPhysarum polycephalum, multinucleate, haploid plasmodia form within clones of uninucleate, haploid amoebae. Analysis of plasmodium development, using time-lapse cinematography, shows that binucleate cells arise from uninucleate cells, by mitosis without cytokinesis. Either one or both daughter cells, from an apparently normal amoebal division, can enter an extended cell cycle (28.7 hours compared to the 11.8 hours for vegetative amoebae) that ends in the formation of a binucleate cell. This long cycle is accompanied by extra growth; cells that become binucleate are twice as big as amoebae at the time of mitosis. Nuclear size also increases during the extended cell cycle: flow cytometric analysis indicates that this is not associated with an increase over the haploid DNA content. During the extended cell cycle uninucleate cells lose the ability to transform into flagellated cells and also become irreversibly committed to plasmodium development. It is shown that commitment occurs a maximum of 13.5 hours before binucleate cell formation and that loss of ability to flagellate precedes commitment by 3–5 hours. Plasmodia develop from binucleate cells by cell fusions and synchronous mitoses without cytokinesis.Abbreviations CL Colonia Leicester - DSDM Dilute semi-defined medium - FKB Formalin killed bacterial suspension - IMT Intermitotic time - LIA Liver infusion agar - SBS Standard bacterial suspension - SDM Semi-defined medium     

Gp63-Like Molecules in <Emphasis Type="Italic">Phytomonas serpens</Emphasis>: Possible Role in the Insect Interaction

In this study, we demonstrated that metallopeptidase inhibitors (EDTA, EGTA, and 1,10-phenanthroline) were able to arrest Phytomonas serpens growth in distinct patterns. This parasite released exclusively metallopeptidases to the extracellular environment, whereas in cellular extracts only cysteine peptidases were detected. In addition, an extracellular polypeptide of 60 kDa reacted in Western blotting probed with polyclonal antibody raised against gp63 of Leishmania amazonensis. In the cellular parasite extract, this antibody recognized bands migrating at 63 and 52 kDa, which partitioned on both aqueous and membrane-rich fractions. Flow cytometry and fluorescence microscopy analyses showed that the gp63-like molecules have a surface location. Moreover, phospholipase C (PLC)-treated parasites reduced the number of gp63-positive cells. The anti-cross-reacting determinant (CRD) and anti-gp63 antibodies recognized the 60-kDa band in the supernatant from PLC-treated cells, suggesting that this protein is glycosylphosphatidylinositol-anchored to the plasma membrane. This is the first report on the presence of gp63-like molecules in members of the Phytomonas genus. The pretreatment of the parasites with anti-gp63 antibody significantly diminished their adhesion index to explanted salivary glands of the phytophagous insect Oncopeltus fasciatus, suggesting a potential involvement of the gp63-like molecules in the adhesive process of this plant trypanosomatid.     

The effect of tunicamycin on the protease activity of GP63 from Leishmania major

The protease activity of gp63 from L. major was studied in relation to tunicamycin induced N-deglycosylation. It was found that after tunicamycin treatment, a N-deglycosylated product of gp63 with protease activity is present at the cell surface of Leishmania promastigote.     

The Ser-Arg-Tyr-Asp region of the major surface glycoprotein of Leishmania mimics the Arg-Gly-Asp-Ser cell attachment region of fibronectin.

The major surface glycoprotein of Leishmania, gp63, a fibronectin-like molecule, plays a key role in parasite-macrophage interaction. Binding of gp63 to macrophage receptors is inhibited by Arg-Gly-Asp-Ser (RGDS)-containing synthetic peptides of fibronectin and by antibodies to these peptides. However, gp63 lacks an RGDS tetrapeptide. We sought to identify the region of gp63 that antigenically and functionally mimics the RGDS-containing region of fibronectin. We thus synthesized on polyethylene rods overlapping tetracosapeptides covering the whole sequence of Leishmania major gp63. gp63 affinity-purified antibodies raised against fibronectin and against the RGDS-containing fibronectin decapeptide RGDSPASSKP bound specifically to gp63 residues 241-264. Subsequently, by the use of smaller peptides, the gp63 tetrapeptide 252-255 (SRYD) was identified as the minimum antibody binding segment. Single residue substitution peptide analogues showed that indeed Tyr and Gly can be alternatively substituted in the SRYD- and RGDS-containing peptides of gp63 and fibronectin, respectively, without major effects on their antibody binding capacity. Subsequently, we investigated the effect of an SRYD peptide on promastigote-macrophage interaction in vitro; treatment of macrophages with an SRYD-containing gp63 octapeptide efficiently inhibited parasite attachment to macrophage receptors. Thus, the conserved among species sequence SRYD of gp63, with significant hydrophilicity, flexibility, and beta-turn propensity features, mimics antigenically and functionally the RGDS sequence of fibronectin. We suggest that this segment constitutes the putative gp63 adhesion site.     

<Emphasis Type="Italic">Gemini pollen 2</Emphasis>, a male and female gametophytic cytokinesis defective mutation

Gametophytic cytokinesis is essential for the development and function of the male and female gametophytes. We have previously described the isolation and characterisation of gemini pollen 1 (gem1) that acts gametophytically to disturb asymmetric division and cytokinesis at pollen mitosis I (PMI) in Arabidopsis. Here we describe the genetic and cytological analysis of an independent gametophytic mutant, gem2, with similar characteristics to gem1, but which maps to a different genetic locus. gem2 shows reduced genetic transmission through both male and female gametes and leads to the production of divided or twin-celled pollen. Developmental analysis revealed that gem2 does not affect karyokinesis at PMI, but leads to repositioning of the cell plate, and partial or complete failure of cytokinesis, resulting in symmetrical divisions or binucleate pollen grains, respectively. Symmetrical divisions lead to altered pollen cell fate with both sister cells displaying vegetative cell fate. Moreover, we demonstrate that the predominant female defect in gem2 is a lack of cellularisation of the embryo sac during megagametogenesis. GEM2 therefore defines an independent genetic locus that is involved in the correct specification of both male and female gametophytic cytokinesis.     

Expression of antigens in virulent and avirulent Indian strains ofLeishmania donovani

Indo-Gen mediated surface labelling with¹²⁵I demonstrated differences in surface oriented antigens between virulent and virulent promastigote ofLeishniania donovani, In case of virulent strains, surface polypeptides with molecular masses of 63, 53, 42 and 38 kDa were found to be labelled with¹²⁵I whereas in the case of aviralent stains 68, 55, 50, 46, 42 and 33 Da, components were iodinated. Further studies by immunoblot assay using different subcellular fractions of virulent and avirulent parasites demonstrated that antibody raised against gp63 cross-reacted with the 63 and 60 kDa antigen of the virulent and avirulentLeishmania donovani strains of Indian origin respectively. It indicates that these two polypeptides are antigenically similar. When virulent and avirulent cells were grown in the presence of varying concentration of tunicarnycin and immunoblot with anti gp63, it was observed that with increasing concentration of tunicamycin the 63 kDa polypeptide of the virulent cells shifted to approximately 58–57 kDa and the 60 kDa polypoptide of the aviruleni cells shifted to 57 kDa. This suggests that glycosylation may play an important role in antigenic variation between virulent and avirulent parasites.     

Cell division in caffeine-induced binucleate protonemal cells ofAdiantum. II. Formation of the preprophase band and cell division in centrifuged and non-centrifuged cells

Organization of microtubules (MTs) in relation to the behavior of nuclei was examined in dividing binucleate cells ofAdiantum capillus-veneris L. To induce binucleate cells, caffeine, an inhibitor of formation of the cell plate, was applied at 4 mM to synchronously dividing protonemal cells during cytokinesis (Murata and Wada 1993). Formation of the preprophase band (PPB) during the next cell cycle was examined in non-centrifuged and centrifuged cells. The two nuclei were separated or associated with one another in both non-centrifuged and centrifuged cells, although the location of the nuclei in the cylindrical protonemal cells was different (Murata and Wada 1993). Irrespective of centrifugation, a single PPB was formed around the nuclei in cells with associated nuclei. Two PPBs were formed in cells with separated nuclei in centrifuged cells. Patterns of mitosis and cytokinesis varied, depending on the location of the PPB and the distribution of the nuclei. The role of the nucleus in formation of the PPB is discussed.     

Characterization and fine-structural localization of actin-and fibronectin-like proteins in planaria (Dugesia lugubris s.l.)

Summary Actin- and fibronectin-like proteins were characterized in the planarian, Dugesia lugubris s.l., by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting analysis using antisera to vertebrate actin and fibronectin. These antisera recognized protein bands of 42 kDa and 220 kDa, respectively. In addition, the immunohistochemical distribution of both actin- and fibronectin-like material was examined by using immuno-electron microscopy. Actin-like protein was localized in myofibrils in various differentiation stages, and in the peripheral cytoplasm and lamellipodia of cells that were migrating. The fibronectin-like component was associated with the extracellular matrix in the fibrillar structures and with the surface of the migrating cells. Our data suggest that similar cellular and molecular mechanisms are involved in cell-matrix interactions and in the morphogenesis of living organisms at different evolutionary levels.     

Co‐localization of β1,6‐branched Oligosaccharides and Coarse Melanin in Macrophage–Melanoma Fusion Hybrids and Human Melanoma Cells In Vitro

Fusion hybrids between normal macrophages and Cloudman S91 melanoma cells were shown earlier to have increased metastatic potential, along with high expression of β1,6‐N‐acetylglucosaminyltransferase  V and β1,6‐branched oligosaccharides. Curiously, hybrids, but not parental melanoma cells, also produced ‘coarse melanin’– autophagic vesicles with multiple melanosomes. As β1,6‐branched oligosaccharides were known to be associated with metastasis, and coarse melanin had been described in invasive human melanomas, we looked for potential relationships between the two. Using lectin‐ and immunohistochemistry, we analyzed cell lines producing coarse melanin for β1,6‐branched oligosaccharides: gp100/pmel‐17 (a melanosomal structural component) and CD63 (a late endosome/lysosome component associated with melanoma and certain other human cancers). Cell lines used in this study were (i) hybrid 94‐H48, a highly metastatic, macrophage–melanoma experimental fusion hybrid; (ii) 6^neo mouse melanoma cells, the weakly metastatic, parental fusion partner; and (iii) SKmel‐23, a human melanoma cell line derived from a metastasis. Coarse melanin granules were prominent both in hybrids and in SKmel‐23 cells, and co‐localized with stains for β1,6‐branched oligosaccharides, gp100/pmel 17, and CD63. This is the first report of this phenotype being expressed in vitro, although co‐expression of β1,6‐branched oligosaccharides and coarse melanin was recently shown to be a common and pervasive characteristic in archival specimens of human melanomas, and was most prominent in metastases. The results suggest that pathways of melanogenesis in melanoma may differ significantly from those in normal melanocytes. In vitro expression of this phenotype provides new biological systems for more detailed analyses of its genesis and regulation at the molecular genetic level.     

Cell division in caffeine-induced binucleate protonemal cells ofAdiantum. I. Asynchronous mitosis of two nuclei in a single cell

Coordination of karyokinesis of two nuclei in individual filamentous binucleate cells of the fern,Adiantum capillus-veneris was investigated. To induce binucleate cells, the protonemata were treated with caffeine, which is known as an inhibitor of plant cytokinesis, during the first synchronous division of cells that was induced by blue light (BL). The next synchronous division of cells in the resultant binucleate cells was analysed. In most cases, the two nuclei were associated with each other and were located in the apical region of the long protonemal cells (approximately 400–600 μm in length, 20 μm in width). In some cells, one nucleus was located in the apical region and the other was located in the middle of the cylinderical region. In such cells, karyokinesis of the apical nucleus preceded that of the basal nucleus, even though karyokinesis of associated nuclei progressed synchronously. Mitotic binucleate cells were centrifuged in order to gather two dissociated heterophasic nuclei. Progression of karyokinesis in the re-associated nuclei became coordinated within 1 h in most cells. These results suggest that mitosis-regulating factor(s) may diffuse to only limited distances inAdiantum protonemata.     

Regulation of cell–cell adhesion during Dictyostelium development

During development of Dictyostelium, four adhesion systems have been identified and adherens junction-like structures have been discovered in the fruiting body. The temporal and spatial expression of cell adhesion molecules (CAMs) is under stringent developmental control, corresponding to major shifts in morphological complexity. Genetic manipulations, including over-expression and knockout mutations, of the adhesion genes, cadA (encoding DdCAD-1), csaA (gp80) and lagC (gp150), have shed light on new roles for cell adhesion molecules in aggregate size regulation, cell-type proportioning, cell differentiation and cell sorting. As cell–cell interactions remain highly dynamic within cell streams and aggregates, mechanisms must exist to facilitate the rapid assembly and disassembly of adhesion complexes. Studies on gp80 have led to a model for the rapid assembly of adhesion complexes via lipid rafts.     

Inhibitory effects of tunicamycin on cell-cell adhesion and cell reaggregation of the cellular slime mold,Polysphondylium pallidum

To assess the role in cell-cell adhesion of gp64, a putative cell-cell adhesion molecule ofPolysphondylium pallidum, we treated the cells with tunicamycin (TM), a known inhibitor of the synthesis of the N-linked oligosaccharide precursor, and examined TM's effect on cell-cell adhesion. The vegetative growth ofPolysphondylium cells was inhibited with TM in a dose-dependent manner. When cells were treated with TM (2.0 μg/ml) during only the first 4 hr of starvation and further starved for 8 hr without TM, the cells dissociated considerably, although even the growth phase cells ofPolysphondylium normally show EDTA-resistant (Ca²⁺-independent) cell adhesions. In parallel with the above effects, the amounts of intact gp64 decreased considerably in time with the lengths of incubation (0 hr>4 hr >8 hr). When TM-treated cells were washed free of TM, and shaken for a further 12 hr, the cells began to aggregate again, accompanied by an increase of gp64. In conclusion, TM affected cell-cell adhesion ofPolysphondylium cells, but we were not able to distinguish whether the inhibition of cell aggregation was due to defects in glycosylation on glycoproteins and/or due to reduced levels of glycoproteins themselves.     

Cell cycle duration and time of DNA synthesis in binucleate cells induced inV. faba meristems by caffeine or isobutyl-methylxanthine

Summary Lateral roots ofVicia faba were treated with a solution of 5-aminouracil (3.93×10^–3M) for 6 hours. After 15 hours roots were recovering from the temporary inhibition of mitosis induced by 5-AU and were approaching peak mitotic indices; they were then treated with 0.1% caffeine or 0.1% isobutylmethylxanthine (IBMX) for 1 hour. Treatment with methylxanthines when the mitotic index was high gave relatively high yields of binucleate cells, 3.8 to 7.5%. DNA synthesis, cell cycle duration and nuclear growth were determined for binucleate cells. Caffeine induced binucleate cells underwent a marked reduction in nuclear volume, from 1,074 m³ at 1+1 hours to 534 m³ at 1+14 hours. Only 15% of these binucleates entered S phase; those that did so were in mitosis or had divided by 1+14 hours. We conclude that 85% of the binucleate cells are so inhibited by caffeine that their G₁ is extended to>14 hours or that they are no longer proliferating cells. IBMX-induced binucleate cells, by contrast, did enter S phase and many of them also divided. Though in IBMX-induced binucleate cells there was also a decrease in nuclear volume up to 1+10 hours, subsequently mean nuclear volume increased e.g. at 1+16 and 1+18 hours. Both caffeine and IBMX treatments resulted in decreases in mean volume of prophase nuclei of mononucleate cells; this is further evidence that both methylxanthines inhibit the macromolecular synthesis required to sustain nuclear growth. It also suggests that nuclear division can be initiated at considerably lower nuclear volumes than those of untreated cells. We suggest that caffeine may act as a mimic of the normal mechanism that regulates the switch from a proliferating to a non-proliferative condition.     

TheCaesalpinia peltophoroides cell cycle under different aeration conditions

Summary Cell cycle parameters were studied inCaesalpinia peltophoroides meristems proliferating under different oxygen tensions. This species has been selected for mixed planting in degraded areas in Brazil, some of which are occasionally flooded. As the species’ adaptation to oxygen deprivation during flooding is not fully understood, the objective of this study was to characterize the meristematic activity of root cells under various oxygen regimes. Synchronous binucleate cells, induced by a pulse of caffeine, showed a cell-cycle time constant under both control (5.6 mg of O₂ per l) and oxygenated conditions (7.9 and 3.2 mg of O₂ per l). The whole cell cycle lasted 10 h, although the relative duration of metaphase and anaphase/early telophase increased in more hypoxic conditions. The species appeared to utilise oxygen diffusing from the shoot to the root system to maintain cell division and root growth.     

Biology of theLeishmania surface: With particular reference to the surface proteinase,gp 63

Summary The protistan parasiteLeishmania is a dimorphic cell that survives as a motile promastigote in the insect digestive tract, and a non-motile, amastigote from within the phagolysosomal compartment of the vertebrate host's phagocytes. The surface ofLeishmania must interface with a range of differing environments and facilitate uptake of nutrients, whilst protecting the parasite from various host defence mechanisms. This review discusses the organization of the leishmanial cell, and the biology of its major surface constituents, the lipophosphoglycan and the surface proteinase, gp 63.     

<Emphasis Type="Italic">FORMOSA</Emphasis> controls cell division and expansion during floral development in <Emphasis Type="Italic">Antirrhinum</Emphasis><Emphasis Type="Italic">majus</Emphasis>

Control of organ size is the product of coordinated cell division and expansion. In plants where one of these pathways is perturbed, organ size is often unaffected as compensation mechanisms are brought into play. The number of founder cells in organ primordia, dividing cells, and the period of cell proliferation determine cell number in lateral organs. We have identified the Antirrhinum FORMOSA (FO) gene as a specific regulator of floral size. Analysis of cell size and number in the fo mutant, which has increased flower size, indicates that FO is an organ-specific inhibitor of cell division and activator of cell expansion. Increased cell number in fo floral organs correlated with upregulation of genes involved in the cell cycle. In Arabidopsis the AINTEGUMENTA (ANT) gene promotes cell division. In the fo mutant increased cell number also correlates with upregulation of an Antirrhinum ANT-like gene (Am-ANT) in inflorescences that is very closely related to ANT and shares a similar expression pattern, suggesting that they may be functional equivalents. Increased cell proliferation is thought to be compensated for by reduced cell expansion to maintain organ size. In Arabidopsis petal cell expansion is inhibited by the BIGPETAL (BPE) gene, and in the fo mutant reduced cell size corresponded to upregulation of an Antirrhinum BPE-like gene (Am-BPE). Our data suggest that FO inhibits cell proliferation by negatively regulating Am-ANT, and acts upstream of Am-BPE to coordinate floral organ size. This demonstrates that organ size is modulated by the organ-specific control of both general and local gene networks. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium infection in the porcine intestine: evidence for caspase-3-dependent and -independent programmed cell death

The normal intestinal epithelium is renewed with a turnover rate of 3–5 days. During Salmonella infection increased cell loss is observed, possibly as a result of programmed cell death (PCD). We have, therefore, studied the effects of Salmonella Typhimurium infection on three elements involved in PCD: caspase-3 activation, c-Jun phosphorylation on serine 63 (both detected by immunocytochemistry), and DNA fragmentation (detected by TUNEL reaction), using a pig jejunal loop model. Additionally, we used nuclear staining for detecting signs of classical apoptosis. Activated caspase-3 was detected in scattered epithelial cells and the number of positive cells increased with increasing times of exposure to Salmonella (P<0.0001). An increase in phospho-c-Jun in epithelial cells was already detectable 5 min after infection and often occurred in cells that appeared not to be invaded by the organism. Changes in caspase-3 activation and c-Jun phosphorylation were most marked in the proximal region of the jejunum. Although rarely observed in the epithelium, proper TUNEL-positive cells were frequently found in the intestinal lumen. Some, but not all, TUNEL-positive cells were also positive for caspase-3, indicating that both caspase-3-dependent and -independent pathways of PCD increased upon infection.     

IgG and IgE immune response against the surface glycoprotein gp200 of Saccharomyces cerevisiae in patients with atopic dermatitis

The heat-stable and soluble glycoprotein gp200 (molecular weight 200 kDa) is part of the cell wall of S. cerevisiae. Recently, an association was shown between IgA and IgG against gp200 and inflammation in Crohn's disease. Gp200 is able to induce a proliferation of human lymphocytes in vitro, together with a natural killer cell associated cytotoxicity. Specific IgE against Saccharomyces cerevisiae (baker's or brewer's yeast) may be detected in approximately 73 %, against Candida albicans in 68% of those patients suffering from severe atopic dermatitis. The aim of this study was to elucidate the possible role of an anti-gp200 immune response for the pathogenesis of atopic dermatitis by immunoblot analysis. Anti-gp200 IgE was found in 55% of healthy individuals, in 67% of individuals with atopic predisposition without eczema, in 63% of the patients with mild atopic dermatitis, and in 86% of patients with severe atopic dermatitis, respectively. On the contrary, anti-gp200 IgG could be shown in 55% of healthy individuals, in 89% of individuals with atopic predisposition but without eczema, in 100% of patients with mild atopic dermatitis, and in 79% with severe atopic dermatitis, respectively. No immunoreactivity was found when an extract of Arxula adeninivorans was used as antigen. These results underline the specificity of the immunoblot results with gp200 from Saccharomyces cerevisiae. It can be concluded that occurrence of specific IgE against Saccharomyces cerevisiae cannot be explained by a cross reactivity, e.g., against Candida albicansallergens. Further investigations with the recombinant gp200 will give information on the role of this glycoprotein both in atopic dermatitis and Morbus Crohn. This revised version was published online in August 2006 with corrections to the Cover Date.     

Immune complex antigens as a tool in serodiagnosis of kala-azar

The 63 kDa surface antigen of Leishmania promastigotes is one of the most important virulent factors in establishing the host parasite relationship. This glycoprotein is revealed by surface iodination study as well as by metabolic labeling and immunoblot methods. In search of this specific antigen for serodiagnosis, immune complexes (ICs) were isolated from kala-azar patient sera and analysed by SDS-PAGE and Western immunoblotting. The immunoblot of kala-azar IC with patient sera, antipromastigote sera and anti gp63 sera detected the major antigen of 55 kDa. This recognition suggests that 55 kDa antigen and gp63 have common antigenic epitope(s). Normal IC did not react with anti gp63 sera indicating absence of this antigen in normal IC. To confirm the parasitic origin of the 55 kDa antigen of kala-azar IC, in vitro IC was formed with parasite antigen and acid dissociated kala-azar IC antibody. This indicated the antigenic similarity of the 55 kDa antigen and gp63 antigen of the parasite. This also suggested that the former antigen may have been processed from gp63. In summary, identification of parasite antigen (55 kDa) in IC of kala-azar patients' sera may be useful in developing a serodiagnostic assay of visceral leishmaniasis. Several other antigens are visualized in kala-azar IC when developed with patient sera. But specificity and efficacy of these antigens have not yet been evaluated in serodiagnosis of the disease.