Lectin Analysis of Surface Saccharides in Two Trichomonas vaginalis Strains Differing in Pathogenicity*

SYNOPSIS. Surface saccharides in 2 Trichomonas vaginalis strains, the moderately pathogenic, JH34A, and the mild, JH162A, were analyzed with the aid of plant lectins. Concanavalin A (Con A), wheat germ agglutinin (WGA), soybean agglutinin (SBA), castor bean agglutinin (CBA), and lectin from the garden pea (GPA) were employed in agglutination tests and in treatment of ultrathin sections for electron microscopy according to the horseradish peroxidase-3,3′-diaminobenzidine method. With Con A and WGA, small quantitative differences were noted between the 2 strains in the results of agglutination and in the reaction-product deposits observed by electron microscopy. Distribution of the binding sites for the 2 lectins was also somewhat different in the JH34A and JH162A trichomonads. In general, the reactions with the more pathogenic strain were slightly stronger. Although the reactions with SBA and CBA lectins were weaker than those with Con A or WGA, they provided the means for qualitative differentiation between the 2 trichomonad strains. SBA alone agglutinated the JH34A strain and formed demonstrable deposits on the cell surfaces. On the other hand, only CBA reacted with JH162A flagellates. The garden pea lectin failed to bind to the surface of either strain. On the basis of results obtained with the control preparations incubated in the presence of specific inhibitors, it was concluded that both strains had α-methyl-D-mannoside and/or α-methyl-D-mannoside-like as well as N-acetyl-D-glucosamine residues on their surfaces. In addition, JH34A strain had D-lactose-containing residues while JH162A trichomonads had residues with D-galactose. Neither strain appeared to possess residues containing N-acetyl-D-galactosamine.     

The Major Surface-Associated Saccharides of Klebsiella pneumoniae Contribute to Host Cell Association

Analysing the pathogenic mechanisms of a bacterium requires an understanding of the composition of the bacterial cell surface. The bacterial surface provides the first barrier against innate immune mechanisms as well as mediating attachment to cells/surfaces to resist clearance. We utilised a series of Klebsiella pneumoniae mutants in which the two major polysaccharide layers, capsule and lipopolysaccharide (LPS), were absent or truncated, to investigate the ability of these layers to protect against innate immune mechanisms and to associate with eukaryotic cells. The capsule alone was found to be essential for resistance to complement mediated killing while both capsule and LPS were involved in cell-association, albeit through different mechanisms. The capsule impeded cell-association while the LPS saccharides increased cell-association in a non-specific manner. The electrohydrodynamic characteristics of the strains suggested the differing interaction of each bacterial strain with eukaryotic cells could be partly explained by the charge density displayed by the outermost polysaccharide layer. This highlights the importance of considering not only specific adhesin:ligand interactions commonly studied in adherence assays but also the initial non-specific interactions governed largely by the electrostatic interaction forces.     

Binding of Lectins to the Cell Surface of Trypanosoma cruzi*

SYNOPSIS. Differences in the composition and distribution of cell membrane carbohydrates were demonstrated in the 3 life cycle forms of 3 Trypanosoma cruzi strains by using lectins with different specificities. The results suggest that lectin binding may be useful in characterization of the parasite strains.     

Specificity of the isolectins from the plant cactusMachaerocereus eruca for oligosaccharides from porcine stomach mucin

Sugar specificity of theMachaerocereus eruca isolectins, MeA_I and MeA_II, has been determined by comparing the capacity of glycans with well defined structures to inhibit their haemagglutinating activity. Both are galactose-specific isolectins with high affinity for O-glycans. However, the twoM. eruca isolectins recognize different oligosaccharidic sequences belonging to O-glycosidically linked glycans from porcine stomach mucin. The minimal structure recognized by MeA_I on the porcine mucin glycans is the O-glycan core Gal1, 3GalNAc-ol, whereas MeA_II has a more extended site and interacts with a biantennary O-glycan possessing the terminal trisaccharide Fuc1,2 (GalNAc1,3) Gal1,4.     

Morphometric Analysis of Anguina amsinckiae from Three Host Species

Amsinckia species (fiddleneck) in the South Coast Ranges of California were surveyed to determine if any of the 12 different California species of Amsinckia are hosts of the nematode, Anguina amsinckiae (Steiner and Scott, 1935) Thorne, 1961. Previously only Amsinckia intermedia Fischer and Meyer was reported as a host of Anguina amsinckiae. The survey established that there are at least two additional hosts of Anguina amsinckiae: Amsinckia lycopsoides Lehmann and Amsinckia gloriosa Suksdorf. Seven sites containing nematode-infected Amsinckia plants were discovered. Every site contained two or more species of Amsinckia; however, only one site contained more than one species of Amsinckia that was galled. Nematode specimens from A. intermedia, A. lycopsoides, and A. gloriosa were used in a morphometric analysis of 14 morphological variables. Stepwise discriminant analysis of the variables to separate the populations by host were successful for females, and the pairwise F-tests showed all three populations to have different group means (P < 0.05). Males from the three hosts were not always separable, however, as only the nematodes from Amsinckia gloriosa had a different group mean (P < 0.05).     

Cell Surface Area and Membrane Folding in Glioblastoma Cell Lines Differing in PTEN and p53 Status

Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo−/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance C_m = 1.9 µF/cm². In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest C_m values of 3.7–4.0 µF/cm², which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.     

Genetic Differences between Blight-Causing Erwinia Species with Differing Host Specificities, Identified by Suppression Subtractive Hybridization

PCR-based subtractive hybridization was used to isolate sequences from Erwinia amylovora strain Ea110, which is pathogenic on apples and pears, that were not present in three closely related strains with differing host specificities: E. amylovora MR1, which is pathogenic only on Rubus spp.; Erwinia pyrifoliae Ep1/96, the causal agent of shoot blight of Asian pears; and Erwinia sp. strain Ejp556, the causal agent of bacterial shoot blight of pear in Japan. In total, six subtractive libraries were constructed and analyzed. Recovered sequences included type III secretion components, hypothetical membrane proteins, and ATP-binding proteins. In addition, we identified an Ea110-specific sequence with homology to a type III secretion apparatus component of the insect endosymbiont Sodalis glossinidius, as well as an Ep1/96-specific sequence with homology to the Yersinia pestis effector protein tyrosine phosphatase YopH.     

Species Richness and Host Specificity among Caterpillar Ensembles on Shrubs in the Andes of Southern Ecuador

Caterpillar ensembles were sampled on 16 species of shrubs from the family Asteraceae and the genus Piper (Piperaceae) in open and forest habitats in the Andean montane forest zone of southern Ecuador between August 2007 and May 2009. Trophic affiliations of caterpillars to the host plants were confirmed in feeding trials. Overall, species richness of herbivorous caterpillars was high (191 species across all plants), but varied strongly between ensembles associated with different plant species (2?C96 lepidopteran species per shrub species). Ensembles on Piper species were characterized by low effective species numbers and high dominance of one or two species of the Geometridae genus Eois Hübner. Low species number and high dominance were also found on latex-bearing Erato polymnioides, whereas ensembles on two other Asteraceae species were far more diverse and less strongly shaped by a few dominant species. The observed diversity patterns fit well to the concept that anti-herbivore defenses of plants are the major factors regulating associated insect ensembles. Local abundance and geographic range of host plants appear to have less influence. Lepidopteran species feeding on Asteraceae were found to be more generalistic than those feeding on Piper species. We conclude that caterpillar ensembles on most, but not all, studied plant species are defined by a small number of dominant species, which usually are narrow host specialists. This pattern was more distinct on Piper shrubs in forest understory, whereas Asteraceae in disturbed habitats had more open caterpillar ensembles.     

Evidence for the Presence of Lectins with Mannose Specificity in the Rat Cerebellum

Two different methods were set up to detect the possible presence of lectin-like molecules with a specificity for mannose-rich glycans in the rat cerebellum. The first, affinity histochemistry, involved the isolation of a particular class of glycoproteins from the cerebella of 11-day-old rats followed by the formation of covalent complexes with horseradish peroxidase and then incubation with cerebellar slices. The second used in vitro interactions between [3H]leucine-labeled proteins, kept in solution, with insolubilized [14C]glucosamine-labeled glycoproteins. The results of both methods are compatible with the presence of lectin-like activities inhibited by high mannose concentrations, but not other sugars. However, the binding sites preferred by these molecules seem to be more than a single mannose residue.     

Amphotericin B-Induced Carbohydrate Changes on the Trypanosoma cruzi Surface Membrane

ABSTRACT Changes in the cell surface carbohydrates of Trypanosoma cruzi epimastigotes induced by Amphotericin B (AmB) were assessed by chemical methods and by agglutination assay employing a panel of highly purified lectins of various sugar specificities, Escherichia coli K12 with mannose-sensitive fimbriae was also used as an agglutination probe. Amphotericin B caused a decrease in the total carbohydrate content of all glycoconjugate fractions isolated. Exposure to AmB strongly affected the mannose/galactose ratio (1:5) in the CHCI3/methanol/H2O soluble fraction. These sugars in 1.4:1 ratio were the major hexose components of control cells. The decrease in the mannose content (48 to 15%) after AmB treatment agrees with the marked decrease in the T. cruzi cell surface receptors for fimbriated E. coli K12. Also, an increase in the galactose content (74%) as compared with control cells (34%) is in agreement with the peanut agglutinin and Euonymus europaeus lectins agglutination results. Differences in the cell surface carbohydrates induced by AmB could be associated with alterations in the membrane structure and organization.     

鱼类三代虫的寄主特异性分析

分析了迄今为止世界范围三代虫的寄主情况。三代虫具有很强的寄主特异性,并且在种、属及科等水平上的特异性均有较明显差异;分析同一种寄主上所寄生的三代虫种类之间的关系,显示大部分寄主只寄生一种三代虫,体现出了三代虫很强的寄主特异性。     

Characterization of Molecular Species Carrying Gross Cell Surface Antigen

The Gross cell surface antigen (GCSA), associated with expression of endogenous Gross-type murine leukemia virus (G-MuLV) in tissues of mice, is defined by the cytotoxic reaction of a C57BL/6 antiserum, anti-AKR spontaneous leukemia K36, with cells of the Gross virus-induced C57BL/6 leukemia, Emale symbolG2. Sequential lactoperoxidase-catalyzed radioiodination of Emale symbolG2 cells, Nonidet P-40 lysis, precipitation with anti-K36 serum, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified molecules with properties of polyproteins encoded by the gag region of the viral genome. These cell surface species could also be labeled by in vitro culturing of Emale symbolG2 with radioactive glucosamine. The viral specificity of these molecules and their participation in the GCSA typing system were established as follows. (i) Absorption of anti-K36 serum with GCSA(+), but not GCSA(-), leukemias led to a marked decrease in precipitation of these proteins. (ii) The same Emale symbolG2 cell surface proteins were also precipitated by antisera against the MuLV virion proteins p30 and p15. (iii) Anti-K36 was shown to possess antibodies against Gross virus p30 and p15. (iv) "Clearing" the Emale symbolG2 lysate of molecules reactive with anti-p30 or anti-p15 sera removed molecules reactive with anti-K36 serum. (v) Absorption of anti-K36 serum with disrupted G-MuLV virions or with Gross p30 or p15 removed GCSA cytotoxic antibodies; partial absorption was achieved with disrupted Rauscher-MuLV (R-MuLV) or with R-MuLV p30, and no absorption was found with R-MuLV p15. These data show that Emale symbolG2 cells express, on their surfaces, MuLV core polyproteins that apparently can be glycosylated and on which the determinants of GCSA are located.     

Thermal Characteristics of C4 Photosynthetic Enzymes from Leaves of Three Sugarcane Species Differing in Cold Sensitivity

Phosphoenolpyruvate carboxylase, NADP-malate dehydrogenase andNADP-malic enzyme in desalted extracts from the leaves of threesugarcane species differing in cold sensitivity were relativelystable at cold temperatures, and their Arrhenius plots appearedas straight lines. Pyruvate,P_i dikinase (PPDK) from the threespecies was cold-inactivated, and its Arrhenius plots exhibiteda clear break-point around 10.6°C. Analysis of cold labilityof PPDK using deuterium oxide and Triton X-100 showed that theinteractions between the subunits possibly involve hydro-phobicbonds which would lead to cold lability. There were no apparentdifferences among the three sugarcane species in the thermalproperties of the four C₄ photosynthetic enzymes. The resultssuggest that the differences in cold sensitivity of sugarcanephotosynthesis may not relate to the thermal properties of C₄photosynthetic enzymes per se. ¹ Present address: Department of Biochemistry, University ofNebraska, Lincoln, NE 68588-0664, U.S.A.     

Eimeria tenella: Host Specificity in Gallinaceous Birds

SYNOPSIS. Eight species representing 8 genera of gallinaceous birds were used: Alectoris graeca; Colinus virginianus; Coturnix coturnix; Gallus gallus; Meleagris gallopavo; Numidia meleagris; Pavo cristatus; Phasianus colchicus. Three-week-old birds were dosed with sporulated oocysts of Eimeria tenella Beltsville strain. At 4, 24, 48, 72, 96, 120, 144, and 168 hr after inoculation, 1-3 infected birds and uninoculated controls of each species were killed by cardiac exsanguination. Pieces of intestines were fixed and examined for stages of E. tenella as stained paraffin sections or indirect fluorescent antibody preparations. Oocyst counts were made in droppings collected for the first 6 days of the patent period. Sporozoites were found in the lamina propria of some birds of 5 species at 4 hr postinoculation, but no stages were found thereafter except in the breeds of G. gallus and A. graeca. At 144 and 168 hr postinoculation, a few macrogametes were found in the ceca of 2 A. graeca , but no oocysts were found in the feces. No statistical difference was found between the number of oocysts produced/bird in the breeds of G. gallus examined. It is evident from these observations that E. tenella did not complete its life cycle in several close phylogenetic relatives of G. gallus , even though in other studies this parasite was found to complete its life cycle in cell cultures derived from the same birds.     

Root Cell Ultrastructure in Developing Aerenchyma Tissue of Three Wetland Species

Patterns of root cortex cell development and ultrastructurewere analysed in Sagittaria lancifolia L., Thalia geniculataL. and Pontederia cordata L. using scanning and transmissionelectron microscopy (SEM, TEM). In all three species, cortexcells were arranged in radial columns extending from the endodermisto the hypodermis/epidermis. During gas space formation, thecortex cells elongated parallel to the root radius and shrankin the plane perpendicular to the radius leaving long and thinrows of cortex cells extending from the endodermis to the epidermis.Although the cortex cells appeared collapsed in tissue withwell-developed gas spaces, TEM revealed that the cortical cellsas well as the epidermal cells maintained intact membranes andmany normal organelles. Formation of root cortex tissue withwell-developed gas spaces does not require cell death in thesespecies. Living cortex cells in root tissue with mature gasspaces could provide a symplastic pathway for transport betweenthe root stele and the living epidermal cells. Copyright 2000Annals of Botany Company Sagittaria lancifolia, Thalia geniculata, Pontederia cordata, aerenchyma, root, wetland, development     

Pathogenicity and Host Specificity of Entomopathogenic Nematodes

The mechanisms of infection and pathogenicity of Steinernematidae and Heterorhabditidae in insect hosts are discussed as factors influencing the host specificity of these nematodes. The invasion and evasion of host defences are important steps in the pathogenic process. The ability of the nematode to penetrate into the insect haemocoel, achieved by the release of proteolytic enzymes, is one specific factor. Another specific factor in the nematode-insect relationship is the ability of the nematode to evade insect defences through failure to be recognized and/or by destruction of insect antibacterial factors. Toxins and extracellular enzymes are important virulence factors released by these nematodes, apparently exhibiting a specific activity against certain insect hosts.     

Immunochemical Analysis of Discoidins I and II at the Cell Surface in Wild Type and Aggregation-Defective Mutants of Dictyostelium discoideum

The endogenous lectins discoidins I and II are believed to be primary components of the morphogenetic cell cohesion system of D discoideum. We have developed two immunochemical methods to analyze the association of the discoidins with the cell surface. One method is a two-stage specific antibody binding assay in which intact cells are incubated on ice with rabbit serum (either control serum or antidiscoidin I and II), washed, then incubated with ¹²⁵I-Protein A. Specific antibody binding is defined as the difference between percent radioactivity bound with antidiscoidin versus control serum during the first stage. Substantial specific binding was observed with developed A3 cells but not with vegetative cells, and nearly all of the activity could be removed by pread-sorption of the antiserum with discoidin-Sepharose. As a complementary method, quantitative immunoadsorption analysis was performed in which we tested the ability of intact cells to remove antibodies reactive with purified ¹²⁵I-discoidin I or II. Developed cells, but not vegetative cells, were capable of adsorbing antibodies reactive with discoidin I as well as those reactive with discoidin II. This represents the first demonstration that both lectins are present on the surface of cohesive cells. These procedures, coupled with other methods to analyze soluble discoidin in cell extracts, were used to study discoidin expression in wild type cells and in two newly isolated aggregation-defective mutants. Strain EB-32 fails to aggregate and displays little or no discoidin in cell extracts or at the cell surface. On the other hand, strain EB-18 forms loose amorphous mounds, and expresses substantial quantities of the discoidins, both in cell extracts and at the cell surface. These mutants should prove valuable in studying the organization and regulation of discoidins I and II at the surface of aggregating cells.     

Extracellular Bacterial Pathogen Induces Host Cell Surface Reorganization to Resist Shear Stress

Bacterial infections targeting the bloodstream lead to a wide array of devastating diseases such as septic shock and meningitis. To study this crucial type of infection, its specific environment needs to be taken into account, in particular the mechanical forces generated by the blood flow. In a previous study using Neisseria meningitidis as a model, we observed that bacterial microcolonies forming on the endothelial cell surface in the vessel lumen are remarkably resistant to mechanical stress. The present study aims to identify the molecular basis of this resistance. N. meningitidis forms aggregates independently of host cells, yet we demonstrate here that cohesive forces involved in these bacterial aggregates are not sufficient to explain the stability of colonies on cell surfaces. Results imply that host cell attributes enhance microcolony cohesion. Microcolonies on the cell surface induce a cellular response consisting of numerous cellular protrusions similar to filopodia that come in close contact with all the bacteria in the microcolony. Consistent with a role of this cellular response, host cell lipid microdomain disruption simultaneously inhibited this response and rendered microcolonies sensitive to blood flow–generated drag forces. We then identified, by a genetic approach, the type IV pili component PilV as a triggering factor of plasma membrane reorganization, and consistently found that microcolonies formed by a pilV mutant are highly sensitive to shear stress. Our study shows that bacteria manipulate host cell functions to reorganize the host cell surface to form filopodia-like structures that enhance the cohesion of the microcolonies and therefore blood vessel colonization under the harsh conditions of the bloodstream.     

Effects of Chilling Temperature on the Activity of Enzymes of Sucrose Synthesis and the Accumulation of Saccharides in Leaves of Three Sugarcane Cultivars Differing in Cold Sensitivity

The effects of short-term exposure to chilling temperature (10 °C) on sucrose synthesis in leaves of the cold-tolerant sugarcane cultivars Saccharum sinense R. cv. Yomitanzan and Saccharum sp. cv. NiF4, and the cold-sensitive cultivar S. officinarum L. cv. Badila were studied. Plants were grown at day/night temperatures of 30/25 °C, and then shifted to a constant day/night temperature of 10 °C. After 52-h exposure to the chilling temperature, sucrose content in the leaves of NiF4 and Yomitanzan showed a 2.5- to 3.5-fold increase relative to that of the control plants that had been left on day/night temperatures of 30/25 °C. No such increase was observed in Badila leaves. Similarly, starch content in the leaves of NiF4 and Yomitanzan was maintained high, but starch was depleted in Badila leaves after the 52-h exposure. During the chilling temperature, sucrose phosphate synthase (SPS; E.C.2.4.1.14) activity was relatively stable in the leaves of NiF4 and Yomitanzan, whereas in Badila leaves SPS activity significantly decreased. There was no significant change in cytosolic fructose-1,6-bisphosphatase activity for the three cultivars at the chilling temperature. This supports the hypothesis that: (1) on exposure to chilling temperature, sucrose content in sugarcane leaves is determined by the photosynthetic rate in the leaves, and is not related to SPS activity; (2) SPS activity in sugarcane leaves at chilling temperature is to be determined by sugar concentration in the leaves.     

Surface Molecules Released by Trypanosoma cruzi Metacyclic Forms Downregulate Host Cell Invasion

BackgroundThe question whether metacylic trypomastigote (MT) forms of different T. cruzi strains differentially release surface molecules, and how they affect host cell invasion, remains to be fully clarified. We addressed that question using T. cruzi strains that differ widely in the ability to invade cells.Conclusion/SignificanceOur data suggest that the surface molecules spontaneously released by MT impair parasite-host cell interaction, gp82 presumably competing with the molecule expressed on MT surface for the host cell receptor, and gp90 further contributing to down modulate invasion.