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.     

Oxidative Enzymes of Ceratocystis fimbriata Isolates Differing in Host Specificity

Eight isolates of Ceratocystis fimbriata differing in pathogenicity on sweet potato roots were compared in vitro for peroxidase, catalase and polyphenol oxidase activities. Pathogenic isolates were generally lower in specific activities of peroxidase and catalase than the nonpathogenic isolates. Cultures of pathogenic isolates were distinguished by black culture liquid compared to the yellow color of nonpathogenic isolates. No consistent differences among the isolates were apparent when the specific activity of polyphenol oxidase preparations were compared on various phenolic substrates. The peroxidase of C. fimbriata had an approximate molecular weight of 81,000 when compared with known protein standards on a column of Sephadex G-100 dextran gel. Its substrate specificity differed from horseradish peroxidase in that it was nonreactive with guaiacol.     

Host Specificity of Pathogenic Pythium Species: Implications for Tree Species Diversity

In the Janzen–Connell hypothesis, host-specific natural enemies enhance species diversity and influence the structure of plant communities. This study tests the explicit assumption of host specificity for soil pathogens of the genus Pythium that cause damping-off disease of germinating seeds and seedlings. We isolated Pythium spp. from soil of a tropical forest in Panama. Then, in an inoculation experiment, we determined the pathogenicity of 75 tropical isolates of unknown pathogenicity and seven pathogenic temperate isolates of Pythium on seeds and/or seedlings of eight tropical tree species. Only three tropical isolates, one identified as P. ultimum and two as P. aphanidermatum , were pathogenic. Tropical pathogenic isolates were pathogenic on 4–6 of eight tree species. Temperate isolates were pathogenic on 0–4 of eight species, indicating that some tropical tree species are susceptible to novel isolates of Pythium . No tree species was susceptible to all isolates and two species were not susceptible to any isolate. Collectively, these results indicate that these Pythium isolates vary widely in their pathogenicity, causing differential mortality of potential host species; likewise, the tree species vary in their susceptibility to a given Pythium isolate. These differences in pathogenicity and susceptibility indicate some support for the Janzen–Connell assumption of host specificity. While they are not restricted to a single species, their intermediate level of specificity suggests that Pythium spp. have the potential to have some effect on forest community structure and diversity.     

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.     

寄主植物的功能性状对桑寄生寄主专一性的影响初探

为探讨寄主多样性和寄主功能性状对桑寄生植物的寄主专一性的影响,调查了西双版纳热带植物园内桑寄生和寄主植物种类。结果表明,桑寄生植物共有2科5属6种1变种,感染隶属于58科190属286种寄主植物1 323株。桑寄生在不同生境的寄生强度具有极显著差异(P0.01),单一种植园的寄生强度最高,而在森林的分布最少。不同种类桑寄生的寄主范围存在较大差异,专一性程度(H′)为1.92～7.05,多度较高的植物更容易被感染。冗余分析表明,寄主植物的胸径、树皮含水量和树皮粗糙度对不同桑寄生的寄主利用差异有显著影响,而木质密度和树皮pH的影响不显著。因此,不同种类桑寄生在热带植物群落的寄主专一性程度有较大差异,对寄主植物特定功能性状的偏好能解释部分差异。     

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.     

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。     

Host Identity Impacts Rhizosphere Fungal Communities Associated with Three Alpine Plant Species

Fungal diversity and composition are still relatively unknown in many ecosystems; however, host identity and environmental conditions are hypothesized to influence fungal community assembly. To test these hypotheses, we characterized the richness, diversity, and composition of rhizosphere fungi colonizing three alpine plant species, Taraxacum ceratophorum, Taraxacum officinale, and Polemonium viscosum. Roots were collected from open meadow and willow understory habitats at treeline on Pennsylvania Mountain, Colorado, USA. Fungal small subunit ribosomal DNA was sequenced using fungal-specific primers, sample-specific DNA tags, and 454 pyrosequencing. We classified operational taxonomic units (OTUs) as arbuscular mycorrhizal (AMF) or non-arbuscular mycorrhizal (non-AMF) fungi and then tested whether habitat or host identity influenced these fungal communities. Approximately 14% of the sequences represented AMF taxa (44 OTUs) with the majority belonging to Glomus groups A and B. Non-AMF sequences represented 186 OTUs belonging to Ascomycota (58%), Basidiomycota (26%), Zygomycota (14%), and Chytridiomycota (2%) phyla. Total AMF and non-AMF richness were similar between habitats but varied among host species. AMF richness and diversity per root sample also varied among host species and were highest in T. ceratophorum compared with T. officinale and P. viscosum. In contrast, non-AMF richness and diversity per root sample were similar among host species except in the willow understory where diversity was reduced in T. officinale. Fungal community composition was influenced by host identity but not habitat. Specifically, T. officinale hosted a different AMF community than T. ceratophorum and P. viscosum while P. viscosum hosted a different non-AMF community than T. ceratophorum and T. officinale. Our results suggest that host identity has a stronger effect on rhizosphere fungi than habitat. Furthermore, although host identity influenced both AMF and non-AMF, this effect was stronger for the mutualistic AMF community.     

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.     

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.     

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.     

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     

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.