Modulation of spore adhesion of the hyperparasitic bacterium Pasteuria penetrans to nematode cuticle

S.B. Sharma and K.G. Davies. 1997. Monoclonal antibodies (mAb) raised to the cuticule surface of second-stage juveniles (J2) of the nematode Heterodern cajani were partially characterized by immunofluorescence and Western blot analysis. Five antigens with relative molecular weights (M_r) 55, 80, 110, 180 and 210 kDa were identified with six mAb. Pasteuria spores, originating from the same population of H. cajani to which the antibodies were raised, were tested for their ability to attach to J2, which had been pretreated with each of the mAb. Monoclonal antibody HC/129 was found to reduce spore attachment by 42%, whereas HC/145 increased spore attachment by 124%. This is the first record of an antibody binding to the cuticle and increasing spore attachment, and suggests that components of the cuticle involved in inhibiting spore attachment may be masking the Pasteuria receptor present on the cuticle.     

Diversity and partial characterization of putative virulence determinants in Pasteuria penetrans, the hyperparasitic bacterium of root-knot nematodes (Meloidogyne spp.)

Antigens recognized by monoclonal antibodies (Mabs) raised to the surface of the obligate nematode hyperparasite Pasteuria penetrans were characterized. Using the attachment of spores of the bacterium to host nematodes to determine the biological variability present on the spore surface greatly underestimated the amount of surface heterogeneity present compared with estimates from immunological techniques. This heterogeneity differed not only between different individual spores from the same population but also between different spore populations. None of the Mabs completely inhibited any spore population from attaching to the nematode cuticle, suggesting that the mechanism of attachment may be more complex than previously supposed. Chemical degradation of one particular epitope recognized by monoclonal antibody PP1/117, and designated ep117, occurred after treatment with NaOH, periodate or Proteinase K, suggesting that an O-linked glycoprotein may be involved. Fibronectin, which had been found to bind to Pasteuria spores through hydrophobic interactions, also prohibited the Mab from recognizing ep117. However, SDS-PAGE of spore extracts followed by immunoblotting showed that none of the Mabs could detect this epitope and so ep117 may be conformational in nature. Thus, the conformation of any particular epitope recognized by a Mab may be important in determining to which nematode a particular spore will attach. The distribution of a particular epitope within a population of spores will in turn therefore determine its virulence on a particular nematode.     

Comparison of two sympatric Pasteuria populations isolated from a tropical vertisol soil

An isolate of Pasteuria (designated PPMJ) recovered from the root-knot nematode Meloidogyne javanica, was characterized using host preference, spore morphometrics, and serology, and compared with another sympatric Pasteuria isolate (designated PPHC) collected from the cyst nematode, Heterodera cajani. PPMJ spores were larger (x 1.5) than the PPHC spores and had a mean diameter of 3.4 m after fixation for electron microscopy. The central body of PPMJ spores was about twice as big as the central body of PPHC spores. The host preference tests, based on spore attachment to the nematode cuticle, revealed that Meloidogyne incognita, M. javanica, M. hapla, Pratylenchus coffeae, and Pratylenchus sp. were hosts of PPMJ but not of PPHC. It was found that males of Radopholus similis were hosts of PPHC. Western blot analysis of spore extracts probed with a polyclonal antiserum raised against PPHC spores showed an antigenic ladder which had similarities to lipopolysaccharide; another antiserum revealed differences in the molecular weight of antigens of the different spore isolates. Population diversity can therefore be vastly altered by the maintenance and culture of the bacterium on a particular host. The implications of these results are discussed in relation to the use of Pasteuria as a biological control agent.K.G. Davies is with IACR-Rothamsted, Harpenden, Hertfordshire, AL5 2JQ, UK S.B. Sharma is with the International Crops Research Institute for the Semi-Aird Tropics (ICRISAT) Asia Center. Patancheru 502324, India.     

Fitting the negative binomial distribution to Pasteuria penetrans spore attachment on root-knot nematodes and predicting probability of spore attachments using a Markov chain model

Pasteuria penetrans controls root-knot nematodes (Meloidogyne spp.) either by preventing invasion or by causing female sterility. The greatest control of P. penetrans occurred when an appropriate quantity of P. penetrans spores attached to the cuticle of a juvenile nematode. The number of spores attaching to juveniles within a given time increased with increasing the time of exposure to spores. Based on this, numbers of encumbered nematodes were recorded 1, 3, 6 and 9 h after placing nematodes in standard P. penetrans spore suspensions. From the count data obtained, P. penetrans attachment was modelled using the Poisson and negative binomial distributions. Attachment count data were observed to be overdispersed with respect to high numbers of spores attaching on each J2 at 6 and 9 h after spore application. It was concluded that the negative binomial distribution was shown to be the most appropriate model to fit the observed data-sets considering that P. penetrans spores are clumped; this could be further refined with a Markov process.     

Understanding the movement of root-knot nematodes encumbered with or without Pasteuria penetrans

Pasteuria penetrans is a naturally occurring bacterial parasite of plant parasitic nematodes showing satisfactory results in a biocontrol strategy of root-knot nematodes (Meloidogyne spp.). The endospores attach to the outside nematode body wall (cuticle) of the infective stage second-stage juveniles (J2) of Meloidogyne populations. Optimal attachment level should be around 5–10 endospores per juvenile, as enough endospores will initiate infection without reducing the ability of the nematode to invade roots. Greater than 15 endospores may disable the nematode in its movements, and invasion may not take place. In this research, evidence is provided that P. penetrans spores disturbed the nematode forward movement by disorganising the nematode's head turns. The results based on Markov chain and Cochran probability model show that even a low number of 5–8 spores of P. penetrans attached to the nematode cuticle have a significant impact on that movement, which plays a role in nematode locomotion.     

The electrostatic nature of the spore of Pasteuria penetrans, the bacterial parasite of root-knot nematodes

The response of spores of Pasteuria penetrans , the Gram-positive obligate nematode hyperparasite, was studied in a direct current electric field and monitored using a microscope attached to a video recorder apparatus. Fluorescence measurements were performed on the spore's surface using HEXCO as a fluorescent probe. The mobilities of the spores and fluorescence measurements were performed in different salt concentrations and at different pH values. The results showed a significant electronegative potential at the spore surface which was dependent on the pH, salt concentration and valency of the cation present in the electrolyte medium. The results of the fluorescence experiments using HEXCO correlated well with the results obtained from the electrophoretic mobility experiments. A polyclonal antiserum raised to spores of P. penetrans affected the surface charge density and the data presented suggest that electrostatic interactions may be important in the binding of spores to the nematode cuticle. The binding of HEXCO to hydrophobic sites on the spores' surfaces suggests the possibility of other attractive forces also being important in the binding process.     

Wheat Germ Agglutinin Bound to the Outer Cuticle of the Seed Gall Nematodes Anguina agrostis and A. tritici

The presence of wheat germ agglutinin (WGA) on the cuticular surface of the seed gall nematodes Anguina agrostis and Anguina tritici was demonstrated, and the nature of its binding was examined. Crude extracts from the cuticles of A. tritici agglutinated human red blood cells, and only N-acetylglucosamine (GlucNAc) inhibited the agglutination. Distribution of the lectin was visualized by treating live infective juveniles (J2) with rabbit anti-WGA antibody and staining with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG. The lectin bound to the outer cuticular surface of the whole body wall. Pretreatment with GlucNAc oligomers did not reduce the fluorescence created by the anti-WGA-WGA binding, indicating at least a partial nonspeciflc adhesion of the WGA to the nematode surface. Proteolytic enzyme pretreatments diminished the fluorescence, whereas lipase and periodate pretreatments increased the fluorescence. Adult females and males were labeled only on the head and tail, whereas eggs were not labeled at all. It was concluded that the WGA on the J2 cuticle originates from the host.     

The axial distribution of plasma membrane molecules in pseudoplasmodia of the cellular slime mold Dictyostelium discoideum

Five plasma membrane molecules were found to be non-uniformly distributed along the axis of the pseudoplasmodium of the cellular slime mold Dictyostelium discoideum and were biochemically characterized. All five of the molecules were distinguished by their ability to bind the lectin wheat germ agglutinin (WGA), and three were recognized by an antibody raised against pseudoplasmodial plasma membranes and exhaustively adsorbed against vegetative cells. The distribution of these molecules corresponded with the pattern of precursors of the terminally differentiated stalk cells and spores before these cells became irreversibly committed to their respective fates. At least four of the molecules appeared in hemispherical aggregates prior to the detection of prestalk or prespore cells, but were not present in undifferentiated vegetative cells.     

Protein glycosylation in the spores of the microsporidia Paranosema (Antonospora) grylli

Long adaptation of microsporidia, a large group of fungi-related protozoa, to intracellular lifestyle has resulted in drastic minimization of a parasite cell. Thus, diversity of carbohydrates in microsporidia glycoproteins and proteoglycans is expected to be restricted by O-linked manno-oligosaccharides because three genes involved in O-mannosylation of proteins and no components of N-linked glycosylation machinery were found in genome of human pathogen Encephalitozoon cuniculi. In this study we investigated glycosylation of spore proteins of microsporidia Paranosema (Antonospora) grylli infecting crickets Gryllus bimaculatus. Using periodic acid-Shiff reagent staining we have demonstrated that some P. grylli spore proteins are highly-glycosylated. The major polar tube protein (PTP1) of 56 kDa was shown as the most intensively decorated band. The experiments with N-glycosidase F and WGA lectin did not reveal any N-glycosylated proteins in P. grylli spores. At the same time, incubation of major spore wall protein of 40 kDa (p40) with mannose specific lectin GNA resulted in specific binding that was reduced by pretreatment of the protein with mannosidases. Interestingly, in spite of PTP1 glycosylation, polar tube proteins extracted from P. grylli spores were not precipitated by GNA-agarose. Since P. grylli and E. cuniculi are distantly related, our data suggest that dramatic reduction of protein glycosylation machinery is a common feature of microsporidia.     

A Rainbow Trout Lectin with Multimeric Structure

A novel lectin has been identified in rainbow trout serum and plasma. The lectin binds to Sepharose (an agarose polymer) in a calcium-dependent manner. Glucose, N-acetyl-glucosamine, mannose, N-acetyl-mannosamine, l-fucose, maltose and α-methyl-mannoside are good inhibitors of this binding, whereas glucosamine and d-fucose inhibits to a lesser degree and mannosamine and galactose do not inhibit the binding to Sepharose. When analysed by SDS-PAGE under non-reducing conditions, the lectin appears as a characteristic ladder of bands with approximately 16 kDa between consecutive bands. Upon reduction, the lectin appears as a 16-kDa band. On size-exclusion chromatography of trout serum and plasma, the protein emerges over a broad range corresponding to sizes from about 2000 kDa to less than 200 kDa. The NH₂-terminal sequence (AAENRNQXPPG) shows no significant homology with known proteins. Because of the characteristic appearance in non-reducing SDS-PAGE and the lectin activity, we propose to name the protein “ladderlectin.”     

Analysis of sugar chain-binding specificity of tomato lectin using lectin blot: recognition of high mannose-type N-glycans produced by plants and yeast

The sugar chain-binding specificity of tomato lectin (LEA) against glycoproteins was investigated qualitatively using lectin blot analysis. Glycoproteins containing tri- and tetra-antennary complex-type N-glycans were stained with LEA. Unexpectedly, glycoproteins containing high mannose-type N-glycans and a horseradish peroxidase were stained with LEA. LEA blot analysis of the glycoproteins accompanied by treatment with exoglycosidase revealed that the binding site of LEA for the complex-type N-glycans was the N-acetyllactosaminyl side chains, whereas the proximal chitobiose core appeared to be the binding site of LEA for high mannose-type N-glycans. Despite these results, the glycoproteins did not inhibit the hemagglutinating activity of LEA. Among the chitin-binding lectins compared, potato tuber lectin showed specificity similar to LEA on lectin blot analysis, while Datura stramonium lectin and wheat germ agglutinin (WGA) did not interact with glycoproteins containing high mannose-type N-glycans, except that RNase B was stained by WGA. Based on these observations, LEA blot analysis was applied to sugar chain analysis of tomato glycoproteins. The most abundant LEA-reactive glycoprotein was purified from the exocarp of ripe tomato fruits, and was identified as the tomato anionic peroxidase1 (TAP1). These results suggest that LEA interacts with glycoproteins produced by tomatoes, which participate in biological activities in tomato plants.     

Development,Distribution, and Host Studies of the Fungus Macrobiotophthoira vermicola (Entomophthorales)

The life cycle and host range of Macrobiotophthora vermicola were studied. Secondary spores produced from forcibly ejected primary spores adhered to the cuticle of Cruznema tripartitum, germinated, and penetrated the cuticle within 30 minutes. New primary spores were produced within 24 hours of initial spore adhesion. In a host range study, species of Rhabditidae, Diplogasteridae, and Aphelenchoidea were hosts, but not species of Bunonematidae, Tripylidae, Cephalobida, or Tylenchina. Numbers of second-stage Meloidogyne incognita juveniles were not decreased when added to soil seeded with infected C. tripartitum. In six Tennessee soybean fields, Macrobiotophthora vermicola was the most commonly encountered nematode-destroying fungus, followed by a sterile, nonseptate fungus and Arthrobotrys conoides. Nematophagous fungi were isolated more frequently from silt loam soils than from clay soils. Addition of C. tripartitum to soil extract plates as a bait nematode did not increase isolations of nematophagous fungi.     

Characterization of zoospore and cyst surface structure in saprophytic and fish pathogenicSaprolegnia species (oomycete fungal protists)

Summary The importance of the surface structure and chemistry in zoospores and cysts of oomycetes is briefly reviewed and the organelle systems associated with encystment described. The surface structure and chemistry of primary and secondary zoospores and cysts ofSaprolegnia diclina (a representative saprophytic species) andS. parasitica (a representative salmonid fish pathogen) were explored using the lectins concanavilin A (Con A) and wheat germ agglutinin (WGA) and monoclonal antibodies (MAbs) raised against a mixed zoospore and cyst suspension ofS. parasitica. The binding of lectins and antibodies to spores was determined using immunofluorescence microscopy with fluorescein isothiocyanate-labelled probes and with electron microscopy with gold-conjugated probes applied to spore suspensions post-fixation. In both species Con A, which is specific for glucose and mannose sugars, bound to both the surface of primary and secondary zoospores (the surface glycocalyx) and their cyst coats and readily induced zoospore encystment. The binding to the cysts appeared to be mainly associated with the matrix material released from the primary and secondary encystment vesicles and which appeared to diminish with time. No binding to germ tube walls was observed with this lectin. The MAb labelling showed a generally similar binding pattern to the primary and secondary cysts to that observed with Con A, although the binding to zoospores was more variable. Primary zoospores bound the antibodies but secondary zoospores appeared less reactive. It is suggested that the MAbs share a common epitope with one or more of the Con A-binding components. In both species WGA, which is specific for amongst other things the sugar N-acetyl glucosamine, bound to localised apical patches on the primary zoospores. This lectin also binds to the ventral groove region of secondary zoospores ofS. diclina, which were induced to encyst by this lectin. In contrast secondary zoospores ofS. parasitica were not induced to encyst by the addition of WGA and showed a patchy dorsal binding with this lectin. WGA also binds to both the inner wall of discharged primary cysts and the young germ tube walls of both species. These observations are discussed both in relation to other oomycete spores and to their possible functional and ecological significance.Abbreviations BSA bovine serum albumin - Con A Concanavalin A - DBA Dolichos biflorus agglutinin - ELISA enzyme-linked immunosorbent assay - EM electron microscope - EV encystment vesicles - FCS foetal calf serum - FITC Fluorescein isothiocyanate - FV peripheral fibrillar vesicles - G+F 0.2% glutaraldehyde and 2.0% formaldehyde primary fixative solution - 2G 2% glutaraldehyde primary fixative - LM light microscopy - MAbs monoclonal antibodies - LPV large peripheral vesicles - PBS phosphate buffered saline - PCV flattened peripheral cisternae - PEV primary encystment vesicle - PIPES piperazine-N,N1-bis(2-ethane sulfonic acid) - PNA Ricinus communis agglutinin - RAM-FITC/Au_10–20 Fluorescein isothiocyanate/gold (10 or 20 nm) labelled rabbit anti-mouse immunoglobulin - RCA Ricinus communis agglutinin - SEM scanning electron micrograph - SBA soybean agglutinin - SEV secondary encystment vesicles - TEM transmission electron micrograph - UEA I Ulex europaeus agglutinin - WGA wheat germ agglutinin     

Peculiarities of glycosylation of proteins in spores of microsporidia Paranosema (Antonospora) grylli

The long-term adaptation of microsporidia, a large group of fungi-related unicellular microorganisms, to intracellular parasitism has led to extreme minimization of the cell functional apparatus. For instance, diversity of carbohydrates in the composition of parasite glycoproteins and proteoglycans seems to be restricted to the presence of O-bound chains composed of mannose residues. This suggestion is based on the discovery in the genome of the human microsporidian Encephalitozoon cuniculi of three genes responsible for the O-mannosylation of proteins with a lack of enzymes participating in N-glycosylation. In the present work, peculiarities of protein glycosylation in spores of the microsporidian Paranosema grylli infecting the fat body of the Mediterranean field cricket Gryllus bimaculatus was studied. SDS-PAGE analysis of spore proteins with subsequent staining by periodate and Schiff reagent has shown that individual glycoproteins of P. grylli are highly glycosylated, while the maximal stain intensity was seen in the major polar-tube protein PTP1. Treatment of the extracted material with N-glycosidase F and hybridization with WGA lectin conjugated with horseradish peroxidase showed no presence of glycosylated proteins in the P. grylli spores. At the same time, the selectively extracted major protein of the exospore p40 was specifically recognized by lectin GNA conjugated with agarose balls. Pretreatment of p40 with α-and β-mannosidases decreased considerably the efficiency of binding. Since lectin GNA is specific towards mannose terminal residues, this indicates the O-mannosylation of the microsporidial exospore major protein. In spite of the intensive PTP1 glycosylation, extracted proteins of the P. grylli polar-tube had no specific binding with GNA-agarose, so the issue of peculiarities of their glycosylation remains an open question. Comparison of the obtained data with results of deciphering of the E. cuniuculi genome allows for the conclusion to be made that the minimization of the glycosylation apparatus of microsporidial proteins is the common peculiarity of this group of parasites.     

Detection of hemicelluloses specific to the cell wall of tracheary elements and phloem cells by fluorescein-conjugated lectins

Summary Binding of fluorescein-conjugated wheat-germ agglutinin (F-WGA) and some other lectins to tissues from various plants were examined by epifluorescence microscopy. F-WGA bound specifically to the walls of tracheary elements (TEs) and phloem cells of pea roots. The binding sites in TEs were localized only in the secondary thickening and became evident at very early stages of differentiation. Fluorescein-conjugated derivatives ofSolanum tuberosum lectin,Lycopersicon esculentum lectin, andDatura stramonium lectin, which bind N-acetylglucosamine residues as WGA, also bound to the secondary thickening of TEs of pea roots. The binding sites for F-WGA were not removed by extraction with hot EDTA and proteinase K, but removed by extraction with an alkali solution. The alkali-extracted binding sites from the roots were precipitated together with hemicelluloses by 80% ethanol. These results indicate that the binding sites are not present on pectins, proteins, or cellulose, but hemicelluloses. Localized distribution of the binding sites for F-WGA in TEs was found also in a variety of angiosperm plants.Abbreviations BSL-II Bandeiraea simplicifolia lectin II - DSL Datura stramonium lectin - F fluorescein-conjugated - LEL Lycopersicon esculentum lectin - MT microtubule - STL Solanum tuberosum lectin - TE tracheary element - WGA wheat-germ agglutinin     

Possible involvement of Ca<Superscript>2+</Superscript>-dependent protein kinases in spore germination of the fern<Emphasis Type="Italic">Osmunda Japonica</Emphasis>

Fern gametophyte is a good model system to investigate signal transduction in plant cells. In this work, we examined whether CDPKs are involved in the mechanisms of spore germination of the fernOsmunda japonica. A protein extract from the spores included four CDPK isoforms with relative molecular weights of 56, 53, 49, and 47 kDa, as detected by immunoblot analysis, and they showed CDPK-like activities, as detected by in-gel protein-kinase assay. It was also found that the inhibitors effective on CDPKs, such as a general protein kinase inhibitor, K252a, and a calmodulin antagonist, W-7, largely suppressed the spore germination, and that many proteins of the spores were phosphorylated in vivo in a calcium dependent manner in the period when the spores require external Ca²⁺ for the germination. Furthermore, we showed that Sr²⁺ and Mn²⁺, which could substitute for Ca²⁺ in the spore germination, were also able to activate theOsmunda CDPKs. From these results, we concluded that CDPKs would participate in the spore germination ofO. japonica.     

Bacterial parasite of a plant nematode: morphology and ultrastructure.

The life cycle of a bacterial endoparasite of the plant-parasitic nematode Meloidogyne incognita was examined by scanning and transmission electron microscopy. The infective stage begins with the attachment of an endospore to the surface of the nematode. A germ tube then penetrates the cuticle, and mycelil colonies form in the pseudocoelom. Sporulation is initiated when terminal cells of the mycelium enlarge to form sporangia. A septum within each sporangium divides the forespore from the basal or parasporal portion of the cell. The forespore becomes enclosed by several laminar coats. The parasporal cell remains attached to the forespore and forms the parasporal microfibers. After the newly formed spores are released into the soil, these microfibers apparently enable a mature spore to attach to the nematode. These results indicate that the endoparasite is a procaryotic organism having structural features that are more common to members of Actinomycetales and to the bacterium Pasteuria ramosa than to the sporozoans or to the family Bacillaceae, as previous investigatios have concluded.     

Vitellin of the sweet potato whitefly,Bemisia tabaci: Biochemical characterization and titer changes in the adult

SDS-PAGE of the sweet potato whitefly (Bemisia tabaci) egg extract showed one major band (approximately 190 kDa) and two minor bands (approximately 75 kDa and 67 kDa). A distinct 190 kDa band was also present in male extract. On SDS gels the vitellin band of the greenhouse whitefly (Trialeurodes vaporarium) was larger, about 220 kDa. The native molecular mass of sweet potato whitefly vitellin was estimated to be 375 kDa using 4–20% native pore-limiting gel electrophoresis. Its isoelectric point was estimated to be 7.3 using isoelectric focusing. Two-dimensional gel electrophoresis and densitometry were used to estimate vitellin subunit composition; the data suggest that the sweet potato whitefly vitellin is likely to be a 380 kDa native molecule formed by two 190 kDa subunits. The two minor bands (75 kDa and 67 kDa) may be breakdown products of the native vitellin. This conclusion was supported by a Western blot of an SDS-PAGE gel of partially degraded female and egg extracts, which showed that polyclonal antiserum raised against the 190 kDa polypeptide recognized the 75 kDa and 67 kDa bands. Seven hybridoma cell lines secreting monoclonal antibodies against the 190 kDa band were screened, and one of them (S1A2G9H2) was mass produced. The antibody recognized the 190 kDa band in a Western blot. All the screened monoclonal antibodies were female and egg-specific by ELISA and/or Western blot, suggesting that the 190 kDa band in male extract was not a vitellin. A sensitive ELISA was established that could detect as little as 1/40 of an egg equivalent of vitellin using the monoclonal antibody from S1A2G9H2. Profiles of female sweet potato whitefly reproductive activities (egg laying, amount of vitellin in the female, and total vitellin produced by a female) within 2 days after eclosion were determined. Arch. Insect Biochem. Physiol. 34:223–237, 1997. © 1997 Wiley-Liss, Inc.     

Effect of Hypoxia on Endothelial Cell Surface Glycoprotein Expression: Modulation of Glycoprotein IIIa and Other Specific Surface Glycoproteins

Exposure to hypoxia alters many aspects of endothelial cell metabolism and function; however, changes in surface glycoconjugates under these conditions have not been extensively evaluated. In the current studies, we examined surface glycoproteins of cultured bovine aortic (BAEC) and pulmonary arterial (BPAEC) endothelial cells under standard culture conditions (21% oxygen) and following exposure to hypoxia (0% oxygen) for varying time periods (30 min to 18 h) using a system of biotinylation, lectin binding (concanavalin A, Con A; Griffonia simplicifolia , GSA; Arachis hypogaea, PNA; Ricinus communis, RCA; or Triticum vulgaris, WGA), subsequent strep-avidin binding, and staining. Using these methods, we identified differences in lectin binding between the two cell types cultured in 21% oxygen with all lectins except PNA. With exposure to 0% oxygen, there was no change in lectin binding to most surface glycoproteins. Several surface glycoproteins, including glycoprotein IIIa on both cell types, demonstrated a time-dependent decrease in lectin binding; in addition, there was an increase in lectin binding to a few specific surface glycoproteins on each cell type within 30-60 min of exposure to 0% oxygen. These changes in specific surface glycoproteins were confirmed in both cell types by ¹²⁵I labeling. Increased lectin binding was observed for Con A binding BAEC glycoproteins at molecular weight (MW) 116, 130, and 205 kDa, GSA binding BAEC glycoproteins at MW 120 and 205 kDa, and RCA binding BPAEC glycoproteins at MW 140 and 205 kDa. Increased binding of WGA or PNA was not observed during exposure to hypoxia. The specificity of lectin binding was further confirmed by competitive inhibition with the appropriate sugar. These studies demonstrate that there are baseline differences between BAEC and BPAEC cell surface glycoproteins and that exposure to hypoxia is associated with little change in lectin binding to most surface glycoproteins. There is, however, increased surface expression of a few glycoproteins that differ depending of the origin of the endothelial cell. Although the mechanism of this increase in lectin binding is not yet clear, subsequent studies suggested that it is due to increased availability of select carbohydrate moieties. The time course of these alterations suggests a possible role in the endothelial cell response to decreases in ambient oxygen tension.     

Characterization of cell surface carbohydrates on asexual spores of the water moldSaprolegnia ferax

Summary In asexual reproduction of the water mold,Saprolegnia ferax, four distinct and sequentially produced spores are involved in dispersal, two of which are motile and two of which are nonmotile. Composition of cell surface glycoproteins may be important in dispersal strategies for each of these stages. Binding patterns of fluorescently labelled lectins were investigated to identify differences in glycoproteins of asexually produced dispersal stages. The pattern of lectin binding to zoospores was diverse. FITC-Con A bound to surfaces of zoospores and membranes of the water expulsion vacuole system, indicating the prescence of mannosyl and glucosyl residues. In zoospores incubated for more than 30 min in FITC-WGA and FITC-GS II. which bind N-acetyl glucosamine, fluorescence was sometimes localized in peripheral, intracellular patches. In shorter incubations, secondary zoospores bound these lectins along the groove region where K-bodies were located. Surfaces of cystospores typically bound FITC-WGA, but not FITC-GS II. FITC-GS II, however, bound to empty cystospore walls, probably because reactive sugars were available at the inner surface of the wall. Germ tubes emerging from cystospores bound labelled WGA and GS II, but not Con A. The same lectin binding pattern was found along discharge papilla of primary cystospores, indicating that modifications in cystospore walls associated with direct germination and zoospore discharge were similar. Thus, glycoproteins involved in early establishment of the hyphal system differ from those forming the cell surface of cystospores. Differences in the binding pattern of lectins to zoospores and cystospores highlight differences between cell surface carbohydrates of motile and nonmotile asexual stages.Abbreviations BPA lectin fromBauhinia purpurea - C₁ primary cystospore - C₂ secondary cystospore - Con A concanavalin A, lectin fromCanavalia ensiformis - DBA lectin fromDolichos biflorus - DIC Nomarski differential interference contrast optics - DS dilute salts - FITC fluorescein isothiocyanate - FUC fucose - Gal galactose - GalNAc N-acetyl galactosamine - Glc glucose - GlcNAc N-acetyl glucosamine - GS I Griffonia simplicifolia lectin I - GS II G. simplicifolia lectin II - Man mannose - MPA lectin fromMaclura pomifera - PC phase contrast optics - PNA lectin fromArachis hypogaea - SBA soybean agglutinin, lectin fromGlycine max - UEA-1 lectin fromUlex europaeus - WGA wheat germ agglutinin fromTriticum vulgare - WV water expulsion vacuole