几种植物生殖细胞质膜表面的凝集素受体荧光标记

为进一步探讨从生殖细胞到精子的发育过程中细胞质膜表面凝集素受体的可能变化,及其与两类对凝集素标记有不同结果的精子的关系,用异硫氰酸荧光素标记的伴刀豆凝集素(Con A)、麦芽凝集素(WGA)和大豆凝集素(SBA)对蚕豆(Vicia faba L．)、鸢尾(Iris tectorium Maxim．)和朱顶红(Hippeastrum vittatum Herb．)的生殖细胞质膜表面的凝集素受体进行标记。结果显示：在不同植物中均有部分生殖细胞不能被凝集素探针标记,且在保持尾状形态的生殖细胞的表面发现有凝集素受体的极性分布。这可能是导致部分精子表面不能被同种凝集素标记的重要原因。此外,同一种凝集素受体在不同物种的生殖细胞上分布不一致,不同的凝集素受体在同一种植物的生殖细胞上的分布模式亦有不同。在蚕豆和鸢尾的生殖细胞表面均有这三种凝集素的受体。在朱顶红生殖细胞的表面有前两种凝集素的受体,分布比较均一,但是没有大豆凝集素的受体。此外,在具尾生殖细胞表面发现有凝集素受体极性分布的现象,为探讨精细胞功能及其表面糖蛋白分布的可能差异提供了重要启示。     

几种植物生殖细胞质膜表面的凝集素受体荧光标记

为进一步探讨从生殖细胞到精子的发育过程中细胞质膜表面凝集素受体的可能变化,及其与两类对凝集素标记有不同结果的精子的关系,用异硫氰酸荧光素标记的伴刀豆凝集素(Con A)、麦芽凝集素(WGA)和大豆凝集素(SBA)对蚕豆(Vicia faba L.)、鸢尾(Iris tectorium Maxim.)和朱顶红(Hippeastrum vittatum Herb.)的生殖细胞质膜表面的凝集素受体进行标记.结果显示:在不同植物中均有部分生殖细胞不能被凝集素探针标记,且在保持尾状形态的生殖细胞的表面发现有凝集素受体的极性分布.这可能是导致部分精子表面不能被同种凝集素标记的重要原因.此外,同一种凝集素受体在不同物种的生殖细胞上分布不一致,不同的凝集素受体在同一种植物的生殖细胞上的分布模式亦有不同.在蚕豆和鸢尾的生殖细胞表面均有这三种凝集素的受体.在朱顶红生殖细胞的表面有前两种凝集素的受体,分布比较均一,但是没有大豆凝集素的受体.此外,在具尾生殖细胞表面发现有凝集素受体极性分布的现象,为探讨精细胞功能及其表面糖蛋白分布的可能差异提供了重要启示.     

Sequestration and Tissue Accumulation of Human Malaria Parasites: Can We Learn Anything from Rodent Models of Malaria?

The sequestration of Plasmodium falciparum-infected red blood cells (irbcs) in the microvasculature of organs is associated with severe disease; correspondingly, the molecular basis of irbc adherence is an active area of study. In contrast to P. falciparum, much less is known about sequestration in other Plasmodium parasites, including those species that are used as models to study severe malaria. Here, we review the cytoadherence properties of irbcs of the rodent parasite Plasmodium berghei ANKA, where schizonts demonstrate a clear sequestration phenotype. Real-time in vivo imaging of transgenic P. berghei parasites in rodents has revealed a CD36-dependent sequestration in lungs and adipose tissue. In the absence of direct orthologs of the P. falciparum proteins that mediate binding to human CD36, the P. berghei proteins and/or mechanisms of rodent CD36 binding are as yet unknown. In addition to CD36-dependent schizont sequestration, irbcs accumulate during severe disease in different tissues, including the brain. The role of sequestration is discussed in the context of disease as are the general (dis)similarities of P. berghei and P. falciparum sequestration.     

Fasciola hepatica: tegumental alterations as a consequence of lectin binding

Adult flukes, Fasciola hepatica, incubated in Hedon - Fleig saline containing concanavalin A (Con A) for 10 and 45 min, respectively, exhibited severe alterations to tegumental morphology involving increased secretory activity, blebbing of the apical plasma membrane, increased total surface area, and swelling of the basal infolds . The effects of Con A were prevented by the addition of alpha-methyl-D-mannoside to the incubating medium. Similar, but less pronounced, effects were caused by wheat germ agglutinin (WGA) binding. Con A and WGA binding indicate the presence of mannose, glucosamine, or glucose moieties and of N-acetylglucosamine. The effects of lectin binding were similar to the early effects of antibody attachment, and it was considered that accelerated membrane turnover was occurring in both cases. Swelling of the basal infolds was thought to be a result of increased apical surface membrane and/or increased permeability due to lectin binding.     

Lectin-Induced Inhibition of Nerve Growth Factor Binding by Receptors of Sympathetic Ganglia

Abstract: Nerve growth factor (NGF) initiates a pleiotypic response in numerous tissues derived from the neural crest by binding to specific plasma membrane receptors. In sympathetic ganglia this receptor has been characterized as a highly asymmetric, minimally hydrophobic, intrinsic membrane protein with a molecular weight of 135,000 (Costrini et al., 1979b). To further characterize this moiety we assessed the effects of lectins on ¹²⁵I-NGF specific binding to preparations of particulate and nonionic detergent-extracted micro-somal receptors of rabbit superior cervical ganglia (SCG). Concanavalin A (Con A) and wheat germ agglutinin (WGA), but not soybean agglutinin or Ulex europaeus I, induced a concentration-related, carbohydrate-specific decrease in ¹²⁵T-NGF binding. Following Con A exposure, ¹²⁵I-NGF specific binding to particulate SCG receptors was maximally reduced to 23% of control values. WGA similarly reduced NGF binding to particulate microsomal receptors to 37% of control values. Scatchard analysis of growth factor binding following Con A exposure indicated that this lectin effect was principally due to a sixfold reduction in maximum receptor affinity. Lectin-associated impairment of NGF binding was also demonstrated by using a Triton X-100 solubilized receptor preparation. These results provide evidence that the high-affinity-state NGF receptor of SCG is a glycoprotein containing N -acetylglucosamine and α-D-mannopyranoside residues. These residues are probably located in close proximity to the growth factor binding region of the NGF receptor.     

Migrating endothelial cells are distinctly hyperglycosylated and express specific migration-associated cell surface glycoproteins

Migration of endothelial cells is one of the first cellular responses in the cascade of events that leads to re-endothelialization of an injured vessel and neovascularization of growing tissues and tumors. To examine the hypothesis that endothelial cells express a specific migration-associated phenotype, we analyzed the cell surface glycoprotein expression of migrating bovine aortic endothelial cell (BAECs). Light microscopic analysis revealed an upregulation of binding sites for the lectins Concanavalin A (Con A), wheat germ agglutinin (WGA), and peanut agglutinin after neuraminidase treatment (N-PNA) on migrating endothelial cells relative to contact-inhibited cells. These findings were confirmed and quantitated with an enzyme-linked lectin assay (ELLA) of circularly scraped BAEC monolayers. The expression of migration-associated cell surface glycoproteins was also analyzed by SDS-PAGE. The overall expression of cell surface glycoproteins was upregulated on migrating BAECs. Migrating BAECs expressed Con A- and WGA-binding glycoproteins with apparent molecular masses of 25 and 48 kD that were not expressed by contact-inhibited BAEC monolayers and, accordingly, disappeared as circularly scraped monolayers reached confluence. Subconfluent BAEC monolayers expressed the same cell surface glycoconjugate pattern as migrating endothelial cells. FACS analysis of circularly scraped BAEC monolayers showed that the phenotypic changes of cell surface glycoprotein expression after release from growth arrest occurred before the recruitment of the cells into the cell cycle (3 vs. 12 h). Suramin, which inhibits endothelial cell migration, abrogated the expression of the migration-associated phenotype and induced the expression of a prominent 28-kD Con A- and WGA-binding cell surface glycoprotein. These results indicate that endothelial cells express a specific migration-associated phenotype, which is characterized by the upregulation of distinct cellular glycoconjugates and the expression of specific migration-associated cell surface glycoproteins.     

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.     

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.     

Postnatal development of lectin binding sites in the rat ventral prostate

Summary Five Fluorescein-isothiocyanate (FITC)-labelled lectins were used to study the postnatal development of carbohydrate constituents in the rat ventral prostate: Concanavalin A (Con A), wheat germ agglutinin (WGA), peanut agglutinin (PNA),Dolichos biflorus agglutinin (DBA) andRicinus communis agglutinin I (RCA-I) With all the lectins, tested, except RCA-I, specific binding sites could be shown for every stage of differentiation in the glandular epithelium. Binding sites for Con A, WGA, PNA and DBA were found from day 10 to 13 post partum onwards. Each lectin showed a characteristic localization. Binding sites for the lectins used changed to different extents during the following two weeks. After the 24th day post partum no further changes in the lectin binding pattern could be found. The development of the lectin binding properties showed that the changes in carbohydrate-containing constituents of the prostate correlate with the beginning of prostatic secretion and to prostatic epithelial differentiation. In the periacinar stroma the development of the lectin binding pattern was similar to that in the glandular epithelium. The changes of stromal binding sites for Con A and WGA during epithelial differentiation may reflect the changes of epithelial-stromal interactions in the prostate.     

Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. I. Histochemical distribution of lectin binding sites in normal alimentary tract as well as in benign and malignant gastric neoplasms

Labeled lectins with binding specificity to the hexose components of mucus glycoproteins (HPA, RCA I, PNA, Con A, WGA, and UEA I) were used to demonstrate structural differences in the glycoprotein composition of various cell types of the normal, benign and malignant gastrointestinal mucosa. While in the RCA I, UEA I, and WGA binding of normal mucus secreting cell types only quantitative differences were observed, the mucus in the surface epithelial cells of gastric mucosa and in the colonic goblet cells was characterized by the absence of PNA, Con A, and PNA, HPA binding sites, respectively. These lectins, however, showed a strong binding to the supranuclear, Golgi-region in the undifferentiated or activated forms of these cells. Even the staining intensity of the luminal membrane surfaces of the non mucinous parietal and chief cells was often stronger by PNA, HPA, and RCA I lectins than that of the mucus secretions in the highly differentiated mucus cells. These results indicate the existence of either heterogeneous glycoprotein components or mucus molecules with variations in the degree of glycosylation of their oligosaccharide chains in the different cells. The latter seems more likely since in benign and malignant alterations lectin binding sites appear in great density, which were found to be characteristic of the undifferentiated mucus cells or for the non mucinous cells of the normal gastric mucosa. Similarly in some gastric cancers which do not stain with the periodic acid-Schiff reaction at all, large amount of free or neuraminic acid substituted PNA binding sites can be detected.     

Fractionation of lymphocytes using affinity chromatography with 9 lectins

Lymphocyte subclasses from normal peripheral blood have been fractionated by affinity chromatography with lectins. Concanavalin A (Con A), Lens culinaris lectin (LC), Pisum sativum lectin (PS), Phaseolus vulgaris lectin (PHA), Dolichos biflours lectin (DB), Glicine max lectin (SBA), Ricinus communis lectin (RCA II), Tetragonolobus purpureus lectin (TP) and Triticum vulgaris lectin (WGA), were coupled to Sepharose 6MB, and lymphocytes labelled with 125I were eluted through the chromatographic columns. The binding of lymphocytes to WGA and SBA lectins was 32% and 13% respectively. The binding to the other lectins tested were found to be between 32% and 13%. When solutions of increasing concentrations of specific sugar were added to the columns a progressive elution of bound lymphocytes was observed. These results indicate the existence of a large range of lymphocyte subclasses, with different binding capacity to lectins, which was a function of the receptor number or/and receptor affinity to each lectin. Furthermore, these two parameters were found to vary in each functional population. Even though all the lymphocytes had lectin receptors, T lymphocytes showed higher affinity for Con A, PHA and TP lectins, while B lymphocytes appeared to be more specific for LC, PS, SBA, DB, RCAII and WGA lectins.     

Heme oxygenase-1 and carbon monoxide suppress the pathogenesis of experimental cerebral malaria

Cerebral malaria claims more than 1 million lives per year. We report that heme oxygenase-1 (HO-1, encoded by Hmox1) prevents the development of experimental cerebral malaria (ECM). BALB/c mice infected with Plasmodium berghei ANKA upregulated HO-1 expression and activity and did not develop ECM. Deletion of Hmox1 and inhibition of HO activity increased ECM incidence to 83% and 78%, respectively. HO-1 upregulation was lower in infected C57BL/6 compared to BALB/c mice, and all infected C57BL/6 mice developed ECM (100% incidence). Pharmacological induction of HO-1 and exposure to the end-product of HO-1 activity, carbon monoxide (CO), reduced ECM incidence in C57BL/6 mice to 10% and 0%, respectively. Whereas neither HO-1 nor CO affected parasitemia, both prevented blood-brain barrier (BBB) disruption, brain microvasculature congestion and neuroinflammation, including CD8(+) T-cell brain sequestration. These effects were mediated by the binding of CO to hemoglobin, preventing hemoglobin oxidation and the generation of free heme, a molecule that triggers ECM pathogenesis.     

Electrophoretic map of boar sperm plasma membrane polypeptides and localization and fractionation of specific polypeptide subclasses

A high resolution, two-dimensional (2-D) electrophoretic map of the plasma membrane (PM) polypeptides from the ejaculated boar spermatozoon is described. 2-D silver-stained polyacrylamide gel electrophoresis (PAGE) gels revealed over 250 polypeptides; Coomassie blue staining revealed more than 100. Fifty Coomassie-staining polypeptides were catalogued and biochemically characterized, with twenty of these designated major sperm PM polypeptides. Cavitation pressures ranging from 50 PSI to 1000 PSI were used to enrich 2-D maps either in head PM (50 PSI) or in flagellar PM (1000 PSI) and provided tentative localization of certain PM polypeptides. Immunoabsorption chromatography showed that most major polypeptides seen in the 2-D map were antigenic. Major polypeptide bands from single dimensional (1-D) gels were excised, antibodies against them were produced in rabbits, and the polypeptides were localized via indirect fluorescein isothiocyanate (FITC) technique. Cross-reacting antigenic determinants in the PAGE PM polypeptides were determined by transblotting and staining the transblots by an indirect peroxidase technique. Cross-reactivity was extensive with several polypeptide groups, but specific enough with others to allow tentative localization. Lectin affinity chromatography using Con A, WGA, RCA-1, PNA, and DBA indicated the lectin specificity of PM polypeptides. These data together with periodic acid-Schiff (PAS) and carbohydrate-specific silver staining permitted identification of glycoproteins in the 2-D maps. FITC coupled to specific lectins showed the regional distribution of these lectins on the sperm surface. The 2-D polypeptide map and the catalogue of properties of major Coomassie-stained PM polypeptides provides a reference for future studies in the boar and other species.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either ¹²⁵I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10⁷ binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

Quantification of lectin receptors on B, T, T-gamma and T-mu lymphocytes. I. Concanavalin A, Pisum sativum, Lens culinaris and wheat-germ agglutinin

The immune system is formed of different lymphocyte subpopulations, each one having a defined role to defend the organism. Their plasma membranes present differences in the glycoproteinic or/and glycolipidic composition, as detected with labelled 125I-lectins. B lymphocytes have a greater number of receptors for the Pisum sativum, Lens culinaris and WGA lectins than T lymphocytes. On the other hand, T lymphocytes bind a greater number of Concanavalin A molecules than B lymphocytes. WGA lectin appeared to be more specific for T mu subpopulation, while Con A and Pisum sativum lectins were bound preferentially to T gamma lymphocytes while no significant differences were observed between both subpopulations for Lens culinaris lectin. From the affinity of each lectin to each lymphocyte population it could be deduced that the receptor structure, conformation and arrangement on the membrane was optimal in B lymphocytes for Con A and WGA binding, and T lymphocytes for Lens culinaris and Pisum sativum binding.     

Chronic ethanol administration alters hepatic surface membranes as evidenced by decreased concanavalin A binding

The effects of chronic ethanol administration on the hepatic surface membrane were examined. The binding of the lectin, concanavalin A (Con A), to isolated hepatocytes was used to ascertain changes in the hepatic plasma membrane, especially in regard to glycoprotein composition, due to chronic ethanol feeding. Hepatocytes, isolated from rats fed ethanol for 5 to 7 weeks, had a decreased ability to bind Con A when compared to hepatocytes from either the pair-fed controls or ad libitum chow-fed rats. Since decreased Con A binding was more apparent at high Con A concentrations, reduced lectin binding likely reflected changes in the composition of surface membrane glycoproteins in the livers of the ethanol-fed rats. When ethanol (50 mM) was added to the incubation medium containing hepatocytes from ethanol-fed rats, pair-fed controls, or chow-fed rats, no effects on Con A binding were observed. These results indicate that chronic ethanol administration induces changes in the oligosaccharide chains of plasma membrane glycoproteins in the liver. Such alterations may play a role in the pathogenesis of alcoholic liver disease.     

Surface alterations during the capacitation of mammalian spermatozoa

Capacitation is the process by which mammalian sperm acquire the ability to undergo the acrosome reaction which, in turn, is a prerequisite for sperm-egg fusion and penetration. Until recently, it was thought that capacitation involved subtle physiological and chemical changes which had no morphological counterparts even at the electron microscopic level. However, it has now been shown by a number of investigators that material associated with the plasma membrane surface is either lost or extensively redistributed during in vitro or in vivo capacitation. We have made use of lectins and antibodies as probes of the sperm surface during capacitation and the acrosome reaction. Concanavalin A (Con A), wheat germ agglutinin (WGA) and soybean agglutinin (SBA) have been used in conjunction with fluorescent tags (FITC) and ultrastructural markers (ferritin, hemocyanin) to study the surface of golden hamster, guinea pig, mouse and human spermatozoa. Con A and WGA label the plasma membrane overlying the acrosomal region quite uniformly on these species. After capacitation there is a specific loss (or masking) of lectin binding sites over the acrosomal region of the sperm head in all species examined. Antibodies prepared against sperm and specific antibodies to a cell surface protein (fibronectin) were also tagged with fluorescent or ultrastructural markers and used to label the surfaces of sperm before and after capacitation. These probes also indicate a specific loss of surface associated material over the acrosomal surface after capacitation. These results are consistent with the notion that there is a general removal of surface components during capacitation and that this denuding of the surface is a prerequisite for the following membrane fusion events involved in the acrosome reaction and sperm-egg fusion.     

Lectin histochemistry of human skeletal muscle

Biotinyl derivatives of seven plant lectins-concanavalin A (Con A), peanut agglutinin (PNA), Ricinus communis agglutinin I (RCA I), Ulex europeus agglutinin I (UEA I), soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), and wheat germ agglutinin (WGA)-were bound to cryostat sections of biopsied normal human muscle and visualized with avidin-horseradish peroxidase conjugates. A distinct staining pattern was observed with each lectin. The most general staining was observed with Con A, RCA I, and WGA, which permitted strong visualization of the plasmalemma-basement membrane unit, tubular profiles in the interior of muscle fibers, blood vessels, and connective tissue. PNA gave virtually no intracellular staining, while SBA and UEA I selectively stained blood vessels. DBA was unique in providing good visualization of myonuclei. In each case, lectin staining could be blocked by appropriate sugar inhibitors. Neuraminidase pretreatment of the cryostat sections altered the pattern of staining by all lectins except UEA I and Con A; staining with RCA I became stronger and that with WGA became less intense, while staining with PNA, SBA and DBA became stronger and more generalized, resembling that of RCA I. These effects of neuraminidase pretreatment are in conformity with the known structure of the oligosaccharide chains of membrane glycoproteins and specificities of the lectins involved.     

Parasite burden and CD36-mediated sequestration are determinants of acute lung injury in an experimental malaria model

Although acute lung injury (ALI) is a common complication of severe malaria, little is known about the underlying molecular basis of lung dysfunction. Animal models have provided powerful insights into the pathogenesis of severe malaria syndromes such as cerebral malaria (CM); however, no model of malaria-induced lung injury has been definitively established. This study used bronchoalveolar lavage (BAL), histopathology and gene expression analysis to examine the development of ALI in mice infected with Plasmodium berghei ANKA (PbA). BAL fluid of PbA-infected C57BL/6 mice revealed a significant increase in IgM and total protein prior to the development of CM, indicating disruption of the alveolar-capillary membrane barrier-the physiological hallmark of ALI. In contrast to sepsis-induced ALI, BAL fluid cell counts remained constant with no infiltration of neutrophils. Histopathology showed septal inflammation without cellular transmigration into the alveolar spaces. Microarray analysis of lung tissue from PbA-infected mice identified a significant up-regulation of expressed genes associated with the gene ontology categories of defense and immune response. Severity of malaria-induced ALI varied in a panel of inbred mouse strains, and development of ALI correlated with peripheral parasite burden but not CM susceptibility. Cd36(-/-) mice, which have decreased parasite lung sequestration, were relatively protected from ALI. In summary, parasite burden and CD36-mediated sequestration in the lung are primary determinants of ALI in experimental murine malaria. Furthermore, differential susceptibility of mouse strains to malaria-induced ALI and CM suggests that distinct genetic determinants may regulate susceptibility to these two important causes of malaria-associated morbidity and mortality.     

Trypanosoma (Nannomonas) congolense: analysis by fluorescein-conjugated plant lectins of surface saccharides of cloned variant antigen types differing in infectivity for mice

Surface saccharides of 4 cloned VATs (variant antigen types) of Trypanosoma (Nannomonas) congolense, AmNats (Amherst Nannomonas antigen types) 1.1, 1.2, 2.1, and 3.1, derived from 3 different stocks, were compared by fluorescein-conjugated, plant lectins using a quantitative fluorescence method. It was ascertained by the ID63 assay that the 4 AmNats differed in their infectivity for mice. The lectins employed for AmNats 1.1, 2.1, and 3.1 were concanavalin A (Con A), wheat germ agglutinin (WGA), soybean agglutinin (SBA), garden pea agglutinin (GPA), and gorse seed (Ulex europaeus) agglutinin (UEA). In view of the results obtained with these 3 AmNats, only Con A, WGA, and GPA were used with AmNat 1.2, which was isolated after the lectin analyses of the other cloned VATs were completed. On the basis of experimental results, we concluded that the amounts of saccharide residues binding the several lectins differed among the 4 AmNats. In each instance, the reaction specificity was controlled by inclusion of an appropriate sugar in the incubation mixture. Although the actual numbers of various specific lectin-binding sites differed among the AmNats 1.1, 2.1, and 3.1, all of them were found to have the following sugars on their surfaces: alpha-D-mannose, N-acetyl-D-glucosamine, D-galactose, alpha-D-glucose, and alpha-L-fucose. AmNat 1.2 treated with Con A, WGA, and GPA only had the first 2 sugars named above and alpha-D-glucose residues. The results of the ID63 assay indicated AmNats 1.1 and 2.1 to be significantly more infective for mice than AmNats 1.2 and 3.1. The lectin analysis revealed that the 2, more infective, cloned VATs incubated with Con A or WGA emitted significantly (approximately 39% to approximately 62%) more fluorescence than the less infective ones. Thus there were significantly more numerous Con A and WGA binding sites on the more infective AmNats. The situation was reversed with regard to GPA. Upon treatment with this lectin, fluorescence emitted by AmNats 1.1 and 2.1 was significantly (approximately 56% to approximately 81%) lower than that recorded for the less infective AmNats 1.2 and 3.1. In light of our results, infectivity of T. congolense cloned VATs was correlated with the presence of higher numbers of alpha-D-mannose and N-acetyl-D-glucosamine residues and of lower numbers of alpha-D-glucose residues on the surface of the bloodstream trypanosomes. There appeared to be no correlation between infectivity and the numbers of D-galactose and alpha-L-fucose residues present on these parasites.