Glycans in the roles of parasitological diagnosis and host-parasite interplay.

The investigation of the glycan repertoire of several organisms has revealed a wide variation in terms of structures and abundance of glycan moieties. Among the parasites, it is possible to observe different sets of glycoconjugates across taxa and developmental stages within a species. The presence of distinct glycoconjugates throughout the life cycle of a parasite could relate to the ability of that organism to adapt and survive in different hosts and environments. Carbohydrates on the surface, and in excretory-secretory products of parasites, play essential roles in host-parasite interactions. Carbohydrate portions of complex molecules of parasites stimulate and modulate host immune responses, mainly through interactions with specific receptors on the surface of dendritic cells, leading to the generation of a pattern of response that may benefit parasite survival. Available data reviewed here also show the frequent aspect of parasite immunomodulation of mammalian responses through specific glycan interactions, which ultimately makes these molecules promising in the fields of diagnostics and vaccinology.

Histochemical differences along the intestine of Corydoras paleatus (Siluriformes: Callichthyidae) / Diferencias histoquímicas a lo largo del intestino de Corydoras paleatus (Siluriformes: Callichthyidae)

AbstractThe Neotropical catfish Corydoras paleatus is a facultative air-breather and the caudal half of the intestine is involved in gas exchange. In South America, air-breathing fishes are found in tropical or sub-tropical freshwaters where the probability of hypoxia is high. The aim of this study was to characterize by traditional histochemical and lectinhistochemical methods the pattern of carbohydrate in the intestinal mucosa. Intestine samples were taken from 25 healthy adult specimens collected in Buenos Aires (Argentina). Samples were fixed by immersion in 10 % buffered formalin and routinely processed and embedded in paraffin wax. Subsequently, these sections were incubated in the biotinylated lectins battery. Labeled Streptavidin-Biotin (LSAB) system was used for detection, diaminobenzidine as chromogen and haematoxylin as a contrast. To locate and distinguish glycoconjugates (GCs) of the globet cells, we used the following histochemical methods: PAS; PAS*S; KOH/ PA*S; PA/Bh/KOH/PAS; KOH/PA*/Bh/PAS; Alcian Blue and Toluidine Blue at different pHs. Microscopically, the general structure of vertebrate intestine was observed and showed all the cell types characteristic of the intestinal epithelium. The cranial sector of catfish intestine is a site of digestion and absorption and its structure is similar to other fish groups. In contrast, enterocytes of the caudal portion are low cuboidal cells; and between these, globet cells and capillaries are observed, these latter may reach the mucosal lumen. Underlying the epithelium, observed a well-developed lamina propria-submucosa made of connective tissue; this layer was highly vascularized and did not exhibit glands. According to histochemistry, the diverse GCs elaborated and secreted in the intestine are associated with specific functions in relation to their physiological significance, with special reference to their role in lubrication, buffering effect and prevention of proteolytic damage to the epithelium together with other biological processes, such as osmoregulation and ion exchange. The lectinhistochemical analysis of the intestinal mucosa reveals the presence of terminal residues of glucose, mannose and galactose. In conclusion, this study has shown that GCs synthesized in the intestine of C. paleatus exhibit a high level of histochemical complexity and that the lectin binding pattern of the intestinal mucosa is characteristic of each species and the variations are related with the multiple functions performed by the mucus in the digestive tract. The information generated here may be a relevant biological tool for comparing and analyzing the possible glycosidic changes in the intestinal mucus under different conditions, such as changes in diet or different pathological stages. Rev. Biol. Trop. 64 (1): 327-340. Epub 2016 March 01.

ResumenEl pez neotropical Corydoras paleatus, de respiración aérea de tipo facultativa, utiliza el sector caudal del intestino para el intercambio gaseoso. En América del Sur, los peces con respiración aérea se encuentran en las aguas dulceacuícolas tropicales y subtropicales, donde la probabilidad de hipoxia es alta. El objetivo de este trabajo fue caracterizar mediante técnicas histoquímicas tradicionales y de lectinhistoquímica el patrón de carbohidratos de la mucosa intestinal. Para ello se utilizaron muestras de intestino de 25 ejemplares sanos adultos recolectados en la provincia de Buenos Aires (Argentina). Las muestras fueron fijadas en formol amortiguado al 10 % y se procesaron para su inclusión en parafina. Posteriormente, los cortes fueron incubados con una batería de lectinas biotiniladas. Se utilizó el sistema de marcado con estreptavidina-biotina (LSAB) para su detección, diaminobencidina como cromógeno y hematoxilina como colorante de contraste. Para localizar y diferenciar los glicoconjugados (GCs) de las células caliciformes, se utilizaron las siguientes técnicas histoquímicas: PAS, PAS*S, PAPS, KOH/PA*S, PA/Bh/KOH/PAS, KOH/PA*/Bh/PAS, Azul Alcian y Azul de Toluidina a diferentes pHs. Microscópicamente, se observa la estructura general del intestino de los vertebrados y el epitelio intestinal presenta todos los tipos celulares característicos de esta región. El sector craneal del intestino de este teleósteo, es el sitio de digestión y absorción, y posee una estructura similar a la de otros grupos de peces. En cambio, los enterocitos de la porción caudal, son células cúbicas bajas, entre ellos se observan células caliciformes y capilares sanguíneas que llegan hasta el lumen de la mucosa. Por fuera del epitelio, se observa una lámina propia-submucosa muy desarrollada compuesta por tejido conectivo, altamente vascularizada que no presenta glándulas. De acuerdo con las técnicas histoquímicas, los diversos GCs elaborados y secretados por la mucosa intestinal se encuentran asociados con funciones específicas de importancia fisiológica, como su rol en la lubricación, su efecto amortiguador y la prevención de daños proteolíticos del epitelio junto con otros procesos biológicos, tales como la osmorregulación y el intercambio iónico. El análisis lectinhistoquímico de la mucosa intestinal revela la presencia de residuos terminales de glucosa, manosa y galactosa. En conclusión, en este estudio se demuestra que los GCs sintetizados en el intestino de C. paleatus muestran un alto nivel de complejidad histoquímica y que el patrón de unión de lectina de la mucosa intestinal es característico para cada especie y las variaciones se hallan relacionadas con las múltiples funciones realizadas por el mucus en el tracto digestivo. La información brindada en este trabajo es una herramienta de relevancia biológica para comparar y analizar los posibles cambios glicosídicos del mucus intestinal bajo diferentes condiciones como los cambios en la dieta o diferentes estados patológicos.

Histochemical differences along the intestine of Corydoras paleatus (Siluriformes: Callichthyidae).

The Neotropical catfish Corydoras paleatus is a facultative air-breather and the caudal half of the intestine is involved in gas exchange. In South America, air-breathing fishes are found in tropical or sub-tropical freshwaters where the probability of hypoxia is high. The aim of this study was to characterize by traditional histochemical and lectinhistochemical methods the pattern of carbohydrate in the intestinal mucosa. Intestine samples were taken from 25 healthy adult specimens collected in Buenos Aires (Argentina). Samples were fixed by immersion in 10 % buffered formalin and routinely processed and embedded in paraffin wax. Subsequently, these sections were incubated in the biotinylated lectins battery. Labeled Streptavidin-Biotin (LSAB) system was used for detection, diaminobenzidine as chromogen and haematoxylin as a contrast. To locate and distinguish glycoconjugates (GCs) of the globet cells, we used the following histochemical methods: PAS; PAS*S; KOH/ PA*S; PA/Bh/KOH/PAS; KOH/PA*/Bh/PAS; Alcian Blue and Toluidine Blue at different pHs. Microscopically, the general structure of vertebrate intestine was observed and showed all the cell types characteristic of the intestinal epithelium. The cranial sector of catfish intestine is a site of digestion and absorption and its structure is similar to other fish groups. In contrast, enterocytes of the caudal portion are low cuboidal cells; and between these, globet cells and capillaries are observed, these latter may reach the mucosal lumen. Underlying the epithelium, observed a well-developed lamina propria-submucosa made of connective tissue; this layer was highly vascularized and did not exhibit glands. According to histochemistry, the diverse GCs elaborated and secreted in the intestine are associated with specific functions in relation to their physiological significance, with special reference to their role in lubrication, buffering effect and prevention of proteolytic damage to the epithelium together with other biological processes, such as osmoregulation and ion exchange. The lectinhistochemical analysis of the intestinal mucosa reveals the presence of terminal residues of glucose, mannose and galactose. In conclusion, this study has shown that GCs synthesized in the intestine of C. paleatus exhibit a high level of histochemical complexity and that the lectin binding pattern of the intestinal mucosa is characteristic of each species and the variations are related with the multiple functions performed by the mucus in the digestive tract. The information generated here may be a relevant biological tool for comparing and analyzing the possible glycosidic changes in the intestinal mucus under different conditions, such as changes in diet or different pathological stages.

Evaluation of glycophenotype in breast cancer by quantum dot-lectin histochemistry.

Cell surface glycoconjugates play an important role in differentiation/dedifferentiation processes and lectins are employed to evaluate them by several methodologies. Fluorescent probes are considered a valuable tool because of their ability to provide a particular view, and are more detailed and sensitive in terms of cell structure and molecular content. The aim of this study was to evaluate and compare the expression and distribution of glycoconjugates in normal human breast tissue, and benign (fibroadenoma), and malignantly transformed (invasive ductal carcinoma) breast tissues. For this, we used mercaptosuccinic acid-coated Cadmium Telluride (CdTe) quantum dots (QDs) conjugated with concanavalin A (Con A) or Ulex europaeus agglutinin I (UEA I) lectins to detect α-D-glucose/mannose and L-fucose residues, respectively. The QD-lectin conjugates were evaluated by hemagglutination activity tests and carbohydrate inhibition assays, and were found to remain functional, keeping their fluorescent properties and carbohydrate recognition ability. Fluorescence images showed that different regions of breast tissue expressed particular types of carbohydrates. While the stroma was preferentially and intensely stained by QD-Con A, ductal cells were preferentially labeled by QD-UEA I. These results indicate that QD-lectin conjugates can be used as molecular probes and can help to elucidate the glycoconjugate profile in biological processes.

Structural and immunological characterization of sulphatides: relevance of sulphate moieties in Trypanosoma cruzi glycoconjugates.

Sulphoglycosphingolipids, present on the surface of diverse cells, participate in the regulation of various cellular events. However, little is known about the structure and the role of sulphoglycosphingolipids in trypanosomatids. Herein, sulphated dihexosylceramide structures - composed mainly of sphingosine as the long chain base acylated with stearic acid - have been determined for the first time in Trypanosoma cruzi epimastigotes by UV-MALDI-TOF-MS analysis. Interestingly, inhibition ELISA assays using cruzipain as antigen and polyclonal rabbit antibodies specific for cruzipain, the major cysteine proteinase of T. cruzi, or for its C-terminal domain, have demonstrated (i) that sulphate epitopes are shared between cruzipain and sulphatides of T. cruzi, (ii) that cross-reactivity maps to the C-terminal domain and (iii) the existence of other antigenic determinants in the glycolipidic structures. These features provide evidence that sulphate groups are antigenic in sulphate-containing parasite glycoconjugates. Furthermore, IgG2 antibody levels inversely correlate with disease severity in chronic Chagas disease patients, suggesting that IgG2 antibodies specific for sulphated epitopes might be associated with protective immunity and might be considered as potential surrogates of the course of chronic Chagas disease.

Glycoconjugates in New World species of Leishmania: polymorphisms in lipophosphoglycan and glycoinositolphospholipids and interaction with hosts.

BACKGROUND: Protozoan parasites of the genus Leishmania cause a number of important diseases in humans and undergo a complex life cycle, alternating between a sand fly vector and vertebrate hosts. The parasites have a remarkable capacity to avoid destruction in which surface molecules are determinant for survival. Amongst the many surface molecules of Leishmania, the glycoconjugates are known to play a central role in host-parasite interactions and are the focus of this review. SCOPE OF THE REVIEW: The most abundant and best studied glycoconjugates are the Lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs). This review summarizes the main studies on structure and biological functions of these molecules in New World Leishmania species. MAJOR CONCLUSIONS: LPG and GIPLs are complex molecules that display inter- and intraspecies polymorphisms. They are key elements for survival inside the vector and to modulate the vertebrate immune response during infection. GENERAL SIGNIFICANCE: Most of the studies on glycoconjugates focused on Old World Leishmania species. Here, it is reported some of the studies involving New World species and their biological significance on host-parasite interaction. This article is part of a Special Issue entitled Glycoproteomics.

Carbohydrate profiling of fungal cell wall surface glycoconjugates of Aspergillus species in brain and lung tissues using lectin histochemistry.

The aim of this study was to evaluate, through lectin histochemistry, the expression of N-acetyl-D-glucosamine, L-fucose, D-galactose and glucose/mannose on the cell wall surfaces of Aspergillus species in histopathological specimens of brain (n = 1) and lung (n = 6) tissues obtained during autopsy of patients diagnosed postmortem as having had invasive aspergillosis. Concanavalin A (Con A), wheat germ agglutinin (WGA), Ulex europeus agglutinin I (UEA-I) and peanut agglutinin (PNA), all conjugated with horseradish peroxidase, were employed. Lectin-binding was visualized using 3,3-diaminobendizine (DAB) and hydrogen peroxide in phosphate buffer solution (PBS). We observed expression of N-acetyl-D-glucosamine and methyl-α-D-mannoside on the cell wall surfaces of all evaluated Aspergillus species, while the expression of L-fucose and D-galactose demonstrated inter and intra-specific variations. The results obtained from this study indicate that the use of WGA and Con A lectins permits visualization of Aspergillus structures such as hyphae, conidial heads and conidia in histopathological specimens of brain and lung tissues.

Carbohydrate profiling of fungal cell wall surface glycoconjugates of Trichophyton tonsurans and other keratinophilic filamentous fungi using lectins.

Various researchers have concluded that lectins are useful reagents for the study of fungal cell wall surface glycoconjugates. In this study, we evaluated the expression of N-acetyl-D-glucosamine, L-fucose, D-galactose and glucose/mannose on the cell wall surface of Trichophyton tonsurans and other keratinophilic filamentous fungi, using a simple lectin-binding protocol. The fungal cultures used were isolated from soils obtained from public parks by the hair-bait technique. The lectin assays used concanavalin A (Con A), wheat germ agglutinin (WGA), Ulex europeus agglutinin I (UEA-I) and peanut agglutinin (PNA), all conjugated with horseradish peroxidase. Adhesive tape was placed sticky-side down over the fungal colony, gently pressed and then removed. The fungal-tape samples were incubated with the lectin for 1 h at 4 °C. Lectin binding was visualised using 3,3-diaminobendizine (DAB) and hydrogen peroxidase. There was a high expression of N-acetyl-D-glucosamine on the cell wall surface of all fungi species tested, whereas the expression of L-fucose, D-galactose and glucose/mannose demonstrated inter-specific variations. The lectin-binding assay presented in this article eliminates many of the laborious steps involved in other protocols. The amount and quality of the mycelium and spores immobilised by the adhesive tapes were suitable for obtaining the carbohydrate profile in glycoconjugates of the cell wall surface of filamentous fungi.

Glycosaminoglycans and glycoconjugates in the adult anuran integument (Lithobates catesbeianus).

Glycosaminoglycans (GAGs) from the integument of Lithobates catesbeianus were biochemically characterized and histochemically localized. Moreover, carbohydrate distribution was investigated using conventional and lectin histochemistry at light microscopy. Hyaluronan (HA), dermatan sulfate (DS) and a heparanoid were found in the integument. Sulfated and carboxylated GAGs were visualized in the Eberth-Katschenko (EK) layer, in the mucous glands, in the hypodermis as well as in the mast cells. Furthermore, glucose and galactose were identified in the integument through thin layer chromatography (TLC) assays. N-Acetyl-beta-glucosamine residues were identified in the mucous glandular cells, between the corneum and spinosum strata, in the subepidermal region, and in the EK layer. N-Acetyl-galactosamine residues were evident in the EK layer, corresponding to a residue of the dermatan sulfate chain, which may be related to the collagenous fiber arrangement. These glycoconjugates occurred as secretory glandular products and as dermal structural elements. Moreover, HA and DS are the predominant GAGs in the L. catesbeianus integument. Considering the importance of glycoconjugates, they play a significant role to the integrity of the skin, providing mechanical support for integument cells. In addition, they are important to the water regulation mechanisms, since L. catesbeianus is preferably aquatic.

Glycosylation patterns of human alpha2-macroglobulin: analysis of lectin binding by electron microscopy.

Human alpha2-macroglobulin (alpha 2M) is a 720 kDa glycoprotein that presents two ultrastructural conformations: slow (S-alpha 2M) and fast (F-alpha 2M). alpha 2M acts mainly as a proteinase scavenger, but an immunomodulatory role was also proposed. This work studies the effect of desialylation and deglycosylation on the structure patterns of alpha 2M by ultrastructural analysis of lectin-induced aggregates, which represents a new approach that had never been previously used. Transmission electron microscopy (TEM) analysis showed the loss of S-alpha 2M conformation after deglycosylation, indicating that glycosidic side-chains contribute to the molecular stability of S-alpha 2M. TEM proved to be an important tool to analyze the effect of biochemical changes on alpha 2M, yielding an objective qualitative control of its morphological state. Certain carbohydrate residues did not vary between the alpha 2M conformations, since both bound similarly ConA and WGA lectins. However, the binding of PNA and BSI-B(4) was slightly lower in F-alpha 2M than in S-alpha 2M. Among the neuraminidases used to desialylate both conformations of alpha 2M that from Arthrobacter ureafaciens was the most effective. Incubation with the lectins ConA or SNA, respectively specific for mannosyl and sialyl residues, led to dose-dependent patterns of aggregation of alpha 2M molecules, mediated by lectin binding and clearly visualized by TEM.

Differences between Naegleria fowleri and Naegleria gruberi in expression of mannose and fucose glycoconjugates.

Naegleria fowleri is the etiologic agent of primary amoebic meningoencephalitis, a rapidly fatal parasitic disease of humans. The adherence of Naegleria trophozoites to the host cell is one of the most important steps in the establishment and invasiveness of this infectious disease. Currently, little is known about the surface molecules that may participate in the interaction of N. fowleri with their target cells. In the present study, we investigated the composition of glycoconjugates present on the surface of trophozoites of the pathogenic N. fowleri and the nonpathogenic Naegleria gruberi. With the use of biotinylated lectins in western blot and flow cytometric analysis, we showed that N. fowleri trophozoites present high levels of surface glycoconjugates that contain alpha-D-mannose, alpha-D-glucose, and terminal alpha-L-fucose residues. A significant difference in the expression of these glycoconjugates was observed between N. fowleri and the nonpathogenic N. gruberi. Furthermore, we suggest that glycoconjugates that contain D-mannose and L-fucose residues participate in the adhesion of N. fowleri and subsequent damage to MDCK cells.

Lectin binding patterns and carbohydrate mediation of sperm binding to llama oviductal cells in vitro.

Sperm binding to oviductal epithelium would be involved in sperm reservoir formation in the utero tubal junction (UTJ). Although in other mammals sperm-oviduct interaction has been proved to be mediated by carbohydrate-recognition mechanisms, the factors implicated in the sperm adhesion to oviductal epithelium of llama are still unknown. In order to assess the role of carbohydrates present in the mucosa surface, we examined the distribution of glycoconjugates in the llama oviduct by confocal lectin-histochemistry. Mannosyl, glucosyl, N-acetylglucosaminyl, galactosyl, N-acetylgalactosaminyl and sialic acid residues were detected in the oviductal mucose glycocalyx. By incubation of UTJ oviductal explants with LCA, DBA, UEA-1 or PNA lectin previous to co-culture with sperm, we observed a significant decrease in sperm binding only with LCA lectin. In the mucosa surface there were numerous d-glucosyl and D-manosyl residues, which were spotted by this lectin. Probably, this fact promotes the whole covering of the oviduct luminal surface by the sugar-lectin complex, preventing sperm access and adhesion of further residues. However, sperm incubation with mannose or glucose does not significantly prevent binding, which means that glucose and mannose would not be involved in a specific sperm-oviduct interaction. On the other hand, we observed a high reduction in sperm binding to UTJ explants with N-acetylgalactosamine and galactose (p<0.001). Coincidentally, binding sites for N-acetylgalactosamine-PAA-FITC conjugate were observed on the whole surface of the sperm, supporting the concept that llama sperm have lectin-like molecules in their surface, as is the case in other mammals. Probably, these lectin-like molecules, by means of N-acetylgalactosamine and galactose recognition, could link the sperm to the oviductal mucosa with the purpose of forming storing sites in the UTJ. Our results support the idea that more than one carbohydrate could participate in sperm reservoir formation in the llama UTJ oviductal segment.

O processo de interação de Leishmania (Leishmania) chagasi com Lutzomyia (Lutzomyia) longipalpis e a importância do lipofosfoglicano (LPG)

Estudos da interação Leishmania-vetor são importantes para o entendimento dos processos dedesenvolvimento e transmissão do parasito. Significativas informações têm sido relatadasprincipalmente para espécies de Leishmania do Velho Mundo. No Novo Mundo, existempoucas informações detalhadas sobre o desenvolvimento de Leishmania chagasi em seu vetornatural Lutzomyia longipalpis. O desenvolvimento desse parasito foi investigado em infecçõesexperimentais do vetor com promastigotas e amastigotas axênicas obtidas por transformação invitro. Ambas as formas do parasito geraram alta porcentagem de flebotomíneos infectados. Foiobservado um decréscimo no número de parasitos por intestino no terceiro dia após o repasto.Entretanto, os parasitos sobreviventes nos dois grupos foram capazes de se multiplicar e sedesenvolver no intestino, apresentando densidade máxima entre seis e sete dias. Todos osmorfotipos de promastigotas foram observados. Infecções iniciadas com promastigotas ouamastigotas axênicas mostraram perfis semelhantes de desenvolvimento e produziram formasinfectantes. Assim, promastigotas podem ser preferencialmente utilizadas em experimentos deinfecção por ser este um processo menos laborioso.

Para muitas espécies de Leishmania temsido sugerido que o lipofosfoglicano (LPG) promova a adesão do parasito ao epitélio intestinaldo flebotomíneo. Análises estruturais do LPG de diferentes espécies de Leishmania têmrevelado que o polimorfismo nesses glicoconjugados se deve a variação dos açúcares dascadeias laterais e "cap". A análise das cadeias laterais da cepa BH46 revelou pela primeira vezum LPG poliglicosilado em L. chagasi. A importância do LPG na sobrevivência dessa espécieem L. longipalpis foi investigada usando parasitos mutantes em LPG. Foram observadasreduções na sobrevivência ou no crescimento desses mutantes no período que antecede adefecação e, em 60 horas, a infecção foi perdida. Em todos os pares naturais Leishmania/vetoranalisados até o momento o LPG é requerido para a adesão do parasito ao intestino para evitarsua expulsão com o bolo fecal. Diferentemente, nós observamos que a síntese de LPG foiessencial para a sobrevivência inicial de L. chagasi no intestino de L. longipalpis,provavelmente protegendo o parasito do ataque enzimático. Aspectos morfológicos dessainteração foram investigados por microscopia óptica, eletrônica de varredura e transmissão.Diferente da adesão altamente especializada previamente proposta para L. chagasi em L.longipalpis, poucos parasitos foram observados com corpo e flagelo em contato com o epitéliointestinal e, esporadicamente, com o flagelo superficialmente inserido entre asmicrovilosidades. Diante de nenhuma vacina efetiva contra a doença e de uma variedadelimitada de drogas para o tratamento, detalhes de todos os aspectos da biologia do parasito sãodesejáveis para a formulação de novas estratégias de controle contra o protozoário e o vetor.

O processo de interação de Leishmania (Leishmania) chagasi com Lutzomyia (Lutzomyia) longipalpis e a importância do lipofosfoglicano (LPG) / The process of interaction of Leishmania (Leishmania) chagasi through Lutzomyia longipalpis (Lutzomyia) and the importance of lipofosfoglicano (LPG)

Estudos da interação Leishmania-vetor são importantes para o entendimento dos processos dedesenvolvimento e transmissão do parasito. Significativas informações têm sido relatadasprincipalmente para espécies de Leishmania do Velho Mundo. No Novo Mundo, existempoucas informações detalhadas sobre o desenvolvimento de Leishmania chagasi em seu vetornatural Lutzomyia longipalpis. O desenvolvimento desse parasito foi investigado em infecçõesexperimentais do vetor com promastigotas e amastigotas axênicas obtidas por transformação invitro. Ambas as formas do parasito geraram alta porcentagem de flebotomíneos infectados. Foiobservado um decréscimo no número de parasitos por intestino no terceiro dia após o repasto.Entretanto, os parasitos sobreviventes nos dois grupos foram capazes de se multiplicar e sedesenvolver no intestino, apresentando densidade máxima entre seis e sete dias. Todos osmorfotipos de promastigotas foram observados. Infecções iniciadas com promastigotas ouamastigotas axênicas mostraram perfis semelhantes de desenvolvimento e produziram formasinfectantes. Assim, promastigotas podem ser preferencialmente utilizadas em experimentos deinfecção por ser este um processo menos laborioso.

Para muitas espécies de Leishmania temsido sugerido que o lipofosfoglicano (LPG) promova a adesão do parasito ao epitélio intestinaldo flebotomíneo. Análises estruturais do LPG de diferentes espécies de Leishmania têmrevelado que o polimorfismo nesses glicoconjugados se deve a variação dos açúcares dascadeias laterais e "cap". A análise das cadeias laterais da cepa BH46 revelou pela primeira vezum LPG poliglicosilado em L. chagasi. A importância do LPG na sobrevivência dessa espécieem L. longipalpis foi investigada usando parasitos mutantes em LPG. Foram observadasreduções na sobrevivência ou no crescimento desses mutantes no período que antecede adefecação e, em 60 horas, a infecção foi perdida. Em todos os pares naturais Leishmania/vetoranalisados até o momento o LPG é requerido para a adesão do parasito ao intestino para evitarsua expulsão com o bolo fecal. Diferentemente, nós observamos que a síntese de LPG foiessencial para a sobrevivência inicial de L. chagasi no intestino de L. longipalpis,provavelmente protegendo o parasito do ataque enzimático. Aspectos morfológicos dessainteração foram investigados por microscopia óptica, eletrônica de varredura e transmissão.Diferente da adesão altamente especializada previamente proposta para L. chagasi em L.longipalpis, poucos parasitos foram observados com corpo e flagelo em contato com o epitéliointestinal e, esporadicamente, com o flagelo superficialmente inserido entre asmicrovilosidades. Diante de nenhuma vacina efetiva contra a doença e de uma variedadelimitada de drogas para o tratamento, detalhes de todos os aspectos da biologia do parasito sãodesejáveis para a formulação de novas estratégias de controle contra o protozoário e o vetor.

Nitrogen-deficient microalgae are rich in cell-surface mannose: potential implications for prey biorecognition by phagotrophic protozoa / Microalgas com deficiência de nitrogênio são enriquecidas com manose na superfície celular: potenciais implicações para reconhecimento de presas por protozoários fagotróficos

Flow cytometry was used to quantify the abundance of mannose-linked glycoconjugates on microalgae precultured using low- or high-nitrate media. Nitrogen-deficient microalgae were richer in cell-surface mannose than nitrogen-sufficient. Findings are discussed in view of recent research which reveals mannose-specific 'feeding receptors' assist prey biorecognition by phagotrophic protozoa that ingest microalgae.

Citometria de fluxo foi usada para quantificar a abundância de glicoconjugados com manose em precultivos de microalgas usando meios com baixo e alto teor de nitrato. Microalgas com deficiências de nitrogênio tinham mais manose na superfície celular do que as com nitrogênio suficiente. Resultados são discutidos com base nas pesquisas recentes que revelam receptores específicos para manose que auxiliam no reconhecimento da presa por protozoários fagotróficos que ingerem microalgas.

Glycoconjugates in the mucosa of the digestive tract of Cynoscion guatucupa: a histochemical study.

This study addresses the histomorphology, the histochemistry and the distribution of glycoconjugates (GCs) in the mucosa of the digestive tract of the weakfish Cynoscion guatucupa. The histological characterization of the buccopharyngeal cavity and the esophagus revealed that they are lined by a stratified epithelium where the mucosa is thrown into longitudinal folds. The stomach is lined with a simple columnar secretory epithelium with tubular glands. GCs were analyzed using a range of histochemical methods. They were identified as oxidizable vicinal diols; sialic acids and some of their chain variants, C7, C8 or C9; sialic acid residues with O-acyl substitution at C8 or C9; carboxyl groups and sulfate groups. Sugar residues in the oligosaccharide side chains of the GCs were investigated using seven biotinylated lectins. Mucous cells from the contents of the buccopharyngeal cavity, esophagus and stomach evidenced neutral, sulphated and sialylated GCs. The latter were substituted mainly in C8. A moderately strong lectin labeling was observed in mucous cells of the three organs studied. Nonetheless, the apical edge of the gastric gland cells showed a strong positive labeling. The presence of different classes of GCs has been associated with different functions, such as lubrication, protection, inhibition of microorganisms proliferation and ionic regulation.

Profiling isoflavone conjugates in different organs of Lupinus exaltatus Zucc.

The profiles of isoflavone conjugates in extracts obtained from different parts of Lupinus exaltatus Zucc. grown in Mexico were compared using HPLC-UV and HPLC-ESI/MSn. Collision-induced dissociation-MSn experiments were performed using an ion trap analyser during HPLC-ESI/MS analyses. Nineteen isoflavone conjugates were identified in samples obtained from air-dried roots, leaves, stems and inflorescences of lupin plants. It was possible to determine the structures of the studied compounds on the basis of the MS recorded. The compounds identified were di- and mono-glucosides of genistein and 2'-hydroxygenistein with a different pattern of C- and O-glycosylation. Some glucosides were acylated with malonic acid. It was not possible to establish the glycosylation sites on the basis of MS alone; however, it was possible to differentiate isoflavone C- and O-glucosides. The highest levels of isoflavones and their conjugates were detected in roots and the lowest in stems. Free aglycones were identified in roots and inflorescences but they were not found in stems and leaves.

Expression pattern of glycoconjugates in the Bidderian and ovarian follicles of the Brazilian toad Bufo ictericus analyzed by lectin histochemistry.

The Bidder's organ and ovary of the Brazilian toad Bufo ictericus were studied by light microscopy, using hematoxylin-eosin (HE) and periodic acid Schiff (PAS) staining. The expression and distribution of carbohydrate moieties was analyzed by lectin histochemistry, using 8 lectins with different carbohydrate specificities: Ulex europaeus (UEA I), Lens culinaris (LCA), Erythrina cristagalli (ECA), Arachis hypogaea (PNA), Ricinus communis (RCA I), Aleuria aurantia (AAA), Triticum vulgaris (WGA), and Glycine maximum (SBA). The results showed that the Bidderian zona pellucida presented alpha-mannose, alpha-L-fucose, beta-D-galactose, N-acetyl-D-glucosamine, and alpha/beta-N-acetyl-galactosamine residues. The Bidderian follicular cells showed the presence of beta-D-galactose and N-acetyl-D-glucosamine. In the extracellular matrix, alpha-mannose and alpha/beta-N-acetyl-galactosamine residues were detected. The ovarian zona pellucida showed alpha-L-fucose, N-acetyl-D-glucosamine, alpha/beta-N-acetyl-galactosamine residues, and alpha-mannose and N-acetyl-D-glucosamine residues were detected in the follicular cells. Thus, the zona pellucida in both organs contains N-acetyl-D-glucosamine, and alpha/beta-N-acetyl-galactosamine residues. alpha-L-fucose residues were detected in the zona pellucida of both organs, using different lectins. Considering that beta-D-galactose residue was absent from ovary but present in the Bidder's organ, this sugar residue may play an important role in follicle development, blocking the Bidderian follicles and preventing further development of the Bidder's organ into a functional ovary.

Expression pattern of glycoconjugates in the Bidderian and ovarian follicles of the Brazilian toad Bufo ictericus analyzed by lectin histochemistry

O órgão do Bidder e o ovário do sapo Bufo ictericus foram analisados por meio de microscopia de luz, utilizando a coloração pela hematoxilina-eosina (HE) e o método do ácido periódico de Schiff (PAS). A expressão e a distribuição de carboidratos foram verificadas por meio da histoquímica com lectinas, tendo sido utilizadas 8 lectinas com diferentes especificidades para carboidratos (Ulex europaeus (UEA I), Lens culinaris (LCA), Erythrina cristagalli (ECA), Arachis hypogaea (PNA), Ricinus communis (RCA I), Aleuria aurantia (AAA), Triticum vulgaris (WGA) e Glycine maximum (SBA). Os resultados mostraram que a zona pelúcida Bidderiana apresenta resíduos de a-mannose, a-L-fucose, b-D-galactose, N-acetilDglicosamine e a/b-N-acetil-galactosamina. As células foliculares Bidderianas mostraram a presença de b-D-galactose e N-acetil-D-glicosamina. Na matriz de extracelular foram detectados resíduos de a-mannose e a/b-N-acetil-galactosamina. Resíduos de a-L-fucose, N-acetyl-D-glicosamina e a/b-N-acetil-galactosamina foram evidenciados na zona pelúcida ovariana, enquanto na célula folicular foi detectado o resíduo de a-mannose e de N-acetil-D-glicosamina. Assim, a zona pelúcida, em ambos os órgãos, contém resíduos de N-acetil-D-glicosamina e a/b-N-acetil-galactosamina. O resíduo de a-L-fucose foi detectado na zona pelúcida de ambos os órgãos, mas utilizando-se diferentes lectinas. Considerando que o resíduo de a-D-galactose é ausente no ovário, mas presente no órgão de Bidder, a a-D-galactose pode ter um papel importante no controle do desenvolvimento folicular, bloqueando o desenvolvimento dos folículos Bidderianos e impedindo que o órgão de Bidder se transforme em um ovário funcional.

[Histochemical modifications of sugar residues in glycoconjugates of human palatine glands during development and maturation]. / Modificaciones histoquimicas de los residuos azucares en los glucoconjugados de las glandulas palatinas humanas durante su desarrollo y maduracion.

ANTECEDENTS: Most of the literature has focused on the morphological and histochemical characteristics of palatine glands during the development histogenesis and maturation process. However, there are no publications regarding protein glycosylation and the distribution of glycoproteins with N and O-glycoside bonds, and their possible functional role. OBJECTIVE: Based on this background we committed ourselves to studying the human palatine glands in different age groups in order to determine the beginning of mucine secretion and the presence of glycoproteins with N and O-glycoside bonds during the processes of differentiation and maturation. MATERIALS AND METHOD: Human palatine glands were obtained from embryos, fetuses, newborn infants, youngsters and adults. They were studied using histochemical methods (PAS, Dane, Alcian blue, Toluidine blue), and lectin histochemistry. RESULTS: The variations observed in glycoconjugates during the development and maturation of human palatine glands show that secretion starts at early stages of prenatal development; it is made up of various sugars that might play an important role in the pre-natal and postnatal periods. CONCLUSIONS: The study of modifications in sugar residues in the human palatine glands during embryogenesis, differentiation and maturation processes allows us to have further knowledge of their histophysiology and possible changes taking place during aging. This study may also help to understand pathological processes in mature salivary glands.