Quantitation of yeast cell-cell fusion using multicolor flow cytometry.

Mating of haploid Saccharomyces cerevisiae cells of opposite sex provides a powerful model system to study the cell-cell fusion. However, a rapid and standardized method is much needed for quantitative assessment of fusion efficiency. The gold standard method relies on counting mating pairs in fluorescence microscopy images. This current method is limited by expectancy bias and it is time consuming, restricting the number of both cell-cell fusion events and strains that can be analyzed at once. Automatic approaches present a solution to these limitations. Here, we describe a novel flow cytometric approach that is able to quickly both identify mating pairs within a mixture of gametes and quantify cell fusion efficiency. This method is based on staining the cell wall of yeast populations with different Concanavalin A-fluorophore conjugates. The mating subpopulation is identified as the two-colored events set and fused and unfused mating pairs are subsequently discriminated by green fluorescent protein bimolecular complementation. A series of experiments was conducted to validate a simple and reliable protocol. Mating efficiency in each sample was determined by flow cytometry and compared with the one obtained with the current gold standard technique. The results show that mating pair counts using both methods produce indistinguishable outcomes and that the flow cytometry-based method provides quantitative relevant information in a short time, making possible to quickly analyze many different cell populations. In conclusion, our data show multicolor flow cytometry-based fusion quantitation to be a fast, robust, and reliable method to quantify the cell-cell fusion in yeast.

Evaluation of WGA and Concanavalin A (Con A) lectin as biomarkers of hepatosplenic schistosomiasis in human biopsies with no evidence of egg-granuloma system.

INTRODUCTION: Colonic lesions are predominant in patients with schistosomiasis. However, carbohydrate alterations in colonic schistosomiasis remain unclear. Lectin-ligands allow us to identify changes in the saccharide patterns of cells. METHODS: Biopsies of descending and rectosigmoid colon of patients were submitted to WGA and Con A lectin histochemistry. RESULTS: WGA stained stroma and gland cells of descending colon and rectosigmoid tissues in a granular strong cytoplasmatic pattern in schistosomiasis specimens differing from normal control and Con A failing to recognize all samples analyzed. CONCLUSIONS: WGA ligands are expressed differently in patients with hepatosplenic schistosomiasis and no evidence of egg-granuloma system.

Characterizing subpopulations of neoplastic cells in serous effusions. The role of immunocytochemistry.

OBJECTIVE: To analyze the role of immunochemistry in serous effusions. STUDY DESIGN: We analyzed cell blocks of 18 pleural and 18 peritoneal effusions diagnosed as malignant (18), benign (14) and suspicious (4). They were immunostained by the avidin-biotin complex method with a panel of four monoclonal antibodies--CEA, Ber-EP4, LeuM1 (CD15) and p53--and, for lectins (Ulex europaeus) UEA-l, ConA and ConBr. RESULTS: Seventeen of the 18 cases of adenocarcinoma were positive for CEA (95%), 12 (66.6%) for Ber-EP4, 11 (61%) for CD15 and 11 (61%) for p53. Twelve of the 18 (66.6%) were positive for UEA-1, CEA, Ber-EP4 and CD15. UEA-1 did not react with mesothelial cells. p53 Gave a positive reaction in only one case, reactive mesothelial cells. ConA and ConBr reacted indiscriminately with benign and malignant cells; thus, it was not useful in distinguishing between these cells. CONCLUSION: In this context no antibody used alone is reliable for corroborating a diagnosis, but the selective use of a small panel of three markers (CEA, Ber-EP4 and LeuM1) can be very useful in solving diagnostic difficulties in the cytodiagnosis of serous effusions.

Regulation of histamine secretion in a mast cell line

La interacción del alergeno con su lg-E específica anclada en la superficie externa de la membrana plasmática del mastocito desencadena la liberación de histamina de dicha célula. Este proceso requiere la presencia de calcio extracelular y de energía bajo la forma de ATP proveniente fundamentalmente de glicólisis donde el lactato es el producto final. La heterogeneidad de los mastocitos ha sido previamente descrita en diversos tejidos de diferentes especies animales. Así, esta línea celular tumoral en ratones LAF1 mostró propiedades similares a las descritas para los mastocitos presentes en la cavidad peritoneal de la rata. Las respuestas metabólicas relacionadas a la secreción de histamina también fueron estudiadas en esta línea de mastocinoma, que exhibieron un comportamiento diferente dependiendo del secretagogo utilizado. En este sentido, una fuerte estimulación de la glicólisis fue observada la presencia de Concanavalina-A en comparación con la estimulación inducida por el A-23187 y el compuesto 48/80. En este modelo experimental se logró establecer relaciones entre la liberación de histamina con el consumo de glucosa, la produción de lactato y la carga adenílica bajo la forma de ATP

[Chemical and nutritional characteristics from grains of five genotypes of Canavalia ensiformis]. / Características químicas y nutricionales del grano de cinco (5) genotipos de Canavalia ensiformis.

This study evaluated the raw meals from grains of five genotypes of Canavalia ensiformis, by means of the chemical composition, the presence of antinutritional factors (Canavanine and hemaglutination activity) and in vitro protein digestibility. The genotypes studied were: Original, Yaracuy, Tovar, Valle de la Pascua and U-02. The results of chemical composition, showed significance difference between them, except moisture content, found the following average values: Protein 31.37%, fiber: 8.10%, ash: 2.93% and moisture: 11.68%. The canavanine content of the genotypes was variable oscillating between 2.02 and 4.86%, the genotype U-02 presented the higher value, respect to the hemaglutination title changed between: +2 and +5. The in vitro protein digestibility of the raw meals showed significance differences between the genotype, it changed between 47.51% and 51.84%, these values were lower than the casein (97.3%).

Aislamiento, purificación y caracterización de concanavalina A a partir de canavalia ensiformis / Isolation, purification and characterization of concanavalin A stemming from Canavalia ensiformis

La concanavalina A es una lectina con múltiples aplicaciones en Medicina, Biología, Hematología, Inmunología y en Bioquímica. Esta lectina representa una valiosa herramienta para el estudio, aislammiento y purificación de diversas proteínas. La concanavalina A se purificó por un método de absorción específica en sephadex G-50, a partir de Canavalia ensiformis cosechada en Cuba. El rendimiento obtenido fue de 1,56 porciento. El producto mostró una actividad hemoaglutinadora de eritrocitos humanos menor que 3,91 µg, y una actividad turbidimétrica de 1,75 mg. Se realizaron estudios de enfoque isoeléctrico y electroforético al producto purificado, y por ambos métodos resultó ser idéntico al de la firma FLOW. La connavallina A se acopló a sefarosa 4B activada con bromuro de cianógeno, y la matriz de afinidad obtenida se utilizó en la purificación de una hormona glicoproteíca marcada radiactivamente con yodo-125 con resultados satisfactorios

Myxo and paramyxovirus surface structures: antigenic and functional studies

Amostras de vírus da Doença de Newcastle (cepas SO-93, B1 e La Sota) e da Influenza (cepa A/PR/8/34 (H1N1) foram analisadas especificamente quanto à presença de carboidratos específicos, de comprovada importância no processamento das atividades biológicas virais. As amostras virais foram concentradas por sedimentaçäo, avaliadas quanto ao seu teor proteico e título hemaglutinante e, entäo, estudadas quanto aos seus resíduos carboidratos. O estudo realizado com uso de lectinas e açúcar específico (inibidor de Concanavalina A) revelou a importância e especificidade do caráter carboidratado de estruturas peptídicas de superfície viral, o que pode ser observado através da inibiçäo do aparecimento das linhas de precipitaçäo entre Concanavalina A e soro específico para vírus da Doença de Newcastle

[Flour from seeds of Canavalia ensiformis L (DC), raw, stored in an alkaline medium, autoclaved or extracted, in diets for growing pigs]. / Harina de granos de Canavalia ensiformis L (DC) cruda, almacenada en medio alcalino, autoclavada o extruída, en dietas para cerdos en crecimiento.

There were made four nutrition experiments using pork in growing process, with an approximate weight of 14.2 kilograms. In each experiment, it was made a substitution in equals parts of corn flour and soy flour for raw Canavalia flour or processed through alkaline storage, autoclaved or extrusion. In the first three experiments, the substitution level of raw Canavalia (RC), stored in alkaline environment (CAMA) or autoclave (CA) were: 0, 5, 10 and 15%. In the fourth one, the including levels of Canavalia Extruida were: 0, 7.5 and 15%. The raw Canavalia as the Extruida drastically reduced the pork's growth. The Canavalia flour autoclaved (121 degrees C/15 psi/90 min) substantially improved the animal answers, even though the growth in all the substitution levels were lower than the one observed in the original portion. The storage of the beans in alkaline environment, made possible better productive behavior in the animals, and it didn't observe differences (p < 0.01) to increase the witness weight, at the substitution level or 5%. The pork's answer to the Canavalia toxin was manifested in the first term, by a drastic reduction in the voluntary consumption of foods. As a whole, the results indicated that none of the methods used were effective to eliminate or to minimize the toxic effects in the raw Canavalia over the productive behavior of growing pork.

Concanavalin A binding sites in fetal, adult, transitional, and malignant rectosigmoid mucosa.

We studied mucin histochemistry in 25 rectosigmoid adenocarcinomas and in the transitional mucosa adjacent to these tumors using standard techniques for the detection of neutral and acid sialomucins and sulfomucins and the paradoxical concanavalin A (Con A) stain. This histochemical procedure selectively detects residues of mannose in glycoproteins exposed to brief steps of oxidation and reduction. Those techniques were also used to study histologically normal mucosa of specimens with carcinoma, normal rectosigmoid mucosa of patients without inflammatory or neoplastic bowel disease, hyperplastic rectal polyps, and rectosigmoid mucosa of human fetuses. Normal mucosa and hyperplastic polyps mainly contained sulfomucins and did not display Con A binding activity with any of the variants of the stain. In contrast, fetal, transitional, and malignant mucosa predominantly showed sialomucins and although not reactive with the standard Con A sequence, displayed binding activity for the lectin after short oxidative-reductive steps. These results provide further evidence that transitional and malignant mucosa produce markedly abnormal mucins whose histochemical patterns represent a re-emergence of the fetal type found during development. The principles of the paradoxic Con A reaction may be applied to unmask lectin binding activity in apparently unreactive sites.