Pedro Hernández

Chemical characterization of the lectin from Amaranthus leucocarpus syn. hypocondriacus by 2-D proteome analysis.

In this work, we characterized chemically the N-acetyl-D-galactosamine specific lectin from Amaranthus leucocarpus syn hypocondriacus lectin (ALL). It is a dimeric glycoprotein composed by three isoforms with pl at 4.8, 4.9, and 5.2. Circular dichroism analysis indicated that the secondary structure of ALL contains 45% of β-sheet and 5% of α-helix. Amino acid sequence of the purified lectin and its isoforms was determined from peptides obtained after trypsin digestion by MALDI-TOF (Matrix assisted laser desorption ionization-time of flight). The tryptic peptides prepared from the purified lectin and the three isoforms showed different degrees (80 to 83%) of identity with the amino acid sequence belonging to a previously described high nutritional value protein from A. hypocondriacus not shown at the time to be a lectin. Furthermore, analysis of tryptic peptides obtained from ALL previously treated with peptide N-glycosidase, revealed a 93% identity with the aforementioned protein. Presence of N-glycosidically linked glycans of the oligomannosidic type and, in minor proportion, of the N-acetyllactosaminic type glycans was determined by affinity chromatography on immobilized Con A.

A comparative study on the purification of the Amaranthus leucocarpus syn. hypocondriacus lectin.

Amaranthus leucocarpus lectin is a homodimeric glycoprotein of 35 kDa per sub-unit, which interacts specifically with N-acetyl-galactosamine. In this work, we compared different glycoproteins that contain Galbeta1-3 GalNAcalpha1-3 Ser/Thr or GalNAcalpha1-3 Ser/Thr in their structure as ligands to purify the A. leucocarpus lectin. From the glycoproteins tested, fetuin was the most potent inhibitor of the hemagglutinating activity and the better ligand for lectin purification; however, the use of desialylated stroma from erythrocytes represented the cheapest method to purify this lectin. O-linked glycans released from the glycoproteins used as affinity matrix and those from different erythrocytes were less inhibitory than parental glycoproteins. The NH2-terminal of the lectin is blocked; moreover, this is the only example of a lectin isolated from this genus to be a glycoprotein. Analysis of the glycoprotein sequences with inhibitory activity for the lectin, showed a different pattern in the O-glycosylation, which confirms that A. leucocarpus lectin recognizes conformation and, probably, distances among O-linked glycans moieties.

Use of Amaranthus leucocarpus Lectin to Differentiate Cervical Dysplasia (CIN)

Abstract Alterations in O‐glycosylation of proteins in cell surfaces can originate disorder in cellular function, as well as in cell transformation and tumoral differentiation. In this work, we investigate changes in O‐glycosylation in cervical intraepithelial dysplasia (CIN) at different stages of differentiation (CIN I, CIN II, and CIN III) using lectins specific for O‐glycosidically linked glycans. Twenty cases with CIN I, CIN II, and CIN III dysplasias each, and 20 normal cases were studied by lectin histochemistry and evaluated under optical microscopy. The lectins from Glycine max and Griffonia simplicifolia showed no differences in their recognition pattern among the different CIN stages and normal tissue. Dolichos Biflorus lectin recognized CIN I dysplasia. Lectin from Amaranthus leucocarpus showed increased reactivity in the presence of CIN II dysplasia, compared with CIN I and CIN III. These results suggest that subtle modifications in the O‐glycosylation pattern could be considered in diagnosis or prognosis of cervical precancerous stages.

O-glycosylation expression in fibroadenoma.

Fibroadenomas are human benign breast tumors characterized by proliferation of epithelial and stroma cells of the terminal ductal unit. Expression of O-glycans seems to contribute to the proliferation and transformation events. With this in mind, we evaluated the expression of glycans in fibroadenoma tissue through immunohistochemistry with antibodies against mucin epitopes (Anti CA15-3 and MUC1), as well as with lectins specific for glycans linked to proteins or lipids, and we compared findings with healthy breast specimens. Our results show positive expression of CA15-3 and MUC1 in fibroadenoma tissue, mainly in duct and stroma cells, whereas, in normal samples, staining was observed in duct cells. The lectin from Glycine max recognized equally well duct and stroma cells; this was the only lectin showing co-localization with anti-CA15-3 in healthy and tumor tissues. Dolichos biflorus, Artocarpus integrifolia, and Griffonia simplicifolia lectins recognized duct cells in control healthy tissues as well as in fibroadenoma tissue. The lectin from Amaranthus leucocarpus recognized only duct cells in control samples, whereas, in fibroadenoma tissue, it recognized duct and some stromal cells, suggesting that O-glycans-type mucin linked to proteins and mucin participate in the development of fibroadenomas.

Expression of antigen tf and galectin-3 in fibroadenoma

BackgroundFibroadenomas are benign human breast tumors, characterized by proliferation of epithelial and stromal components of the terminal ductal unit. They may grow, regress or remain unchanged, as the hormonal environment of the patient changes. Expression of antigen TF in mucin or mucin-type glycoproteins and of galectin-3 seems to contribute to proliferation and transformations events; their expression has been reported in ductal breast cancer and in aggressive tumors.FindingsLectin histochemistry, immunohistochemistry, and immunofluorescence were used to examine the expression and distribution of antigen TF and galectin-3. We used lectins from Arachis hypogaea, Artocarpus integrifolia, and Amaranthus lecuocarpus to evaluate TF expression and a monoclonal antibody to evaluate galectin-3 expression. We used paraffin-embedded blocks from 10 breast tissues diagnosed with fibroadenoma and as control 10 healthy tissue samples. Histochemical and immunofluorescence analysis showed positive expression of galectin-3 in fibroadenoma tissue, mainly in stroma, weak interaction in ducts was observed; whereas, in healthy tissue samples the staining was also weak in ducts. Lectins from A. leucocarpus and A. integrifolia specificaly recognized ducts in healthy breast samples, whereas the lectin from A. hypogaea recognized ducts and stroma. In fibroadenoma tissue, the lectins from A. integrifolia, A. Hypogaea, and A. leucocarpus recognized mainly ducts.ConclusionsOur results suggest that expression of antigen TF and galectin-3 seems to participate in fibroadenoma development.

GLYCOSYLATION PATTERN IN THE APPENDIX TESTIS IN CHILDREN WITH CRYPTORCHIDISM

In humans, at about week 6, sex cords develop within the forming testes. Testes normally descend to the scrotum; cryptorchidism occurs when one or two testes do not descend to scrotum and in some case are accompanied by the appendix testis. The appendix testis is a small sessile or polypoid structure located at the antero superior pole of the testis, adjacent to the head of the epididymis. Glycans can be involved in development of the appendix testis and cryptorchidism. In this work, lectin histochemistry was used to evaluate glycans expression in appendix testis in children with cryptorchidism. Our results showed that lectin from Lens culinaris, Ulex europaeus I., Canavalia ensiformis, Artocarpus integrifolia, Glycine max, and Griffonia simplicifolia recognizes epithelial and estromal cells. Not interaction was observed with lectin from Amaranthus leucocarpus, while lectin from Dolichus biflorus lectin only recognizes epithelial cells. Our results suggest that O-glycans linked in some glycoproteins represent important elements in appendix testis development.

Higroma quístico cervical en el primer trimestre. Resultados perinatales

Resumen Objetivos Valorar la evolucion antenatal y perinatal de fetos con diagnostico de higroma quistico cervical. Material y metodos Se estudio a 33 gestantes con diagnostico de higroma quistico cervical fetal. A las mujeres que decidieron seguir el embarazo se les realizo estudio cromosomico, valoracion morfologica y seguimiento del higroma. Resultados En nuestra casuistica, encontramos una evidente asociacion con cromosomopatia (7 [37%] de 19 fetos, con cariotipo confirmado), aunque probablemente el numero de fetos con anomalias cromosomicas sea mayor. Por el contrario, unicamente 10 (30%) gestaciones con el diagnostico de higroma quistico cervical tuvieron un resultado perinatal satisfactorio. Conclusiones En el caso del higroma quistico cervical, su relativamente facil diagnostico, la extension de los estudios ecograficos y su asociacion frecuente a cromosomopatias conducen a la mayoria de las parejas a la interrupcion voluntaria del embarazo; no obstante, si se hace el diagnostico de ausencia de anomalias cromosomicas, ausencia de malformaciones y regresion, se podria considerar un resultado perinatal satisfactorio.