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Introduction: In Mexico, there are two recorded living species of Cassiduloida: Cassidulus caribaearum and Rhyncholampas pacifica. Most of the taxonomic studies on cassiduloids have used external morphology, pedicellariae and morphometric characters; however, the intraspecific variation of quantitative and qualitative characters has been poorly evaluated. Objective: To compare the basic morphology of R. pacifica and C. caribaearum. Methods: We examined a total of 2 158 specimens of R. pacifica and C. caribaearum, selecting 50 to evaluate shape and size with linear regression and Principal Component analysis. We selected an additional 62 specimens per species to identify significant character correlations and morphological groups within species. Results: There is a direct relationship between Test length and Test width. Test height/Test width, and Total length (oral view)/Distance from the ambitus to the peristome apex, are the two main ratios to distinguish both species. C. caribaearum is more dorsoventrally compressed and has a round peristome base; versus R. pacifica has a tall and triangular one. There are four morphological groups of C. caribaearum and two groups for R. pacifica. Conclusions: These two species can be distinguished with reliable morphological characters, in which peristome shape suggests that R. pacifica is more adapted to burrowing deeper into certain types of substratum.
Introducción: En México, hay dos especies vivientes registradas de Cassiduloida: Cassidulus caribaearum y Rhyncholampas pacifica. La mayoría de los estudios taxonómicos sobre casiduloides han utilizado morfología externa, pedicelarios y caracteres morfométricos; sin embargo, la variación intraespecífica de caracteres cuantitativos y cualitativos ha sido poco evaluada. Objetivo: Comparar la morfología básica de R. pacifica y C. caribaearum. Métodos: Examinamos un total de 2 158 especímenes de R. pacifica y C. caribaearum, seleccionando 50 para evaluar la forma y el tamaño con regresión lineal y análisis de componentes principales. Seleccionamos 62 especímenes adicionales por especie para identificar correlaciones significativas de caracteres y grupos morfológicos dentro de las especies. Resultados: Existe una relación directa entre la longitud de la testa y el ancho de la testa. La Altura de la testa / Anchura de la testa y la Longitud total (vista oral) / Distancia desde el ambitus hasta el ápice del peristoma, son las dos proporciones principales para distinguir ambas especies. C. caribaearum está más comprimido dorsoventralmente y tiene una base del peristoma redonda; versus R. pacifica que tenía una alta y triangular. Hay cuatro grupos morfológicos de C. caribaearum y dos grupos de R. pacifica. Conclusiones: Estas dos especies se pueden distinguir con caracteres morfológicos confiables, en los que la forma del peristoma sugiere que R. pacifica está más adaptada para excavar más profundamente en ciertos tipos de sustratos.
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Animales , Estrellas de Mar/anatomía & histología , Equinodermos/clasificación , MéxicoRESUMEN
The production of olive oil is an important economic engine in the Mediterranean area. Nowadays, olive oil is obtained mainly by mechanical processes, by using the whole fruit as the primary raw material. Although the mesocarp is the main source of lipids contributing to olive oil formation, the seed also contributes to the olive oil composition and attributes. The olive seed is also becoming an interesting emerging material itself when obtained after alternative processing of the olive fruit. Such seed is used for the production of differential oil and a unique flour among other bioactive products, with increasing uses and applications in cosmetics, nutrition, and health. However, olive seed histology has been poorly studied to date. A complete description of its anatomy is described for the first time in the present study by using the 'Picual' cultivar as a model to study the development of the different tissues of the olive seed from 60 to 210 days after anthesis. A deep analysis of the seed coats, endosperm storage tissue and the embryo during their development has been performed. Moreover, a panel of other olive cultivars has been used to compare the weight contribution of the different tissues to the seed, seed weight variability and the number of seeds per fruit. In addition to the histological features, accumulation of seed storage proteins of the 7S-type (ß-conglutins) in the seed tissues has been assessed by both biochemical and immunocytochemical methods. These hallmarks will help to settle the basis for future studies related to the location of different metabolites along the olive seed and mesocarp development, and therefore helping to assess the appropriate ripening stage for different commercial and industrial purposes.
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Molecular evidence on the heterogeneity present in the Ole e 1 allergen of the olive pollen is emerging. Such polymorphism is dependent on the cultivar origin of pollen, which also determines wide differences in the expression of this protein. Determination of biochemical and molecular characteristics of Ole e 1 pollen allergen in two Iranian olive cultivars, namely 'Rowghani' and 'Zard' is necessary to assess their allergenicity potential. SDS-PAGE and immunoblotting analysis of pollen extracts showed that both cultivars present high and low expression of Ole e 1, respectively. These protein levels correlated with similarly different levels of transcripts, as determined by RT-PCR. Two-dimensional protein profiles also showed conspicuous differences in the distribution and the level of expression of those spots reacting to an anti-Ole e 1 antibody. Bioinformatic analysis of four Ole e 1 sequences corresponding to 'Rowghani' and two sequences for 'Zard', showed numerous heterogeneities when compared with those Ole e 1 and Ole e 1-like sequences present in databases. Nucleotide substitutions resulted in many cases in changes over the predicted amino acid sequences. A cladistic analysis of the sequences showed Iranian entries in a central position between West-European sequences, and Ole e 1-like sequences from other Oleaceae species. Moreover, amino acid changes affected key epitopes of the protein involved in the recognition of the protein by the human immune system. Putative implications of polymorphism in both the biological role and the allergic reactivity of Ole e 1 are discussed.
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Alérgenos/genética , Antígenos de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Olea/genética , Proteínas de Plantas/genética , Polen/genética , Polimorfismo Genético , Alérgenos/clasificación , Alérgenos/inmunología , Secuencia de Aminoácidos , Anticuerpos/química , Antígenos de Plantas/clasificación , Antígenos de Plantas/inmunología , Secuencia de Bases , Western Blotting , Humanos , Irán , Datos de Secuencia Molecular , Olea/clasificación , Olea/inmunología , Filogenia , Extractos Vegetales/química , Proteínas de Plantas/clasificación , Proteínas de Plantas/inmunología , Polen/clasificación , Polen/inmunología , ARN Mensajero/genética , ARN Mensajero/inmunología , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Standardization of pollen protein extracts is essential in order to ensure efficiency and safety in allergy diagnosis and immunotherapy. In this paper, we have optimized a multiplex Western blotting method for the simultaneous detection of four olive pollen allergens (Ole e 1, Ole e 2, Ole e 5, and Ole e 9) on a single blot using a monoclonal antibody from mouse and three polyclonal antibodies raised in rabbit. We utilized unconjugated Fab antibody fragments for blocking rabbit primary antibodies, and fluorescence-based detection. These changes allowed an accurate and reliable comparative quantitation of these allergens among pollen-protein samples from six olive cultivars. In addition, we also tested the IgE-binding capacity of these pollen extracts by reprobing the same blot with a pool of sera from eight patients allergic to olive and detection with enzyme conjugated antibodies. A noticeable variability regarding allergen content and IgE-reactivity was found among the olive cultivars analyzed. Moreover, we could easily confirm the identity of some of the IgE-binding proteins by simply overlapping both fluorescence and chemiluminescence images. This method is versatile since it can be applied to other allergogenic plant species and extended to other allergens.
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Alérgenos/análisis , Western Blotting/métodos , Proteínas de Plantas/análisis , Polen/inmunología , Alérgenos/química , Alérgenos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Ratones , Olea/química , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Polen/química , Conejos , Rinitis Alérgica Estacional/sangre , Pruebas Serológicas/métodos , Pruebas Serológicas/normasRESUMEN
The structural changes occurred in differentiating olive cotyledon cells into mesophyll cells are described. Using histological and immunocytological methods as well as microscopic observations, we showed that in the cells of mature embryo, large electron-dense proteins bodies (PBs) are surrounded by numerous oil bodies (OBs). After 3 days of in vitro germination, the presence of large PBs originated by fusion of smaller PBs was observed. It was also detected a close spatial proximity between PBs and OBs, likely as a reflection of interconnected metabolic pathways. Between the 3rd and the 12th day of germination, the formation of a large vacuolar compartment takes place accompanied by a decrease in the PBs and OBs number. This was coincident with a progressive decrease in the amount of the 11S-type seed storage proteins (SSPs), showed in situ and after Western blot analysis of crude protein extracts. After 26 days germination, the cellular organization became typical for a leaf mesophyll cell, with well-differentiated chloroplasts surrounding a large central vacuole. Our results suggest that the olive cotyledon storage reserves are mobilized gradually until the seedling becomes autotrophic. Moreover, the specific accumulation of storage proteins in the intravacuolar material suggests that these structures may operate as a shuttle for SSPs and/or products of their degradation into the cytoplasm, where finally they supply amino acids for the differentiating mesophyll cells.
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Cotiledón/citología , Germinación , Olea/crecimiento & desarrollo , Proteínas de Almacenamiento de Semillas/metabolismo , Plantones/crecimiento & desarrollo , Semillas/crecimiento & desarrollo , Western Blotting , Diferenciación Celular , Cloroplastos/metabolismo , Cotiledón/crecimiento & desarrollo , Cotiledón/metabolismo , Inmunohistoquímica , Células del Mesófilo/citología , Células del Mesófilo/metabolismo , Olea/citología , Olea/metabolismo , Células Vegetales/metabolismo , Plantones/citología , Plantones/metabolismo , Semillas/citología , Semillas/metabolismo , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Vacuolas/metabolismoRESUMEN
BACKGROUND: Pollens from different olive (Olea europaea L.) cultivars have been shown to differ significantly in their content in Ole e 1 and in their overall allergenicity. This allergen is, in addition, characterized by a high degree of polymorphism in its sequence. The purpose of this study is to evaluate the putative presence of divergences in Ole e 1 sequences from different olive cultivars. RESULTS: RNA from pollen individually collected from 10 olive cultivars was used to amplify Ole e 1 sequences by RT-PCR, and the sequences were analyzed by using different bioinformatics tools. Numerous nucleotide substitutions were detected throughout the sequences, many of which resulted in amino acid substitutions in the deduced protein sequences. In most cases variability within a single variety was much lower than among varieties. Key amino acid changes in comparison with "canonical" sequences previously described in the literature included: a) the substitution of C19-relevant to the disulphide bond structure of the protein-, b) the presence of an additional N-glycosylation motif, and c) point substitutions affecting regions of Ole e 1 already described like relevant for the immunogenicity/allergenicity of the protein. CONCLUSION: Varietal origin of olive pollen is a major factor determining the diversity of Ole e 1 variants. We consider this information of capital importance for the optimal design of efficient and safe allergen formulations, and useful for the genetic engineering of modified forms of the allergen among other applications.