Sonia Morales

Characterization of Profilin Polymorphism in Pollen with a Focus on Multifunctionality

Profilin, a multigene family involved in actin dynamics, is a multiple partners-interacting protein, as regard of the presence of at least of three binding domains encompassing actin, phosphoinositide lipids, and poly-L-proline interacting patches. In addition, pollen profilins are important allergens in several species like Olea europaea L. (Ole e 2), Betula pendula (Bet v 2), Phleum pratense (Phl p 12), Zea mays (Zea m 12) and Corylus avellana (Cor a 2). In spite of the biological and clinical importance of these molecules, variability in pollen profilin sequences has been poorly pointed out up until now. In this work, a relatively high number of pollen profilin sequences have been cloned, with the aim of carrying out an extensive characterization of their polymorphism among 24 olive cultivars and the above mentioned plant species. Our results indicate a high level of variability in the sequences analyzed. Quantitative intra-specific/varietal polymorphism was higher in comparison to inter-specific/cultivars comparisons. Multi-optional posttranslational modifications, e.g. phosphorylation sites, physicochemical properties, and partners-interacting functional residues have been shown to be affected by profilin polymorphism. As a result of this variability, profilins yielded a clear taxonomic separation between the five plant species. Profilin family multifunctionality might be inferred by natural variation through profilin isovariants generated among olive germplasm, as a result of polymorphism. The high variability might result in both differential profilin properties and differences in the regulation of the interaction with natural partners, affecting the mechanisms underlying the transmission of signals throughout signaling pathways in response to different stress environments. Moreover, elucidating the effect of profilin polymorphism in adaptive responses like actin dynamics, and cellular behavior, represents an exciting research goal for the future.

Polymorphisms in the transforming growth factor‐β1 gene (TGF‐β1) and the risk of advanced alcoholic liver disease

Abstract: Background/aims: There are wide interindividual differences in the risk of developing alcoholic cirrhosis. Transforming growth factor β1 (TGF‐β1) is the main cytokine involved in liver fibrogenesis. The TGF‐β1 gene is polymorphic at several sites and these polymorphisms are probably related to differences in the rate of TGF‐β1 synthesis. Our aim has been to analyse the influence of the TGF‐β1 gene polymorphisms in the predisposition to advanced alcoholic liver disease (ALD) in ethanol abusers.

Olive pollen profilin (Ole e 2 allergen) co-localizes with highly active areas of the actin cytoskeleton and is released to the culture medium during in vitro pollen germination

Pollen allergens offer a dual perspective of study: some of them are considered key proteins for pollen physiology, but they are also able to trigger allergy symptoms in susceptible humans after coming in contact with their tissues. Profilin (Ole e 2 allergen) has been characterized, to some extent, as one of the major allergens from Olea europaea L. pollen, a highly allergenic species in the Mediterranean countries. In order to obtain clues regarding the biological role of this protein, we have analyzed both its cellular localization and the organization of actin throughout pollen hydration and early pollen tube germination. The localization of the cited proteins was visualized by confocal laser scanning microscopy immunofluorescence using different antibodies. Upon pollen hydration and pollen germination, a massive presence of profilin was detected close to the site of pollen tube emergence, forming a ring‐like structure around the ‘effective’ apertural region. Profilin was also detected in the pollen exine of the germinating pollen grains and in the germination medium. After using a permeabilization‐enhanced protocol for immunolocalization, profilin was also localized in the cytoplasm of the pollen tube, particularly at both the proximal and apical ends. Noticeable accumulations of actin were observed in the cytoplasm of the pollen tube; particularly, in both the apical region and the area immediately close to the aperture. Actin filaments were not observed, probably due to the need of further enhanced fixation procedures. The ultrastructural localization of profilin showed the presence of the protein in the cytoplasm of both the mature pollen grain and the pollen tube.

Nicotinamide Inhibits the Lysosomal Cathepsin b-like Protease and Kills African Trypanosomes

Background: Nicotinamide is a soluble compound of the vitamin B3 group with antimicrobial activity. Results: Nicotinamide causes disruption of the lysosome and inhibits the cathepsin b-like enzyme, an essential lysosomal protease in trypanosomatids. Conclusion: Nicotinamide kills African trypanosomes by targeting the cathepsin b-like protease. Significance: These results demonstrate for the first time the inhibitory effect of nicotinamide on a protease. Nicotinamide, a soluble compound of the vitamin B3 group, has antimicrobial activity against several microorganisms ranging from viruses to parasite protozoans. However, the mode of action of this antimicrobial activity is unknown. Here, we investigate the trypanocidal activity of nicotinamide on Trypanosoma brucei, the causative agent of African trypanosomiasis. Incubation of trypanosomes with nicotinamide causes deleterious defects in endocytic traffic, disruption of the lysosome, failure of cytokinesis, and, ultimately, cell death. At the same concentrations there was no effect on a cultured mammalian cell line. The effects on endocytosis and vesicle traffic were visible within 3 h and can be attributed to inhibition of lysosomal cathepsin b-like protease activity. The inhibitory effect of nicotinamide was confirmed by a direct activity assay of recombinant cathepsin b-like protein. Taken together, these data demonstrate that inhibition of the lysosomal protease cathepsin b-like blocks endocytosis, causing cell death. In addition, these results demonstrate for the first time the inhibitory effect of nicotinamide on a protease.

Pentosidina: un nuevo biomarcador de las complicaciones en la diabetes mellitus

Diabetes mellitus causes an increase of morbidity and mortality. Advanced glycosilation end products (AGE) are formed by non-enzymatic glycation between proteins and reducing sugars as glucose. Oxidative reactions (glycoxidations) are essential for the formation of some AGE, for example pentosidine. Increased concentrations of pentosidine can be found in pathological conditions associated with hyperglycaemia and also related to increased oxidative stress. In individuals with diabetes mellitus, pentosidine formation and accumulation is developed at an accelerated rate in cells without insulin control for glucose uptake. Pentosidine has a pivotal role in diabetic complications, probably as a consequence of the diverse properties of this compound, which alters the structure and function of molecules in biological systems. The following review discusses the alterations in the concentration of pentosidine in the body, particularly in relation to changes occurring in diabetes and its complications such as vascular and bone disease, nephropathy, neuropathy and retinopathy. Novel therapeutic approaches which can prevent or ameliorate the toxic effects of AGE in the initiation and progression of diabetic complications are reviewed.

A novel multiplex method for the simultaneous detection and relative quantitation of pollen allergens

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.

Pollen Allergenicity is Highly Dependent on the Plant Genetic Background: The “Variety”/“Cultivar” Issues

© 2012 de Dios Alche et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pollen Allergenicity is Highly Dependent on the Plant Genetic Background: The “Variety”/“Cultivar” Issues

Differential Immune-Reactivity and Subcellular Distribution Reveal the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism

© 2012 Jimenez-Lopez et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Differential Immune-Reactivity and Subcellular Distribution Reveal the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism

Clustering of Olive Pollens into Model Cultivars on the Basis of Their Allergenic Content

This work was supported by the Spanish Ministry of Science and Innovation (MICINN) (ERDF-cofinanced projects AGL2008-00517, BFU2011-22779 and PIE-200840I186) and the Junta de Andalucia (ERDF-cofinanced projects P2010-CVI5767 and P2010-AGR6274).

Involvement of Climatic Factors in the Allergen Expression in Olive Pollen

Olive allergen concentrations in the pollen grain are critical in allergic response of atopic patients. Existing evidence shows that climatic factors can influence pollen allergen content. To date, the influences of temperature and precipitations over the content of birch and olive pollens in their respective major allergens have been analyzed. Bet v 1, the major allergen of birch pollen, increased its expression (Buters et al. 2008) and presented a higher allergenicity (Ahlholm et al. 1998) while temperatures were elevated. Differently, the content in the major olive pollen allergen Ole e 1 showed no apparent correlation with either temperatures or precipitations (Fernandez Caldas et al. 2007). However, a positive correlation between total allergenicity and rainfall occurring in winter months was found. This correlation was not analyzed independently for each pollen allergen.