1-L polyethylene glycol + ascorbic acid versus sodium picosulfate + magnesium citrate bowel preparations for colonoscopy: effectiveness and safety

Background: adequate bowel preparation is crucial for the protective effect of colonoscopy. Commonly used preparation regimens like polyethylene glycol (PEG) or sodium picosulfate with magnesium citrate (SPMC) have shown similar results in clinical trials, but low-volume PEG + ascorbic acid (1-L PEG + ASC) versus SPMC have never been compared in a real-life setting. Aim: to evaluate the effectiveness and safety of 1-L PEG + ASC versus SPMC in a real-life setting for the overall population, for patients aged ≥ 65 years, and males versus females. Methods: out-patients aged ≥ 18 years who underwent colonoscopy for any indication were randomly assigned to the 1-L PEG + ASC or SPMC group. Using the Boston Bowel Preparation Scale (BBPS), the primary endpoints were the bowel cleansing success of the overall colon and right colon, as well as high-quality (HQ) cleansing. Furthermore, the effectiveness and safety outcomes for age groups and males versus females were compared. Results: 1-L PEG + ASC showed significantly better bowel cleansing success than SPMC. Particularly remarkable is the HQ cleansing reached with 1-L PEG + ASC compared with SPMC (55.5 % versus 25.4 % in the overall colon, and 58.7 % versus 27.2 % in the right colon). 1-L PEG + ASC was equally effective for men and women while SPMC showed significant differences between genders (men had worse bowel cleansing). Age did not affect the cleansing effectiveness. 1-L PEG + ASC versus SPMC showed significant differences in tolerance and safety; women also had significantly worse tolerance than men for both solutions, but these did not affect the quality of bowel cleansing. Conclusions: in our real-life setting, 1-L PEG + ASC offered better adequate and HQ bowel cleansing than SPMC, achieving excellent cleansing quality, regardless of gender or tolerance. (AU)

Allogeneic Adipose-Derived Mesenchymal Stem Cells (Horse Allo 20) for the Treatment of Osteoarthritis-Associated Lameness in Horses: Characterization, Safety, and Efficacy of Intra-Articular Treatment.

Osteoarthritis commonly causes lameness in the horse and has a great impact in performance animals. Due to the limitations of current medical therapies, allogenic mesenchymal stem cells (MSCs) may become an alternative method to control inflammation, reduce tissue damage and pain, and therefore improve lameness. We present the results of a regulatory clinical trial testing adipose-derived MSCs (Horse Allo 20) in veterinary (Agencia Española del Medicamento y Productos Sanitarios, Spanish Medicines Agency, Reference number 325/ECV) involving a total number of 80 participants and with 90 days of follow-up period. The manufacturing process of Horse Allo 20 was robust with no influence of the adipose tissue donor (gender, age, or breed), sample origin (intraperitoneal or subcutaneous), or storage conditions (fresh vs. frozen product presentations) on the quality, safety, and efficacy of the drug product. An in vivo safety study showed that local and systemic tolerance was safe even after repeated intra-articular administration (three injections). An in vivo efficacy study demonstrated the efficacy of the treatment after one or two injections by a reduction in lameness (P < 0.05) for an extended period of time (90 days), decreasing the need for prolonged local and/or systemic anti-inflammatory therapies and their well-known deleterious effects and toxicities.

Erradicación de la infección por Helicobacter pylori con una nueva terapia cuádruple basada en bismuto en la práctica clínica / Eradication of Helicobacter pylori infection with a new bismuth-based quadruple therapy in clinical practice

Introducción: La erradicación de la infección por Helicobacter pylori representa un desafío clínico. Objetivo: Evaluar la eficacia y seguridad de la terapia cuádruple con esomeprazol más una cápsula 3 en 1 que contiene subcitrato de bismuto, metronidazol y tetraciclina, más probióticos en pacientes diagnosticados de infección por H. pylori en la práctica clínica habitual. Métodos: Estudio prospectivo, intervencional, unicéntrico y abierto realizado en pacientes consecutivos con indicación confirmada de erradicación de infección por H. pylori. Los pacientes fueron tratados con 3 cápsulas de Pylera(R) 4veces al día (desayuno, comida, merienda y cena), más 40mg de esomeprazol, 2veces al día durante 10días (30min antes de desayuno y cena) y probióticos durante 30días. La erradicación de la infección por H. pylori se confirmó mediante la prueba del aliento con urea marcada realizada al menos 28días después del final del tratamiento. Resultados: Un total de 100 pacientes fueron incluidos consecutivamente. Veinticinco (25,0%) pacientes tenían historia previa de tratamiento de su infección por H. pylori. En la población por intención de tratar, las tasas de erradicación fueron del 90,7% (68/75) y del 80,0% (20/25) en los pacientes tratados con Pylera(R) como primera línea o como terapia de rescate, respectivamente. Dieciocho pacientes (18%) presentaron, al menos, un acontecimiento adverso, la mayoría (89%) leves. Conclusión: Diez días de tratamiento con un régimen cuádruple de bismuto, metronidazol y tetraciclina más esomeprazol y probióticos es una estrategia eficaz y segura en pacientes con infección por H. pylori (AU)

Introduction: The eradication of Helicobacter pylori infection represents a clinical challenge. Objective: To evaluate the efficacy and safety of quadruple therapy with esomeprazole plus a 3-in-1 capsule containing bismuth subcitrate, metronidazole and tetracycline, plus probiotics in patients diagnosed with H. pylori infection in routine clinical practice. Methods: A prospective, interventional, single-centre and open-label study in consecutive patients with a confirmed indication for eradication of H. pylori infection. Patients were treated with three capsules of Pylera(R) four times a day (breakfast, lunch, afternoon snack and dinner), plus 40mg of esomeprazole twice daily for 10 days (30min before breakfast and dinner) and probiotics for 30 days. Eradication of H. pylori infection was confirmed by labelled urea breath test performed at least 28 days after the end of treatment. Results: A total of 100 patients were consecutively enrolled. Twenty-five patients (25.0%) had a prior history of treatment for their H. pylori infection. In the intention-to-treat population, eradication rates were 90.7% (68/75) and 80.0% (20/25) in patients treated with Pylera(R) as the first line or as rescue therapy, respectively. Eighteen patients (18%) had at least one adverse event, most of which (89%) were mild. Conclusion: Ten days of treatment with a quadruple regimen of bismuth, metronidazole and tetracycline plus esomeprazole and probiotics is an effective and safe strategy in patients with H. pyloriinfection (AU)

Eradication of Helicobacter pylori infection with a new bismuth-based quadruple therapy in clinical practice. / Erradicación de la infección por Helicobacter pylori con una nueva terapia cuádruple basada en bismuto en la práctica clínica.

INTRODUCTION: The eradication of Helicobacter pylori infection represents a clinical challenge. OBJECTIVE: To evaluate the efficacy and safety of quadruple therapy with esomeprazole plus a 3-in-1 capsule containing bismuth subcitrate, metronidazole and tetracycline, plus probiotics in patients diagnosed with H. pylori infection in routine clinical practice. METHODS: A prospective, interventional, single-centre and open-label study in consecutive patients with a confirmed indication for eradication of H. pylori infection. Patients were treated with three capsules of Pylera® four times a day (breakfast, lunch, afternoon snack and dinner), plus 40mg of esomeprazole twice daily for 10 days (30min before breakfast and dinner) and probiotics for 30 days. Eradication of H. pylori infection was confirmed by labelled urea breath test performed at least 28 days after the end of treatment. RESULTS: A total of 100 patients were consecutively enrolled. Twenty-five patients (25.0%) had a prior history of treatment for their H. pylori infection. In the intention-to-treat population, eradication rates were 90.7% (68/75) and 80.0% (20/25) in patients treated with Pylera® as the first line or as rescue therapy, respectively. Eighteen patients (18%) had at least one adverse event, most of which (89%) were mild. CONCLUSION: Ten days of treatment with a quadruple regimen of bismuth, metronidazole and tetracycline plus esomeprazole and probiotics is an effective and safe strategy in patients with H. pylori infection.

The Pollen Coat Proteome: At the Cutting Edge of Plant Reproduction.

The tapetum is a single layer of secretory cells which encloses the anther locule and sustains pollen development and maturation. Upon apoptosis, the remnants of the tapetal cells, consisting mostly of lipids and proteins, fill the pits of the sculpted exine to form the bulk of the pollen coat. This extracellular matrix forms an impermeable barrier that protects the male gametophyte from water loss and UV light. It also aids pollen adhesion and hydration and retains small signaling compounds involved in pollen-stigma communication. In this study, we have updated the list of the pollen coat's protein components and also discussed their functions in the context of sexual reproduction.

Olive seed protein bodies store degrading enzymes involved in mobilization of oil bodies.

The major seed storage reserves in oilseeds are accumulated in protein bodies and oil bodies, and serve as an energy, carbon, and nitrogen source during germination. Here, the spatio-temporal relationships between protein bodies and several key enzymes (phospholipase A, lipase, and lipoxygenase) involved in storage lipid mobilization in cotyledon cells was analysed during in vitro seed germination. Enzyme activities were assayed in-gel and their cellular localization were determined using microscopy techniques. At seed maturity, phospholipase A and triacylglycerol lipase activities were found exclusively in protein bodies. However, after seed imbibition, these activities were shifted to the cytoplasm and the surface of the oil bodies. The activity of neutral lipases was detected by using α-naphthyl palmitate and it was associated mainly with protein bodies during the whole course of germination. This pattern of distribution was highly similar to the localization of neutral lipids, which progressively appeared in protein bodies. Lipoxygenase activity was found in both the protein bodies and on the surface of the oil bodies during the initial phase of seed germination. The association of lipoxygenase with oil bodies was temporally correlated with the appearance of phospholipase A and lipase activities on the surface of oil bodies. It is concluded that protein bodies not only serve as simple storage structures, but are also dynamic and multifunctional organelles directly involved in storage lipid mobilization during olive seed germination.

A protocol for protein extraction from lipid-rich plant tissues suitable for electrophoresis.

Plant tissues contain high levels of nonprotein contaminants such as lipids, phenolic compounds, and polysaccharides among others, which interfere with protein extraction and electrophoretic separation. Preparation of good-quality protein extracts is a critical issue for successful electrophoretic analysis. Here, we describe a three-step method for protein extraction from lipid-rich plant tissues, which is suitable for both 1-D and 2-D electrophoresis and is compatible with downstream applications. The protocol includes prefractionation, filtration, and TCA/acetone precipitation steps prior to protein resolubilization.

Proteomics profiling reveals novel proteins and functions of the plant stigma exudate.

Proteomic analysis of the stigmatic exudate of Lilium longiflorum and Olea europaea led to the identification of 51 and 57 proteins, respectively, most of which are described for the first time in this secreted fluid. These results indicate that the stigmatic exudate is an extracellular environment metabolically active, participating in at least 80 different biological processes and 97 molecular functions. The stigma exudate showed a markedly catabolic profile and appeared to possess the enzyme machinery necessary to degrade large polysaccharides and lipids secreted by papillae to smaller units, allowing their incorporation into the pollen tube during pollination. It may also regulate pollen-tube growth in the pistil through the selective degradation of tube-wall components. Furthermore, some secreted proteins were involved in pollen-tube adhesion and orientation, as well as in programmed cell death of the papillae cells in response to either compatible pollination or incompatible pollen rejection. Finally, the results also revealed a putative cross-talk between genetic programmes regulating stress/defence and pollination responses in the stigma.

Somatic MYH7, MYBPC3, TPM1, TNNT2 and TNNI3 mutations in sporadic hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a clinically heterogeneous genetic heart disease characterized by left ventricular hypertrophy in the absence of another disease that could explain the wall thickening. Elucidation of the genetic basis of HCM lead to the identification of several genes encoding sarcomeric proteins, such as MYH7, MYBPC3, TPM1, TNNT2, and TNNI3. Sarcomeric genes are mutated in approximately 40% of HCM patients and a possible explanation for the incomplete yield of mutation-positive HCM may be somatic mutations. METHODS AND RESULTS: We studied 104 unrelated patients with non-familial HCM. Patients underwent clinical evaluation and mutation screening of 5 genes implicated in HCM (MYH7, MYBPC3, TPM1, TNNT2, and TNNI3) in genomic DNA isolated from resected cardiac tissue; 41 of 104 were found to carry a mutation, but as several patients carried the same mutations, the total amount of different mutations was 37; 20 of these mutations have been previously described, and pathogenicity has been assessed. To determine the effect of the 17 new mutations an in silico assay was performed and it predicted that 4 variants were damaging mutations. All identified variants were also seen in the DNA isolated from the corresponding blood, which demonstrated the absence of somatic mutations. CONCLUSIONS: Somatic mutations in MYH7, MYBPC3, TPM1, TNNT2, and TNNI3 do not represent an important etiologic pathway in HCM.

Electrophoretic profiling and immunocytochemical detection of pectins and arabinogalactan proteins in olive pollen during germination and pollen tube growth.

BACKGROUND AND AIMS: Cell wall pectins and arabinogalactan proteins (AGPs) are important for pollen tube growth. The aim of this work was to study the temporal and spatial dynamics of these compounds in olive pollen during germination. METHODS: Immunoblot profiling analyses combined with confocal and transmission electron microscopy immunocytochemical detection techniques were carried out using four anti-pectin (JIM7, JIM5, LM5 and LM6) and two anti-AGP (JIM13 and JIM14) monoclonal antibodies. KEY RESULTS: Pectin and AGP levels increased during olive pollen in vitro germination. (1 → 4)-ß-d-Galactans localized in the cytoplasm of the vegetative cell, the pollen wall and the apertural intine. After the pollen tube emerged, galactans localized in the pollen tube wall, particularly at the tip, and formed a collar-like structure around the germinative aperture. (1 → 5)-α-l-Arabinans were mainly present in the pollen tube cell wall, forming characteristic ring-shaped deposits at regular intervals in the sub-apical zone. As expected, the pollen tube wall was rich in highly esterified pectic compounds at the apex, while the cell wall mainly contained de-esterified pectins in the shank. The wall of the generative cell was specifically labelled with arabinans, highly methyl-esterified homogalacturonans and JIM13 epitopes. In addition, the extracellular material that coated the outer exine layer was rich in arabinans, de-esterified pectins and JIM13 epitopes. CONCLUSIONS: Pectins and AGPs are newly synthesized in the pollen tube during pollen germination. The synthesis and secretion of these compounds are temporally and spatially regulated. Galactans might provide mechanical stability to the pollen tube, reinforcing those regions that are particularly sensitive to tension stress (the pollen tube-pollen grain joint site) and mechanical damage (the tip). Arabinans and AGPs might be important in recognition and adhesion phenomena of the pollen tube and the stylar transmitting cells, as well as the egg and sperm cells.

Differential expression and sequence polymorphism of the olive pollen allergen Ole e 1 in two Iranian cultivars.

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.

New insights into the early steps of oil body mobilization during pollen germination.

In some plants, pollen grains accumulate storage lipids that serve as energy supply during germination. Here, three enzymes involved in early steps of oil body mobilization in the male gametophyte were functionally characterized for the first time. The effect of extracellular sugars on pollen performance and oil body dynamics was also analysed. Olive pollen oil bodies showed phospholipase A, lipase, and lipoxygenase activities on their surface. Enzyme activity levels increased during germination with a maximum after 3h. Removal of extracellular sugars from the germination medium did not affect pollen performance but increased enzyme activity rates and sped up oil body mobilization. Inhibitors seriously hampered pollen germination and pollen tube growth, leading to a characteristic accumulation of oil bodies in the germinative aperture. It can be concluded that storage lipids are sufficient for proper olive pollen germination. A lipase and a lipoxygenase are likely involved in oil body mobilization. Extracellular sugars may modulate their function, while a phospholipase A may promote their access to the storage lipids.

Cellular localization and levels of pectins and arabinogalactan proteins in olive (Olea europaea L.) pistil tissues during development: implications for pollen-pistil interaction.

Cell wall components in the pistil are involved in cell-cell recognition, nutrition and regulation of pollen tube growth. The aim of this work was to study the level, whole-organ distribution, and subcellular localization of pectins and arabinogalactan proteins (AGPs) in the olive developing pistil. Western blot analyses and immunolocalization with fluorescence and electron microscopy were carried out using a battery of antibodies recognizing different types of pectin epitopes (JIM7, JIM5, LM5, and LM6) and one anti-AGPs antibody (JIM13). In the olive pistil, highest levels of acid esterified and de-esterified pectins were observed at pollination. Moreover, pollination was accompanied by a slight decrease of the galactose-rich pectins pool, whereas arabinose-rich pectins were more abundant at that time. An increased expression of AGPs was also observed during pollination, in comparison to the pistil at the pre-anthesis stage. After pollination, the levels of pectins and AGPs declined significantly. Inmunofluorescence localization of pectins showed their different localization in the olive pistil. Pectins with galactose residues were located mainly in the cortical zones of the pistil, similar to the neutral pectins, which were found in the parenchyma and epidermis. In turn, the neutral pectins, which contain arabinose residues and AGPs, were localized predominantly in the stigmatic exudate, in the cell wall of secretory cells of the stigma, as well as in the transmitting tissue of the pistil during the pollination period. The differences in localization of pectins and AGPs are discussed in relation to their roles during olive pistil developmental course.

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination.

BACKGROUND AND AIMS: A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized. METHODS: The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods. KEY RESULTS: Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles. CONCLUSIONS: In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.

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.

Whole-organ analysis of calcium behaviour in the developing pistil of olive (Olea europaea L.) as a tool for the determination of key events in sexual plant reproduction.

BACKGROUND: The pistil is a place where multiple interactions between cells of different types, origin, and function occur. Ca(2+) is one of the key signal molecules in plants and animals. Despite the numerous studies on Ca(2+) signalling during pollen-pistil interactions, which constitute one of the main topics of plant physiology, studies on Ca(2+) dynamics in the pistil during flower formation are scarce. The purpose of this study was to analyze the contents and in situ localization of Ca(2+) at the whole-organ level in the pistil of olive during the whole course of flower development. RESULTS: The obtained results showed significant changes in Ca(2+) levels and distribution during olive pistil development. In the flower buds, the lowest levels of detectable Ca(2+) were observed. As flower development proceeded, the Ca(2+) amount in the pistil successively increased and reached the highest levels just after anther dehiscence. When the anthers and petals fell down a dramatic but not complete drop in calcium contents occurred in all pistil parts. In situ Ca(2+) localization showed a gradual accumulation on the stigma, and further expansion toward the style and the ovary after anther dehiscence. At the post-anthesis phase, the Ca(2+) signal on the stigmatic surface decreased, but in the ovary a specific accumulation of calcium was observed only in one of the four ovules. Ultrastructural localization confirmed the presence of Ca(2+) in the intracellular matrix and in the exudate secreted by stigmatic papillae. CONCLUSIONS: This is the first report to analyze calcium in the olive pistil during its development. According to our results in situ calcium localization by Fluo-3 AM injection is an effective tool to follow the pistil maturity degree and the spatial organization of calcium-dependent events of sexual reproduction occurring in developing pistil of angiosperms. The progressive increase of the Ca(2+) pool during olive pistil development shown by us reflects the degree of pistil maturity. Ca(2+) distribution at flower anthesis reflects the spatio-functional relationship of calcium with pollen-stigma interaction, progamic phase, fertilization and stigma senescence.

Characterization of a caleosin expressed during olive (Olea europaea L.) pollen ontogeny.

BACKGROUND: The olive tree is an oil-storing species, with pollen being the second most active site in storage lipid biosynthesis. Caleosins are proteins involved in storage lipid mobilization during seed germination. Despite the existence of different lipidic structures in the anther, there are no data regarding the presence of caleosins in this organ to date. The purpose of the present work was to characterize a caleosin expressed in the olive anther over different key stages of pollen ontogeny, as a first approach to unravel its biological function in reproduction. RESULTS: A 30 kDa caleosin was identified in the anther tissues by Western blot analysis. Using fluorescence and transmission electron microscopic immunolocalization methods, the protein was first localized in the tapetal cells at the free microspore stage. Caleosins were released to the anther locule and further deposited onto the sculptures of the pollen exine. As anthers developed, tapetal cells showed the presence of structures constituted by caleosin-containing lipid droplets closely packed and enclosed by ER-derived cisternae and vesicles. After tapetal cells lost their integrity, the caleosin-containing remnants of the tapetum filled the cavities of the mature pollen exine, forming the pollen coat. In developing microspores, this caleosin was initially detected on the exine sculptures. During pollen maturation, caleosin levels progressively increased in the vegetative cell, concurrently with the number of oil bodies. The olive pollen caleosin was able to bind calcium in vitro. Moreover, PEGylation experiments supported the structural conformation model suggested for caleosins from seed oil bodies. CONCLUSIONS: In the olive anther, a caleosin is expressed in both the tapetal and germ line cells, with its synthesis independently regulated. The pollen oil body-associated caleosin is synthesized by the vegetative cell, whereas the protein located on the pollen exine and its coating has a sporophytic origin. The biological significance of the caleosin in the reproductive process in species possessing lipid-storing pollen might depend on its subcellular emplacement. The pollen inner caleosin may be involved in OB biogenesis during pollen maturation. The protein located on the outside might rather play a function in pollen-stigma interaction during pollen hydration and germination.

Development of the cotyledon cells during olive (Olea europaea L.) in vitro seed germination and seedling growth.

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.

Screening mutations in myosin binding protein C3 gene in a cohort of patients with Hypertrophic Cardiomyopathy.

BACKGROUND: MyBPC3 mutations are amongst the most frequent causes of hypertrophic cardiomyopathy, however, its prevalence varies between populations. They have been associated with mild and late onset disease expression. Our objectives were to establish the prevalence of MyBPC3 mutations and determine their associated clinical characteristics in our patients. METHODS: Screening by Single Strand Conformation Polymorphisms (SSCP) and sequencing of the fragments with abnormal motility of the MyBPC3 gene in 130 unrelated consecutive HCM index cases. Genotype-Phenotype correlation studies were done in positive families. RESULTS: 16 mutations were found in 20 index cases (15%): 5 novel [D75N, V471E, Q327fs, IVS6+5G>A (homozygous), and IVS11-9G>A] and 11 previously described [A216T, R495W, R502Q (2 families), E542Q (3 families), T957S, R1022P (2 families), E1179K, K504del, K600fs, P955fs and IVS29+5G>A]. Maximum wall thickness and age at time of diagnosis were similar to patients with MYH7 mutations [25(7) vs. 27(8), p = 0.16], [46(16) vs. 44(19), p = 0.9]. CONCLUSIONS: Mutations in MyBPC3 are present in 15% of our hypertrophic cardiomyopathy families. Severe hypertrophy and early expression are compatible with the presence of MyBPC3 mutations. The genetic diagnosis not only allows avoiding clinical follow up of non carriers but it opens new possibilities that includes: to take preventive clinical decisions in mutation carriers than have not developed the disease yet, the establishment of genotype-phenotype relationship, and to establish a genetic diagnosis routine in patients with familial HCM.

Cellular localization of ROS and NO in olive reproductive tissues during flower development.

BACKGROUND: Recent studies have shown that reactive oxygen species (ROS) and nitric oxide (NO) are involved in the signalling processes taking place during the interactions pollen-pistil in several plants. The olive tree (Olea europaea L.) is an important crop in Mediterranean countries. It is a dicotyledonous species, with a certain level of self-incompatibility, fertilisation preferentially allogamous, and with an incompatibility system of the gametophytic type not well determined yet. The purpose of the present study was to determine whether relevant ROS and NO are present in the stigmatic surface and other reproductive tissues in the olive over different key developmental stages of the reproductive process. This is a first approach to find out the putative function of these signalling molecules in the regulation of the interaction pollen-stigma. RESULTS: The presence of ROS and NO was analyzed in the olive floral organs throughout five developmental stages by using histochemical analysis at light microscopy, as well as different fluorochromes, ROS and NO scavengers and a NO donor by confocal laser scanning microscopy. The "green bud" stage and the period including the end of the "recently opened flower" and the "dehiscent anther" stages displayed higher concentrations of the mentioned chemical species. The stigmatic surface (particularly the papillae and the stigma exudate), the anther tissues and the pollen grains and pollen tubes were the tissues accumulating most ROS and NO. The mature pollen grains emitted NO through the apertural regions and the pollen tubes. In contrast, none of these species were detected in the style or the ovary. CONCLUSION: The results obtained clearly demonstrate that both ROS and NO are produced in the olive reproductive organs in a stage- and tissue- specific manner. The biological significance of the presence of these products may differ between early flowering stages (defence functions) and stages where there is an intense interaction between pollen and pistil which may determine the presence of a receptive phase in the stigma. The study confirms the enhanced production of NO by pollen grains and tubes during the receptive phase, and the decrease in the presence of ROS when NO is actively produced.