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)-β-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.     

Interspecific Introgression in Cetaceans: DNA Markers Reveal Post-F1 Status of a Pilot Whale

Visual species identification of cetacean strandings is difficult, especially when dead specimens are degraded and/or species are morphologically similar. The two recognised pilot whale species (Globicephala melas and Globicephala macrorhynchus) are sympatric in the North Atlantic Ocean. These species are very similar in external appearance and their morphometric characteristics partially overlap; thus visual identification is not always reliable. Genetic species identification ensures correct identification of specimens. Here we have employed one mitochondrial (D-Loop region) and eight nuclear loci (microsatellites) as genetic markers to identify six stranded pilot whales found in Galicia (Northwest Spain), one of them of ambiguous phenotype. DNA analyses yielded positive amplification of all loci and enabled species identification. Nuclear microsatellite DNA genotypes revealed mixed ancestry for one individual, identified as a post-F1 interspecific hybrid employing two different Bayesian methods. From the mitochondrial sequence the maternal species was Globicephala melas. This is the first hybrid documented between Globicephala melas and G. macrorhynchus, and the first post-F1 hybrid genetically identified between cetaceans, revealing interspecific genetic introgression in marine mammals. We propose to add nuclear loci to genetic databases for cetacean species identification in order to detect hybrid individuals.     

Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype,induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells

Background

EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines.

Methods

EPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts.

Results

Our study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts.

Conclusions

Taken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.

     

Affective Priming Using Facial Expressions Modulates Liking for Abstract Art

We examined the influence of affective priming on the appreciation of abstract artworks using an evaluative priming task. Facial primes (showing happiness, disgust or no emotion) were presented under brief (Stimulus Onset Asynchrony, SOA = 20ms) and extended (SOA = 300ms) conditions. Differences in aesthetic liking for abstract paintings depending on the emotion expressed in the preceding primes provided a measure of the priming effect. The results showed that, for the extended SOA, artworks were liked more when preceded by happiness primes and less when preceded by disgust primes. Facial expressions of happiness, though not of disgust, exerted similar effects in the brief SOA condition. Subjective measures and a forced-choice task revealed no evidence of prime awareness in the suboptimal condition. Our results are congruent with findings showing that the affective transfer elicited by priming biases evaluative judgments, extending previous research to the domain of aesthetic appreciation.     

Selective Vulnerability of Spinal and Cortical Motor Neuron Subpopulations in delta7 SMA Mice

Loss of the survival motor neuron gene (SMN1) is responsible for spinal muscular atrophy (SMA), the most common inherited cause of infant mortality. Even though the SMA phenotype is traditionally considered as related to spinal motor neuron loss, it remains debated whether the specific targeting of motor neurons could represent the best therapeutic option for the disease. We here investigated, using stereological quantification methods, the spinal cord and cerebral motor cortex of ∆7 SMA mice during development, to verify extent and selectivity of motor neuron loss. We found progressive post-natal loss of spinal motor neurons, already at pre-symptomatic stages, and a higher vulnerability of motor neurons innervating proximal and axial muscles. Larger motor neurons decreased in the course of disease, either for selective loss or specific developmental impairment. We also found a selective reduction of layer V pyramidal neurons associated with layer V gliosis in the cerebral motor cortex. Our data indicate that in the ∆7 SMA model SMN loss is critical for the spinal cord, particularly for specific motor neuron pools. Neuronal loss, however, is not selective for lower motor neurons. These data further suggest that SMA pathogenesis is likely more complex than previously anticipated. The better knowledge of SMA models might be instrumental in shaping better therapeutic options for affected patients.     

Assessment of the efficacy of the utilisation of conventional and electric toothbrushes by the older adults

Objective: To evaluate the efficacy of electric and conventional toothbrushes for a group of elderly individuals. Background: Although the electric toothbrush has been recommended for elderly individuals, there had previously never been a study regarding its efficacy. Material and methods: Sixty independent elders of both genders with different oral conditions from the Center Adult Day Vitória, Espírito Santo, Brazil, were randomly divided into two groups of 30 individuals. One group received the Oral B CrossAction Power electric toothbrush, whereas the other received a conventional Bitufo Class 32 soft toothbrush to perform oral hygiene. The bacterial plaque index (O’Leary Plaque Index) and DMFT index were assessed as a measure of oral hygiene and oral health. The data were analysed using the Shapiro–Wilk, Mann–Whitney and Wilcoxon tests. Results: The results of the efficacy of the Oral B Cross Action Power electric toothbrush demonstrated that on the 7th and 15th days, the bacterial plaque indexes were 24.91 ± 12.81 and 22.11 ± 14.46, respectively, which corresponds to a 50.24% removal of bacterial plaque on the 7th and 55.83% on the 15th days. Although the electric toothbrush removed more bacterial plaque than the conventional toothbrush, the difference was not statistically significant. Conclusion: Both the conventional and the electric toothbrushes were effective in removing bacterial plaque within the elderly group. More studies are necessary to test the efficacy of electric toothbrushes in relation to conventional toothbrushes for elderly patients.     

Genetic and developmental analyses of chaetae pattern formation in Drosophila tergites

Summary The role of the achaete-scute complex and extramacrochaetae, Notch, Delta, Enhancer of split and Hairless genes in chaeta patterning in Drosophila tergites was studied in genetic mosaics and in mutant combinations. The mutant phenotypes of different alleles of each gene can be ordered in characteristic topographical seriations. These seriations are related to the pattern of proliferation of histoblasts and the time of singularization of sensory organ mother cells from surrounding epidermal cells. Genetic mosaics of lethal alleles show that these genes are fundamentally involved in this singularization and subsequent differentiation. The study of mutant combinations of alleles of these genes reveals specific relationships of epistasis and synergism between them. The results suggest that spatial and temporal variations in achaete-scute complex functional products in cells, modulated by the activity of other genes involved in signal transduction, define the patterned differentiation of sensory organs in tergites. Offprint requests to: A. García-Bellido     

Grazing history influences biodiversity: a case study on ground-dwelling arachnids (Arachnida: Araneae, Opiliones) in the Natural Park of Alpi Marittime (NW Italy)

Alpine pastures are typical examples of “high nature value farmlands”, representing important habitats harbouring unique biological communities. Disturbance induced by overgrazing influences significantly ecosystem processes, in which invertebrates play a major role. To develop new models of sustainable management of pastures, more knowledge is needed of the animal communities, essential to the ecological functioning of pasture ecosystems. The apparent poor ecological state of several pastures in the Natural Regional Park of Alpi Marittime (NW-Italy) lead us to evaluate the influence of grazing history on biodiversity, using spider and harvestmen assemblages as key groups. Four different pastoral types characterized by four different grazing histories were identified using the Daget-Poissonet method. Spiders and harvestmen were collected by means of pitfall traps. Arachnid assemblages were characterized in terms of composition, abundance, species richness, richness of endemic species and taxonomic relatedness. Generalized linear mixed models (GLMM) were used to test differences among assemblages occurring in each pastoral type. Furthermore, we tested differences in terms of plant communities (species richness and percentage of zoogenic species). Specificity and fidelity of every spider and harvestmen species within pastoral types were explored by the IndVal (Indicator Value) procedure. Fifty-eight species of spiders and seven species of harvestmen were collected (2,304 individuals). Pastoral types related to intensive grazing were characterized by the dominance of diurnal wanderer spiders (namely Lycosidae) while, conversely, nocturnal wanderers (mainly Gnaphosidae) were more abundant in extensive pastoral types. Results show that both species richness and spider abundance were higher in abandoned areas of extensive grazing, while endemic assemblages were richer in extensive grazed areas, which also hosted the most diverse plant community. Furthermore, most of the indicator species of spiders of this type were endemic, characterized by more demanding ecological requirements.     

Modeling human osteosarcoma in mice through 3AB-OS cancer stem cell xenografts

Osteosarcoma is the second leading cause of cancer‐related death for children and young adults. In this study, we have subcutaneously injected—with and without matrigel—athymic mice (Fox1nu/nu) with human osteosarcoma 3AB‐OS pluripotent cancer stem cells (CSCs), which we previously isolated from human osteosarcoma MG63 cells. Engrafted 3AB‐OS cells were highly tumorigenic and matrigel greatly accelerated both tumor engraftment and growth rate. 3AB‐OS CSC xenografts lacked crucial regulators of beta‐catenin levels (E‐cadherin, APC, and GSK‐3beta), and crucial factors to restrain proliferation, resulting therefore in a strong proliferation potential. During the first weeks of engraftment 3AB‐OS‐derived tumors expressed high levels of pAKT, beta1‐integrin and pFAK, nuclear beta‐catenin, c‐Myc, cyclin D2, along with high levels of hyperphosphorylated‐inactive pRb and anti‐apoptotic proteins such as Bcl‐2 and XIAP, and matrigel increased the expression of proliferative markers. Thereafter 3AB‐OS tumor xenografts obtained with matrigel co‐injection showed decreased proliferative potential and AKT levels, and undetectable hyperphosphorylated pRb, whereas beta1‐integrin and pFAK levels still increased. Engrafted tumor cells also showed multilineage commitment with matrigel particularly favoring the mesenchymal lineage. Concomitantly, many blood vessels and muscle fibers appeared in the tumor mass. Our findings suggest that matrigel might regulate 3AB‐OS cell behavior providing adequate cues for transducing proliferation and differentiation signals triggered by pAKT, beta1‐integrin, and pFAK and addressed by pRb protein. Our results provide for the first time a mouse model that recapitulates in vivo crucial features of human osteosarcoma CSCs that could be used to test and predict the efficacy in vivo of novel therapeutic treatments. J. Cell. Biochem. 113: 3380–3392, 2012. © 2012 Wiley Periodicals, Inc.