Loss of sialic acid binding domain redirects protein σ1 to enhance M cell-directed vaccination

Ovalbumin (OVA) genetically fused to protein sigma 1 (pσ1) results in tolerance to both OVA and pσ1. Pσ1 binds in a multi-step fashion, involving both protein- and carbohydrate-based receptors. To assess the relative pσ1 components responsible for inducing tolerance and the importance of its sialic binding domain (SABD) for immunization, modified OVA-pσ1, termed OVA-pσ1(short), was deleted of its SABD, but with its M cell targeting moiety intact, and was found to be immunostimulatory and enhanced CD4(+) and CD8(+) T cell proliferation. When used to nasally immunize mice given with and without cholera toxin (CT) adjuvant, elevated SIgA and serum IgG responses were induced, and OVA-pσ1(s) was more efficient for immunization than native OVA+CT. The immune antibodies (Abs) were derived from elevated Ab-forming cells in the upper respiratory tissues and submaxillary glands and were supported by mixed Th cell responses. Thus, these studies show that pσ1(s) can be fused to vaccines to effectively elicit improved SIgA responses.     

TRFLP analysis reveals that fungi rather than bacteria are associated with premature yeast flocculation in brewing

Premature yeast flocculation (PYF) is a sporadic fermentation problem in the brewing industry that results in incomplete yeast utilization of fermentable sugars in wort. Culture-independent, PCR-based fingerprinting techniques were applied in this study to identify the associations between the occurrence of the PYF problem during brewery fermentation with barley malt-associated microbial communities (both bacteria and fungi). Striking differences in the microbial DNA fingerprint patterns for fungi between PYF positive (PYF +ve) and negative (PYF ?ve) barley malts were observed using the terminal restriction fragment length polymorphism (TRFLP) technique. The presence of terminal restriction fragments (TRFs) of 360–460 bp size range, for fungal HaeIII restriction enzyme-derived TRFLP profiles appeared to vary substantially between PYF +ve and PYF ?ve samples. The source of the barley malt did not influence the fungal taxa implicated in PYF. TRFLP analysis indicates bacterial taxa are unlikely to be important in causing PYF. Virtual digestion of fungal sequences tentatively linked HaeIII TRFs in the 360–460 bp size range to a diverse range of yeast/yeast-like species. Findings from this study suggest that direct monitoring of barley malt samples using molecular methods could potentially be an efficient and viable alternative for monitoring PYF during brewery fermentations.     

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.     

Genetic diversity of the expansive grass Brachypodium pinnatum in a changing landscape: Effect of habitat age

Perennial grasses constitute a major group of species showing a dramatic decline of biodiversity in successional plant communities. Using AFLP markers, we examined 12 populations of the expansive grass Brachypodium pinnatum differing in habitat age (30–50, ca. 100 and >300 years old) in order to determine whether clonal diversity of populations, genetic variation, and the relative importance of clonal propagation versus sexual reproduction change with grassland age. Five AFLP primer combinations gave a total of 517 bands, 79% of which were polymorphic. 314 different multilocus lineages were distinguished among the 453 samples analyzed. The number of genotypes (G) and clonal richness (R) decreased with habitat age, while the distribution of the frequency of genets changed from many clones of similar size to dominance by one or a few large clones. We consider these results to give evidence of significant role of sexual reproduction in the early phases of colonization and prevalence of clonal growth and competitive exclusion of less adapted genotypes in the later ones. However, habitat age had only marginal effect on genetic diversity, as percentage of polymorphic loci (PPL) within all the populations analyzed was similar, viz. 38.6–43.5%.     

Myriocin, an inhibitor of serine palmitoyl transferase, impairs the uptake of transferrin and low-density lipoprotein in mammalian cells

The role of sphingolipids in clathrin-mediated endocytosis is only poorly understood in mammalian cells. Thus the relationship between sphingolipid de novo synthesis and clathrin-mediated endocytosis of transferrin were studied in L929 fibroblasts and two other cell lines. Endocytosis was measured using live cell imaging with fluorescent transferrin or (125)I-transferrin. Lipids were primarily measured using electrospray ionization tandem mass spectrometry. At physiological temperature, transferrin uptake was significantly decreased by the inhibitor of serine palmitoyl transferase myriocin. Myriocin inhibited also the uptake of low-density lipoproteins. The endocytosis inhibition by myriocin could be released by the addition of sphingoid base and by the protein phosphorylation effectors phorbol-12-myristate, 13-acetate (PMA) and okadaic acid. Myriocin influenced not only sphingolipids but also the glycerophospholipid profile. The study of phosphatidylcholine species shows adaptations to more saturated, alkylated and longer fatty acid moieties. The reported results imply that in mammalian cells, at 37°C, sphingolipid de novo synthesis is required for clathrin-mediated endocytosis.     

Chloroplast biogenesis — Correlation between structure and function

Chloroplast biogenesis is a multistage process leading to fully differentiated and functionally mature plastids. Complex analysis of chloroplast biogenesis was performed on the structural and functional level of its organization during the photoperiodic plant growth after initial growth of seedlings in the darkness. We correlated, at the same time intervals, the structure of etioplasts transforming into mature chloroplasts with the changes in the photosynthetic protein levels (selected core and antenna proteins of PSI and PSII) and with the function of the photosynthetic apparatus in two plant species: bean (Phaseolus vulgaris L.) and pea (Pisum sativum L). We selected these plant species since we demonstrated previously that the mature chloroplasts differ in the thylakoid organization. We showed that the protein biosynthesis as well as photosynthetic complexes formation proceeds gradually in both plants in spite of periods of darkness. We found that both steady structural differentiation of the bean chloroplast and reformation of prolamellar bodies in pea were accompanied by a gradual increase of the photochemical activity in both species. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.