The use of quantitative imaging to investigate regulators of membrane trafficking in Arabidopsis stomatal closure

Expansion of gene families facilitates robustness and evolvability of biological processes but impedes functional genetic dissection of signalling pathways. To address this, quantitative analysis of single cell responses can help characterize the redundancy within gene families. We developed high‐throughput quantitative imaging of stomatal closure, a response of plant guard cells, and performed a reverse genetic screen in a group of Arabidopsis mutants to five stimuli. Focussing on the intersection between guard cell signalling and the endomembrane system, we identified eight clusters based on the mutant stomatal responses. Mutants generally affected in stomatal closure were mostly in genes encoding SNARE and SCAMP membrane regulators. By contrast, mutants in RAB5 GTPase genes played specific roles in stomatal closure to microbial but not drought stress. Together with timed quantitative imaging of endosomes revealing sequential patterns in FLS2 trafficking, our imaging pipeline can resolve non‐redundant functions of the RAB5 GTPase gene family. Finally, we provide a valuable image‐based tool to dissect guard cell responses and outline a genetic framework of stomatal closure.      

Adaptation to developmental diet influences the response to selection on age at reproduction in the fruit fly

Experimental evolution (EE) is a powerful tool for addressing how environmental factors influence life‐history evolution. While in nature different selection pressures experienced across the lifespan shape life histories, EE studies typically apply selection pressures one at a time. Here, we assess the consequences of adaptation to three different developmental diets in combination with classical selection for early or late reproduction in the fruit fly Drosophila melanogaster. We find that the response to each selection pressure is similar to that observed when they are applied independently, but the overall magnitude of the response depends on the selection regime experienced in the other life stage. For example, adaptation to increased age at reproduction increased lifespan across all diets; however, the extent of the increase was dependent on the dietary selection regime. Similarly, adaptation to a lower calorie developmental diet led to faster development and decreased adult weight, but the magnitude of the response was dependent on the age‐at‐reproduction selection regime. Given that multiple selection pressures are prevalent in nature, our findings suggest that trade‐offs should be considered not only among traits within an organism, but also among adaptive responses to different—sometimes conflicting—selection pressures, including across life stages.     

Conformational solution studies of the SDS micelle-bound 11-28 fragment of two Alzheimer's beta-amyloid variants (E22K and A21G) using CD, NMR, and MD techniques

The beta-amyloid (Abeta) is the major peptide constituent of neuritic plaques in Alzheimer's disease (AD) and its aggregation is believed to play a central role in the pathogenesis of the disease. Naturally occurring mutations resulting in changes in the Abeta sequence (pos. 21-23) are associated with familial AD-like diseases with extensive cerebrovascular pathology. It was proved that the mutations alter the aggregation ability of Abeta and its neurotoxicity. Among five mutations at positions 21-23 there are two mutations with distinct clinical characteristics and potentially distinct pathogenic mechanism-the Italian (E22K) and the Flemish (A21G) mutations. In our studies we have examined the structures of the 11-28 fragment of the Italian and Flemish Abeta variants. The fragment was chosen because it has been shown to be the most important for amyloid fibril formation. The detailed structure of both variants Abeta(11-28) was determined using CD, 2D NMR, and molecular dynamics techniques under water-SDS micelle conditions. The NMR analysis revealed two distinct sets of proton resonances for the peptides. The studies of both peptides pointed out the existence of well-defined alpha-helical conformation in the Italian mutant, whereas the Flemish was found to be unstructured with the possibility of a bent structure in the central part of the peptide.     

The structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O36 containing 3-deoxy-d-manno-oct-2-ulosonic acid

An oligosaccharide that corresponds to the repeating unit of the O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O36. Structural studies of the oligosaccharide and O-deacylated lipopolysaccharide were performed using sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, and H-detected (1)H,(13)C HSQC and HMBC experiments. It was found that the O-polysaccharide is built up of linear trisaccharide repeating units containing 2-acetamido-2-deoxyglucose, 6-deoxy-l-talose (l-6dTal), and 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and has the following structure. [structure: see text]     

Structure of the O-polysaccharide and serological cross-reactivity of the lipopolysaccharide of Providencia alcalifaciens O32 containing N-acetylisomuramic acid

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O32 and studied by sugar and methylation analyses, solvolysis with triflic acid, 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY, TOCSY, ROESY, H-detected 1H,13C HSQC and HMBC experiments. It was found that the polysaccharide has a branched tetrasaccharide repeating unit containing 2-acetamido-3-O-[(S)-1-carboxyethyl]-2-deoxy-D-glucose (D-GlcNAc3Slac, N-acetylisomuramic acid) with the following structure: [STRUCTURE: SEE TEXT]. Serological studies with O-antisera showed antigenic relationships between P. alcalifaciens O32 and O29 as well as several other Providencia and Proteus strains sharing putative epitopes on the O-polysaccharides.     

Molecular phylogenetics of representative Paramecium species

The genus Paramecium has been known to science for 250 years and contains some of the most widely studied species of ciliates. At present, the basic research object for phylogenetic studies is the genome of various paramecia. One of the most widely used markers are genes coding for various rRNA's. Comparative analyses of sequences coding rRNA were applied for resolving the systematic position of some paramecia species and also for the establishment of an accurate taxonomy of Paramecium. Paramecia were also model organisms for their systematic group in more general studies in a comparative analysis among ciliates, fungi, plants and multicellular animals, illustrating the evolutionary relationships between Archaebacteria and Eucaryota. A new, revolutionary genealogy proposed the shifting of presumptively advanced groups towards more primitive ones, and traditionally primitive forms were located closer to highly specialized taxa, but rRNA analysis did not unambiguously resolve associations within the studied groups. Because of the aforementioned concerns, the number of molecular markers used for alternative studies is growing, such as genes coding proteins from the Hsp family or histone proteins. Other promising candidate markers may be hemoglobin genes or genes coding á-tubulins. In case of comparative analyses ofnucleotide sequences, the outcome of the research usually depends upon a subjective choice of DNA. One of the directions of research in molecular phylogenetics include indirect methods that allow for an estimation of entire genomes, for example RAPD-PCR-fingerprinting.     

Effect of steroids on basal and LH-stimulated prostaglandins F(2alpha) and E(2) release and cyclooxygenase-2 expression in cultured porcine endometrial stromal cells

Ovarian steroids modulate uterine receptivity in domestic species. Luteinizing hormone (LH) stimulates prostaglandin (PG)F(2alpha) release from the porcine endometrium. However, the combined action of LH and steroids on PGs secretion has not yet been studied in pigs. The aim of the present study was to examine the effect of estradiol (E(2)) and progesterone (P(4)) on basal and LH-stimulated PGF(2alpha) and PGE(2) secretion and cyclooxygenase-2 (COX-2) protein expression in porcine endometrial stromal cells obtained on days 12-13 of the estrous cycle. Cells were cultured for 48 h in a medium containing charcoal-stripped newborn calf serum alone or supplemented with 10 nM E(2) and/or 50 nM P(4). Then, the cells were incubated for 6 h in the presence or absence of LH (20 ng/ml). Long exposure of stromal cells to steroids had no effect on PGF(2alpha) secretion, but PGE(2) release increased in the presence of E(2) plus P(4) (p<0.05). Pre-incubation of cells with E(2) plus P(4) resulted in enhanced PGF(2alpha) (p<0.05) and PGE(2) (p<0.001) secretion. Moreover, LH increased PG(2alpha) secretion in control (p<0.05) and E(2)-treated stromal cells (p<0.01). LH tended (p=0.07) to elevate PGE(2) release only in cells pre-exposed to E(2) plus P(4). The expression of COX-2 protein was increased by LH (p<0.05), but not by steroids. These results confirm the stimulatory effect of LH on PGF(2alpha) secretion and COX-2 expression in porcine stromal cells before luteolysis. PG release from porcine endometrium seems to be controlled by ovarian steroids, however only E(2)-treated-treated cells responded to LH.     

Regulation of autophagy in bovine mammary epithelial cells

The bovine mammary gland undergoes intensive remodelling during the lactation cycle, and the escalation of this process is observed during dry periods. The main type of cell death responsible for bovine mammary gland involution is apoptosis; however, there are also a lot of cells exhibiting morphological features of autophagy during drying off. Our in vitro and in vivo studies of bovine mammary gland physiology suggest that the enhanced process of autophagy, observed at the end of lactation and during dry periods, is the result of: (1) decreased level of lactogenic hormones (GH, IGF-I), (2) decreased GH-R and IGF-IR alpha expression, (3) increased expression of auto/paracrine apoptogenic peptides (IGFBPs, TGFbeta), (4) increased influence of sex steroids (17beta-estradiol and progesterone) and (5) enhanced competition between the between the intensively developing fetus and the mother organism for nutritional and bioactive compounds. The above conditions may create a state of temporary malnutrition of mammary epithelial cells, which forces the cells to the induction of autophagy, as a mechanism for stabilizing intracellular supplies of energy and amino acids, especially during the enhanced activity of apoptogenic factors.     

ROS and Phytohormones in Plant-Plant Allelopathic Interaction

Allelopathy refers to plant-plant interference mediated mostly by plant released products of secondary metabolism. It was recently suggested that allelochamicals may influence growth of neighboring plants by induction of oxidative stress. We have focused on the role of reactive oxygen species (ROS) and phytohormons (ABA and ethylene) in the biochemical and molecular regulation of plant response to sunflower phytotoxins.Key Words: ABA, allelopathy, ethylene, H₂O₂, reactive oxygen species, seed germination