Involvement of a protein kinase C and protein phosphatases in adhesion of CD4+ T cells to and detachment from extracellular matrix proteins.

For immune surveillance and function to be effective, T lymphocytes constantly recirculate via lymph and blood between lymphoid organs and body tissues. To enable efficient cell movement and migration, cell adhesion to components of the basement membrane and the extracellular matrix (ECM) must be a rapid and transitory process. Whether phosphorylation and dephosphorylation of cellular proteins are involved in this phenomena was explored by monitoring the adhesion of T cells to immobilized ECM proteins. A short exposure of 51Cr-labeled human CD4+ T cells to phorbol esters in vitro induced a rapid beta 1-integrin-mediated adhesion to both fibronectin and laminin, as determined by inhibition with anti-integrin antibodies. Adhesion was reversible; detachment from the immobilized ECM ligands occurred between 20 and 120 min without further intervention. This T cell adhesion was regulated by the activation of protein kinase C because (a) staurosporine and H-7 inhibitors of protein kinase C suppressed T cell adhesion, and (b) PMA-induced down-regulation of intracellular levels of protein kinase C was associated with the abrogation of the T cell adhesiveness to fibronectin and laminin. Furthermore, inhibition of protein phosphatases activity by okadaic acid delayed the detachment of the T cells from fibronectin or laminin. Thus, we suggest that T cell-ECM interactions such as adhesion and detachment are regulated, respectively, by protein kinase C and protein phosphatases.     

Phenology and polyploidy in annual Brachypodium species (Poaceae) along the aridity gradient in Israel

Local adaptation of plants along environmental gradients provides strong evidence for clinal evolution mediated by natural selection. Plants have developed diverse strategies to mitigate stress, for example, drought escape is a phenological strategy to avoid drought stress, while polyploidy was proposed as a genomic adaptation to stress. Polyploidy as an adaptation to aridity (an environmental parameter integrating temperature and precipitation) was previously documented in annual Brachypodium spp. (Poaceae) in the Western Mediterranean. Here, we examined whether polyploidy or phenology are associated with aridity in annual Brachypodium spp. along the aridity gradient in the Eastern Mediterranean. Using flow cytometry, we determined ploidy levels of plants from natural populations along the Israeli gradient, spanning ∼424 km from mesic Mediterranean to extreme desert climates. In a common garden we recorded time of seedling emergence, flowering and senescence. We tested whether the proportion of allotetraploids in the populations and phenological traits were associated with aridity. Contrary to a previous study in the Western Mediterranean, we found no effect of aridity on the proportion of allotetraploids and diploids within populations. Interestingly, phenology was associated with aridity: time of emergence was later, while flowering and senescence were earlier in desert plants. Our results indicate that in the Eastern Mediterranean, adaptation of Brachypodium to aridity is mediated mainly by phenology, rather than ploidy level. Therefore, we suggest that genome duplication is not the main driver of adaptation to environmental stress; rather, phenological change as a drought escape mechanism may be the major adaptation.     

An innovative approach to grape metabolomics: stilbene profiling by suspect screening analysis

Suspect screening analysis is a targeted metabolomics approach in which identification of compounds relies on specific available information such as their molecular formula and isotopic pattern. This method was applied to the study of grape metabolomics with an UPLC/MS high-resolution Q-TOF mass spectrometer (nominal resolution 40,000) coupled with a Jet Stream ionization source. The present paper describes the detailed qualitative and quantitative study of grape stilbenes, the principal polyphenols associated with the beneficial effects of drinking wine. For identification of compounds, a new database was expressly constructed from the molecular information of potential metabolites of grape and wine from the literature and other electronic databases. Currently, GrapeMetabolomics contains about a thousand putative grape compounds. If untargeted analysis of a sample provides identification of a new compound with a sufficiently confident score, it is added to the database. Thus, by increasing the number of samples studied, GrapeMetabolomics can be expanded. This method is effective for identification of the molecular formulae of several hundred metabolites in two runs (positive and negative ionization) with minimal sample preparation, and can also be used to analyse some single classes of compounds involved in cell and tissue metabolism. With this approach, a total of 18 stilbene derivatives was identified in two grape samples (Raboso Piave and Primitivo) on the basis of accurate mass measurements and isotopic patterns, and identification was confirmed by MS/MS analysis. The approach can also potentially be applied to the metabolomics of other plant varieties.     

Distinct functional roles for hopanoid composition in the chemical tolerance of Zymomonas mobilis

Hopanoids are a class of membrane lipids found in diverse bacterial lineages, but their physiological roles are not well understood. The ethanol fermenter Zymomonas mobilis features the highest measured concentration of hopanoids, leading to the hypothesis that these lipids can protect against the solvent toxicity. However, the lack of genetic tools for manipulating hopanoid composition in this bacterium has limited their further functional analysis. Due to the polyploidy (>50 genome copies per cell) of Z. mobilis, we found that disruptions of essential hopanoid biosynthesis (hpn) genes act as genetic knockdowns, reliably modulating the abundance of different hopanoid species. Using a set of hpn transposon mutants, we demonstrate that both reduced hopanoid content and modified hopanoid polar head group composition mediate growth and survival in ethanol. In contrast, the amount of hopanoids, but not their head group composition, contributes to fitness at low pH. Spectroscopic analysis of bacterial‐derived liposomes showed that hopanoids protect against several ethanol‐driven phase transitions in membrane structure, including lipid interdigitation and bilayer dissolution. We propose that hopanoids act through a combination of hydrophobic and inter‐lipid hydrogen bonding interactions to stabilize bacterial membranes during solvent stress.     

Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress

Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H₂DCFDA‐staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into ‘low‐ROS’ and ‘high‐ROS’ subpopulations. Pollen germination assays following fluorescence‐activated cell sorting revealed that the high‐ROS pollen germinated with a frequency that was 35‐fold higher than the low‐ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose‐dependent alterations in DCF‐fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry‐based approaches to investigate metabolic changes during stress responses in pollen.     

FolM, a new chromosomally encoded dihydrofolate reductase in Escherichia coli

Escherichia coli (thyA DeltafolA) mutants are viable and can grow in minimal medium when supplemented with thymidine alone. Here we present evidence from in vivo and in vitro studies that the ydgB gene determines an alternative dihydrofolate reductase that is related to the trypanosomatid pteridine reductases. We propose to rename this gene folM.