Both MHC class II and its GPI-anchored form undergo hop diffusion as observed by single-molecule tracking

Previously, investigations using single-fluorescent-molecule tracking at frame rates of up to 65 Hz, showed that the transmembrane MHC class II protein and its GPI-anchored modified form expressed in CHO cells undergo simple Brownian diffusion, without any influence of actin depolymerization with cytochalasin D. These results are at apparent variance with the view that GPI-anchored proteins stay with cholesterol-enriched raft domains, as well as with the observation that both lipids and transmembrane proteins undergo short-term confined diffusion within a compartment and long-term hop diffusion between compartments. Here, this apparent discrepancy has been resolved by reexamining the same paradigm, by using both high-speed single-particle tracking (50 kHz) and single fluorescent-molecule tracking (30 Hz). Both molecules exhibited rapid hop diffusion between 40-nm compartments, with an average dwell time of 1-3 ms in each compartment. Cytochalasin D hardly affected the hop diffusion, consistent with previous observations, whereas latrunculin A increased the compartment sizes with concomitant decreases of the hop rates, which led to an ∼50% increase in the median macroscopic diffusion coefficient. These results indicate that the actin-based membrane skeleton influences the diffusion of both transmembrane and GPI-anchored proteins.     

Influence of the hypogaeic army ant<Emphasis Type="Italic"> Dorylus (Dichthadia) laevigatus</Emphasis> on tropical arthropod communities

The majority of army ant species forage hypogaeically. Due to the difficulties in observing these ants, their potential influence on hypogaeic and epigaeic arthropod communities has not yet been investigated. As the first hypogaeically foraging army ant studied in detail, we attracted Dorylus laevigatus to areas monitored for their arthropod diversity. Here, for the first time, the same sites were sampled before and after an army ant raid. Furthermore, interactions between D. laevigatus and the five most common ground-nesting ant species were noted and their life-history traits compared, allowing first inferences on possible mechanisms of their coexistence. The occurrence of D. laevigatus within a study plot had no evident effect on the number of arthropod taxa or individuals collected with epigaeic and hypogaeic pitfall traps. Likewise, juvenile arthropods, which are less mobile and thus are potentially easier prey for D. laevigatus, showed no differences in their collected numbers before and after the army ant had visited a plot. However, significantly fewer ant species were collected with hypogaeic traps after D. laevigatus had been within the study plots, indicating a possible predation of D. laevigatus especially on two Pseudolasius and one Pheidole species. The five most common ground-foraging ant species demonstrated their ability to avoid, kill, and even prey on the army ant. The reaction of Lophomyrmex bedoti towards D. laevigatus indicated the former to be a potential prey species, while Pachycondyla sp. 2 showed signs of "enemy specification." Odontoponera diversus and O. transversa actively preyed on D. laevigatus, while Pheidologeton affinis fought with D. laevigatus over resources. All ant species could co-occur with D. laevigatus at palm oil baits. Adding to the differences detected in previous studies between D. laevigatus and epigaeically foraging army ant species, the occurrence of this hypogaeic army ant seems to have less devastating effects on arthropod community compositions than those of epigaeically mass raiding species.     

Crystal structure of Schizosaccharomyces pombe riboflavin kinase reveals a novel ATP and riboflavin-binding fold

The essential redox cofactors riboflavin monophosphate (FMN) and flavin adenine dinucleotide (FAD) are synthesised from their precursor, riboflavin, in sequential reactions by the metal-dependent riboflavin kinase and FAD synthetase. Here, we describe the 1.6A crystal structure of the Schizosaccharomyces pombe riboflavin kinase. The enzyme represents a novel family of phosphoryl transferring enzymes. It is a monomer comprising a central beta-barrel clasped on one side by two C-terminal helices that display an L-like shape. The opposite side of the beta-barrel serves as a platform for substrate binding as demonstrated by complexes with ADP and FMN. Formation of the ATP-binding site requires significant rearrangements in a short alpha-helix as compared to the substrate free form. The diphosphate moiety of ADP is covered by the glycine-rich flap I formed from parts of this alpha-helix. In contrast, no significant changes are observed upon binding of riboflavin. The ribityl side-chain might be covered by a rather flexible flap II. The unusual metal-binding site involves, in addition to the ADP phosphates, only the strictly conserved Thr45. This may explain the preference for zinc observed in vitro.     

High-level expression of the mycobacterial porin MspA in Escherichia coli and purification of the recombinant protein

MspA is the prototype of a new family of tetrameric porins and provides the main general diffusion pathway for hydrophilic compounds through the outer membrane of Mycobacterium smegmatis. Structural analysis was hampered by the scarce amount of pure protein. After replacement of the GC-rich codons of the mspA gene by codons optimal for high-level expression in Escherichia coli, the mature MspA protein was overproduced in E. coli. The recombinant MspA (rMspA) monomer (M(r) 20000) was purified by anion exchange and hydrophobic interaction chromatography yielding 2.6 mg pure protein per liter of culture. This exceeded the yield of the native protein 10-fold. Circular dichroism revealed that rMspA is folded in a native-like structure. rMspA assembled partially to the channel-forming tetramer both during expression in E. coli and after purification in vitro. Thus, overexpression in E. coli and chromatographic purification are key steps towards a high resolution structure of MspA.     

Lipids in blood-brain barrier models in vitro I: Thin-layer chromatography and high-performance liquid chromatography for the analysis of lipid classes and long-chain polyunsaturated fatty acids

The objectives of this study were to optimize a sensitive high-performance liquid chromatography (HPLC) method for fatty acid (FA) analysis for the quantification of polyunsaturated FAs (PUFAs) in cell lipid extracts and to analyze the lipid and FA patterns of three cell lines used in blood-brain barrier (BBB) models: RBE4, ECV304, and C6. Thin-layer chromatographic analysis revealed differences in the phosphatidylcholine-phosphatidylethanolamine (PC:PE) ratios and the triglyceride (TG) content. The PC:PE ratio was <1 for RBE4 cells but >1 for ECV304 and C6 cells. ECV304 cells displayed up to 9% TG depending on culture time, whereas the other cell lines contained about 1% TG. The percentages of docosahexaenoic acid were 9.4 +/- 1.7% of the unsaturated FAs in RBE4 cells (n = 5; 4 d in culture; 9.9% after 10 d), 8.1 +/- 2.0% in ECV304 cells (n = 11; 10 to 14 d), and 6.7 +/- 0.6% in C6 cells (n = 6; 10 to 14 d) and were close to the published values for rat brain microvascular endothelium. The percentage of arachidonic acid (C20:4) was about half that in vivo. ECV304 cells contained the highest fraction of C20:4, 17.8 +/- 2.2%; RBE4 cells contained 11.6 +/- 2.4%; and C6 cells 15.8 +/- 1.9%. It is concluded that a sensitive HPLC method for FAs is now optimized for the analysis of long-chain PUFAs. The results provide a useful framework for studies on the effects of lipid modulation and give reference information for the development of further BBB models.     

Dual Role of GdmH in Producer Immunity and Secretion of the Staphylococcal Lantibiotics Gallidermin and Epidermin

The biosynthetic gene clusters of the staphylococcal lantibiotics epidermin and gallidermin are distinguished by the presence of the unique genes epiH and gdmH, respectively. They encode accessory factors for the ATP-binding cassette transporters that mediate secretion of the antimicrobial peptides. Here, we show that gdmH also contributes to immunity to gallidermin but not to nisin. gdmH alone affected susceptibility to gallidermin only moderately, but it led to a multiplication of the immunity level mediated by the FEG immunity genes when cloned together with the gdmT gene, suggesting a synergistic activity of the H and FEG systems. gdmH-related genes were identified in the genomes of several bacteria, indicating an involvement in further cellular functions.     

Dynamic imaging of protease activity with fluorescently quenched activity-based probes

Protease activity is tightly regulated in both normal and disease conditions. However, it is often difficult to monitor the dynamic nature of this regulation in the context of a live cell or whole organism. To address this limitation, we developed a series of quenched activity-based probes (qABPs) that become fluorescent upon activity-dependent covalent modification of a protease target. These reagents freely penetrate cells and allow direct imaging of protease activity in living cells. Targeted proteases are directly identified and monitored biochemically by virtue of the resulting covalent tag, thereby allowing unambiguous assignment of protease activities observed in imaging studies. We report here the design and synthesis of a selective, cell-permeable qABP for the study of papain-family cysteine proteases. This probe is used to monitor real-time protease activity in live human cells with fluorescence microscopy techniques as well as standard biochemical methods.     

Metabolic profiling of rhizomes of native populations of the strictly endemic Croatian species Iris adriatica

Iris adriatica Trinajsti? ex Miti? (Iridaceae L.) is a strictly endemic taxon from Croatia. It is a rhizomatous dwarf plant from the I. pumila complex with a distribution area limited to the Croatian part of the Mediterranean area, mainly central Dalmatia. The genus Iris is known for its richness in isoflavonoids which also play a significant role in chemotaxonomy and biological activity. Hence, in the current study, different plant batches of I. adriatica collected in early spring of 2016 were analysed for their phytochemical profiles and qualitatively compared. UHPLC-PDA-ESI-MS analyses of methanolic rhizome extracts were performed. Altogether, 36 compounds, representing isoflavonoids (including 6,7-methylendioxy derivatives), benzophenones and xanthones were found as aglycones or in glycosidically bound form to be the main constituent groups of I. adriatica rhizomes. Qualitative results were identical between different batches of plant material from collection sites in central Dalmatia, they differed only in quantity. For some phenolic compounds of I. adriatica, chemotaxonomic relevance was detected.     

Green light: a signal to slow down or stop

Light has a profound effect on plant growth and development. Red and blue light best drive photosynthetic metabolism, so it is no surprise that these light qualities are particularly efficient in advancing the developmental characteristics associated with autotrophic growth habits. Photosynthetically inefficient light qualities also impart important environmental information to a developing plant. For example, far-red light reverses the effect of phytochromes, leading to changes in gene expression, plant architecture, and reproductive responses. Recent evidence shows that green light also has discrete effects on plant biology, and the mechanisms that sense this light quality are now being elucidated. Green light has been shown to affect plant processes via cryptochrome-dependent and cryptochrome-independent means. Generally, the effects of green light oppose those directed by red and blue wavebands. This review examines the literature where green light has been implicated in physiological or developmental outcomes, many not easily attributable to known sensory systems. Here roles of green light in the regulation of vegetative development, photoperiodic flowering, stomatal opening, stem growth modulation, chloroplast gene expression and plant stature are discussed, drawing from data gathered over the last 50 years of plant photobiological research. Together these reports support a conclusion that green light sensory systems adjust development and growth in orchestration with red and blue sensors.     

Acute Fluoxetine Treatment Induces Slow Rolling of Leukocytes on Endothelium in Mice

ObjectiveActivated platelets release serotonin at sites of inflammation where it acts as inflammatory mediator and enhances recruitment of neutrophils. Chronic treatment with selective serotonin reuptake inhibitors (SSRI) depletes the serotonin storage pool in platelets, leading to reduced leukocyte recruitment in murine experiments. Here, we examined the direct and acute effects of SSRI on leukocyte recruitment in murine peritonitis.MethodsC57Bl/6 and Tph1−/− (Tryptophan hydroxylase1) mice underwent acute treatment with the SSRI fluoxetine or vehicle. Serotonin concentrations were measured by ELISA. Leukocyte rolling and adhesion on endothelium was analyzed by intravital microscopy in mesentery venules with and without lipopolysaccharide challenge. Leukocyte extravasation in sterile peritonitis was measured by flow cytometry of abdominal lavage fluid.ResultsPlasma serotonin levels were elevated 2 hours after fluoxetine treatment (0.70±0.1 µg/ml versus 0.27±0.1, p = 0.03, n = 14), while serum serotonin did not change. Without further stimulation, acute fluoxetine treatment increased the number of rolling leukocytes (63±8 versus 165±17/0.04 mm²min⁻¹) and decreased their velocity (61±6 versus 28±1 µm/s, both p<0.0001, n = 10). In Tph1−/− mice leukocyte rolling was not significantly influenced by acute fluoxetine treatment. Stimulation with lipopolysaccharide decreased rolling velocity and induced leukocyte adhesion, which was enhanced after fluoxetine pretreatment (27±3 versus 36±2/0.04 mm², p = 0.008, n = 10). Leukocyte extravasation in sterile peritonitis, however, was not affected by acute fluoxetine treatment.ConclusionsAcute fluoxetine treatment increased plasma serotonin concentrations and promoted leukocyte-endothelial interactions in-vivo, suggesting that serotonin is a promoter of acute inflammation. E-selectin was upregulated on endothelial cells in the presence of serotonin, possibly explaining the observed increase in leukocyte-endothelial interactions. However transmigration of neutrophils in sterile peritonitis was not affected by higher serotonin concentrations, indicating that the effect of fluoxetine was restricted to early steps in the leukocyte recruitment. Whether SSRI use in humans alters leukocyte recruitment remains to be investigated.