Profiling of Arabidopsis secondary metabolites by capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry

Large-scale metabolic profiling is expected to develop into an integral part of functional genomics and systems biology. The metabolome of a cell or an organism is chemically highly complex. Therefore, comprehensive biochemical phenotyping requires a multitude of analytical techniques. Here, we describe a profiling approach that combines separation by capillary liquid chromatography with the high resolution, high sensitivity, and high mass accuracy of quadrupole time-of-flight mass spectrometry. About 2000 different mass signals can be detected in extracts of Arabidopsis roots and leaves. Many of these originate from Arabidopsis secondary metabolites. Detection based on retention times and exact masses is robust and reproducible. The dynamic range is sufficient for the quantification of metabolites. Assessment of the reproducibility of the analysis showed that biological variability exceeds technical variability. Tools were optimized or established for the automatic data deconvolution and data processing. Subtle differences between samples can be detected as tested with the chalcone synthase deficient tt4 mutant. The accuracy of time-of-flight mass analysis allows to calculate elemental compositions and to tentatively identify metabolites. In-source fragmentation and tandem mass spectrometry can be used to gain structural information. This approach has the potential to significantly contribute to establishing the metabolome of Arabidopsis and other model systems. The principles of separation and mass analysis of this technique, together with its sensitivity and resolving power, greatly expand the range of metabolic profiling.     

Amphetamine modulates human incentive processing

Research suggests that psychostimulants can physiologically alter dopamine kinetics in the ventral striatum (VS) and psychologically enhance mood and attention. Using event-related functional magnetic resonance imaging (fMRI), we conducted a within-subject, double-blind, placebo (PLAC)-controlled study of the effects of oral dextroamphetamine (AMPH, 0.25 mg/kg) treatment on brain activity and affect during incentive processing. In two counterbalanced scanning sessions 60-180 min after ingesting AMPH or PLAC, 8 healthy volunteers played a game involving anticipation and receipt of monetary gains and losses. Group and volume of interest analyses suggested that by enhancing tonic over phasic activation, AMPH treatment "equalized" levels of VS activity and positive arousal during anticipation of both gain and loss. These findings suggest that therapeutic effects of amphetamine on incentive processing may involve reducing the difference between anticipation of gains and losses.     

Cell Penetrating Peptides and Cationic Antibacterial Peptides: TWO SIDES OF THE SAME COIN*

Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs.     

Polymerisation of chemically cross-linked actin:thymosin beta(4) complex to filamentous actin: alteration in helical parameters and visualisation of thymosin beta(4) binding on F-actin.

The beta-thymosins are intracellular monomeric (G-)actin sequestering proteins forming 1:1 complexes with G-actin. Here, we analysed the interaction of thymosin beta(4) with F-actin. Thymosin beta(4) at 200 microM was chemically cross-linked to F-actin. In the presence of phalloidin, the chemically cross-linked actin:thymosin beta(4) complex was incorporated into F-actin. These mixed filaments were of normal appearance when inspected by conventional transmission electron microscopy after negative staining. We purified the chemically cross-linked actin:thymosin beta(4) complex, which polymerised only when phalloidin and the gelsolin:2-actin complex were present simultaneously. Using scanning transmission electron microscopy, the mass-per-length of control and actin:thymosin beta(4) filaments was found to be 16.0(+/-0.8) kDa/nm and 18.0(+/-0.9) kDa/nm, respectively, indicating an increase in subunit mass of 5.4 kDa. Analysis of the helical parameters revealed an increase of the crossover spacing of the two right-handed long-pitch helical strands from 36.0 to 40.5 nm. Difference map analysis of 3-D helical reconstruction of control and actin:thymosin beta(4) filaments yielded an elongated extra mass. Qualitatively, the overall size and shape of the difference mass were compatible with published data of the atomic structure of thymosin beta(4). The deduced binding sites of thymosin beta(4) to actin were in agreement with those identified previously. However, parts of the difference map might represent subtle conformational changes of both proteins occurring upon complex formation.     

Information processing in the adaptation of Saccharomyces cerevisiae to osmotic stress: an analysis of the phosphorelay system

Cellular signaling is key for organisms to survive immediate stresses from fluctuating environments as well as relaying important information about external stimuli. Effective mechanisms have evolved to ensure appropriate responses for an optimal adaptation process. For them to be functional despite the noise that occurs in biochemical transmission, the cell needs to be able to infer reliably what was sensed in the first place. For example Saccharomyces cerevisiae are able to adjust their response to osmotic shock depending on the severity of the shock and initiate responses that lead to near perfect adaptation of the cell. We investigate the Sln1–Ypd1–Ssk1-phosphorelay as a module in the high-osmolarity glycerol pathway by incorporating a stochastic model. Within this framework, we can imitate the noisy perception of the cell and interpret the phosphorelay as an information transmitting channel in the sense of C.E. Shannon’s “Information Theory”. We refer to the channel capacity as a measure to quantify and investigate the transmission properties of this system, enabling us to draw conclusions on viable parameter sets for modeling the system.     

Prevalence of hematozoans in lions (Panthera leo) and cheetah (Acinonyx jubatus) in Serengeti National Park and Ngorongoro Crater, Tanzania

Lions (Panthera leo) and cheetah (Acinonyx jubatus) from the Serengeti National Park and Ngorongoro Crater Conservation Area, Tanzania were examined for the presence of blood protozoans. Twenty-eight percent of the lions were infected with Trypanosoma sp. and the prevalence of trypanosome infection varied significantly between adjacent habitats. All of the animals were infected with Hepatozoon sp. and a Theileria sp.-like piroplasm that was morphologically indistinguishable from Theileria felis.     

Human neurocysticercosis: in vivo expansion of peripheral regulatory T cells and their recruitment in the central nervous system

Human neurocysticercosis (NC) is caused by Taenia solium larvae lodged in the central nervous system. Most cases occur with no, or mild, neurological symptoms. However, in some patients, neuroinflammation is exacerbated, leading to severe forms of the disease. Considering the critical role of regulatory T cells (Tregs) in balancing inflammation in chronic diseases, their participation in restraining the inflammatory response in NC was explored in the present study. The frequency of Tregs and their relationship with the level of the proliferative response, the level of activated lymphocytes, and the cytokines expressed were determined in severe NC patients compared with those from healthy donors. Significantly increased peripheral Tregs (CD4(+)CD25(high) and CD4(+)CD25(high)FoxP3(+), CD4(+)CD25(high)CTLA4(+), and CD4(+)CD25(high) IL10(+)) and a significant decrease in activated (CD38(+) and CD69(+)) T cells were observed in 19 NC patients versus 10 healthy subjects. Significantly increased Tregs in NC are accompanied by a depressed specific, and non-specific, lymphocyte proliferative response, and they negatively correlate with activated CD4(+)CD69(+) lymphocytes. Treg frequencies were also determined in cerebral spinal fluid for 8 of the 19 NC patients. A positive significant correlation between peripheral and local Tregs was observed. Here, we report for the first time data that support the possible contribution of local and systemic Tregs in limiting neuroinflammation in NC.     

How to connect an IgE-driven response with CTL activity?

One of the goals of cell-based immune therapy in cancer is the induction of tumor-specific cytotoxic T-lymphocyte (CTL) responses. To achieve this objective, the ability of dendritic cells (DC) to cross-present tumor antigens can be exploited. One of the most efficient pathways for the induction of CTLs by cross-presentation is mediated by immunoglobulins of the IgG class, which are used by DCs to sample antigen in the form of immune complexes via Fc-gamma receptors. Could DCs use an IgE-mediated cross-presentation mechanism in a comparable manner to induce CTLs? We here discuss the potential of two human IgE Fc receptors, FcεRI and FcεRII, to serve as antigen uptake receptors for IgE-mediated cross-presentation. We conclude that the existence of an IgE-mediated cross-presentation pathway would provide a direct link between IgE-driven immune responses and CTL activity.