Breaking Caenorhabditis elegans the easy way using the Balch homogenizer: an old tool for a new application

The biochemical quantification of sterols in insects has been difficult because only small amounts of tissues can be obtained from insect bodies and because sterol metabolites are structurally related. We have developed a highly specific and sensitive quantitative method for determining of the concentrations of seven sterols—7-dehydrocholesterol, desmosterol, cholesterol, ergosterol, campesterol, stigmasterol, and β-sitosterol—using a high performance liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry (HPLC/APCI-MS/MS). The sterols were extracted from silkworm larval tissues using the Bligh and Dyer method and were analyzed using HPLC/APCI-MS/MS with selected reaction monitoring, using cholesterol-3,4-¹³C₂ as an internal standard. The detection limits of the method were between 12.1 and 259 fmol. The major sterol in most silkworm larval tissues was cholesterol, whereas only small quantities of the dietary sterols were detected. Thus, a simple, sensitive, and specific method was successfully developed for the quantification of the sterol concentrations in each tissue of an individual silkworm larva. This method will be a useful tool for investigating to molecular basis of sterol physiology in insects, facilitating the quantification of femtomole quantities of sterols in biological samples.     

Adenosine A(2A) receptor activation limits chronic granulomatous disease-induced hyperinflammation

Chronic granulomatous disease (CGD) is caused by defects in the NADPH oxidase complex and is characterized by an increased susceptibility to infection. Other significant complications of CGD include autoimmunity and non-infectious hyperinflammatory disorders. We show that a gp91^phox deficiency leads to the development of phenotypically altered T lymphocytes in mice and that this abnormal, hyperactive phenotype can be modulated by activation of the adenosine A_2A receptor. T cells isolated from CGD mice produce significantly higher levels of the pro-inflammatory cytokines IFN-γ, IL-2, TNF-α, IL-4 and IL-13 than do WT cells after TCR-mediated activation; treatment with the selective adenosine A_2A receptor agonist, CGS21680, potently inhibits this response. Additionally, the over exuberant inflammatory response elicited by thioglycollate challenge in gp91^phox deficient mice is attenuated by CGS21680. These data suggest that treatment with A_2AR agonists may be an effective therapy by which to regulate the immune system hyperactivity that results from a gp91^phox deficiency.     

Zero methane emission bogs: extreme rhizosphere oxygenation by cushion plants in Patagonia

? Vascular wetland plants may substantially increase methane emissions by producing root exudates and easily degradable litter, and by providing a low-resistance diffusion pathway via their aerenchyma. However, model studies have indicated that vascular plants can reduce methane emission when soil oxygen demand is exceeded by oxygen released from roots. Here, we tested whether these conditions occur in bogs dominated by cushion plants. ? Root-methane interactions were studied by comparing methane emissions, stock and oxygen availability in depth profiles below lawns of either cushion plants or Sphagnum mosses in Patagonia. ? Cushion plants, Astelia pumila and Donatia fascicularis, formed extensive root systems up to 120 cm in depth. The cold soil (< 10°C) and highly decomposed peat resulted in low microbial activity and oxygen consumption. In cushion plant lawns, high soil oxygen coincided with high root densities, but methane emissions were absent. In Sphagnum lawns, methane emissions were substantial. High methane concentrations were only found in soils without cushion plant roots. ? This first methane study in Patagonian bog vegetation reveals lower emissions than expected. We conclude that cushion plants are capable of reducing methane emission on an ecosystem scale by thorough soil and methane oxidation.     

Palmitoylation of MIR2 is required for its function

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two RING finger E3 ubiquitin ligases (MIR1 and MIR2) that mediate ubiquitination and degradation of cellular proteins important for the establishment of an efficient antiviral immune response. MIR1 and MIR2 share 30% sequence identity; however, their substrate preferences are varied. MIR1 has been shown to primarily downregulate major histocompatibility complex class I (MHC-I), whereas MIR2 can downregulate a wide range of cell surface proteins. Many of the MIR substrates are thought to be present in lipid raft microdomains, a subregion of the plasma membrane known to be important for a wide range of signal transduction events. Palmitoylation is a posttranslational modification that increases recruitment of transmembrane proteins to lipid rafts. In this study, we investigated the importance of palmitoylation for MIR function. We present evidence that MIR2-mediated downregulation of MHC-I and platelet endothelial cell adhesion molecule 1 (PECAM-1) but not other substrates is inhibited in the presence of the drug 2-bromohexadecanoic acid (2-Br), a chemical inhibitor of palmitoylation. Biochemical analysis indicates that MIR2 is directly palmitoylated on cysteine 146. Mutation of this cysteine to a phenylalanine prevents MIR2 palmitoylation and blocks the ability of MIR2 to downregulate MHC-I and PECAM-I but not B7.2 and intercellular adhesion molecule 1 (ICAM-I), consistent with the phenotype observed after 2-Br treatment. Unpalmitoylated MIR2 does not interact with MHC-I and is thus unable to ubiquitinate and downregulate MHC-I from the cell surface. Furthermore, we observed that MIR2 is palmitoylated in vivo during lytic infection. Palmitoylation may act to regulate MIR2 function and localization during viral infection by allowing MIR2 to properly interact with and downregulate multiple substrates known to play an important role in the host immune response.     

Rheological properties of ovalbumin hydrogels as affected by surfactants addition

The gel properties of ovalbumin mixtures with three different surfactants (sodium perfluorooctanoate, sodium octanoate and sodium dodecanoate) have been studied by rheological techniques. The gel elasticities were determined as a function of surfactant concentration and surfactant type. The fractal dimension of the formed structures was evaluated from plots of storage modulus against surfactant concentration. The role of electrostatic, hydrophobic and disulfide SS interactions in these systems has been demonstrated to be the predominant. The viscosity of these structures tends to increase with surfactant concentration, except for the fluorinated one. Unfolded ovalbumin molecules tend to form fibrillar structures that tend to increase with surfactant concentration, except for the fluorinated one. This fact has been related to the particular nature of this molecule.     

Apo- and holo-lactoferrin are both internalized by lactoferrin receptor via clathrin-mediated endocytosis but differentially affect ERK-signaling and cell proliferation in Caco-2 cells

Lactoferrin (Lf) is a major iron-binding and multi-functional protein in exocrine fluids such as breast milk and mucosal secretions. The functions of Lf appear dependent upon the iron saturation of the Lf protein and are postulated to be mediated through Lf internalization by a Lf receptor (LfR). However, mechanisms by which LfR mediates Lf internalization in enterocytes are unknown. We now demonstrate that a LfR previously cloned from the small intestine mediates Lf endocytosis in a human enterocyte model (Caco-2 cells). LfR was detected at the plasma membrane by cell surface biotinylation; both apo-Lf and holo-Lf uptake were significantly inhibited in cells transfected with LfR siRNA. Treatments of hypertonic sucrose and clathrin siRNA and co-immunoprecipitation of LfR with clathrin adaptor AP2 indicate that LfR regulates Lf endocytosis via clathrin-mediated endocytosis. Although both iron-free Lf (apo-Lf) and iron-saturated Lf (holo-Lf) enter Caco-2 cells via a similar mechanism and no significant differences were observed in the binding and uptake of apo- and holo-Lf in Caco-2 cells, apo-Lf but not holo-Lf stimulates proliferation of Caco-2 cells. Interestingly, apo-Lf stimulated extracellular signal-regulated mitogen-activated protein kinase (ERK) cascade to a significantly greater extent than holo-Lf and the apo-Lf induced proliferation was significantly inhibited by an ERK cascade inhibitor (U0126) and clathrin siRNA. Taken together, our data suggest that LfR is a major pathway through which Lf is taken up by enterocytes, which occurs independently of iron saturation through clathrin-mediated endocytosis. The differential effects of apo- and holo-Lf are not due to differences in cellular internalization mechanisms.     

Malate decarboxylases: evolution and roles of NAD(P)-ME isoforms in species performing C(4) and C(3) photosynthesis

In the C(4) pathway of photosynthesis two types of malate decarboxylases release CO(2) in bundle sheath cells, NADP- and NAD-dependent malic enzyme (NADP-ME and NAD-ME), located in the chloroplasts and the mitochondria of these cells, respectively. The C(4) decarboxylases involved in C(4) photosynthesis did not evolve de novo; they were recruited from existing housekeeping isoforms. NADP-ME housekeeping isoforms would function in the control of malate levels during hypoxia, pathogen defence responses, and microspore separation, while NAD-ME participates in the respiration of malate in the tricarboxylic acid cycle. Recently, the existence of three enzymatic NAD-ME entities in Arabidopsis, occurring by alternative association of two subunits, was described as a novel mechanism to regulate NAD-ME activity under changing metabolic environments. The C(4) NADP-ME is thought to have evolved from a C(3) chloroplastic ancestor, which in turn would have evolved from an ancient cytosolic enzyme. In this way, the C(4) NADP-ME would have emerged through gene duplication, acquisition of a new promoter, and neo-functionalization. In contrast, there would exist a unique NAD-ME in C(4) plants, which would have been adapted to perform a dual function through changes in the kinetic and regulatory properties of the C(3) ancestors. In addition to this, for the evolution of C(4) NAD-ME, insertion of promoters or enhancers into the single-copy genes of the C(3) ancestors would have changed the expression without gene duplication.     

Identification of a novel Bcl-2-interacting mediator of cell death (Bim) E3 ligase, tripartite motif-containing protein 2 (TRIM2), and its role in rapid ischemic tolerance-induced neuroprotection

We have previously shown that the cell death-promoting protein Bcl-2-interacting mediator of cell death (Bim) is ubiquitinated and degraded following a neuroprotection-conferring episode of brief ischemia (preconditioning). Here, we identify the E3 ligase that ubiquitinates Bim in this model, using a proteomics approach. Using phosphorylated GST-Bim as bait, we precipitated and identified by mass spectrometry tripartite motif protein 2 (TRIM2), a RING (really interesting new gene) domain-containing protein. The reaction between TRIM2 and Bim was confirmed using co-immunoprecipitation followed by immunoblotting. We show that TRIM2 binds to Bim when it is phosphorylated by p42/p44 MAPK but does not interact with a nonphosphorylatable Bim mutant (3ABim). 12-O-tetradecanoylphorbol-13-acetate activation of p42/p44 MAPK drives Bim ubiquitination in mouse embryonic fibroblast cells and is associated with an increased interaction between TRIM2 and Bim. One hour following preconditioning ischemia, the binding of Bim to TRIM2 increased, consistent with the time window of enhanced Bim degradation. Blocking p42/p44 MAPK activation following preconditioning ischemia with U0126 or using the nonphosphorylatable 3ABim reduced the binding between Bim and TRIM2. Immunodepletion of TRIM2 from cell lysates prepared from preconditioned cells reduced Bim ubiquitination. Finally, suppression of TRIM2 expression, using lentivirus transduction of shRNAmir, stabilized Bim protein levels and blocked neuroprotection observed in rapid ischemic tolerance. Taken together, these data support a role for TRIM2 in mediating the p42/p44 MAPK-dependent ubiquitination of Bim in rapid ischemic tolerance.     

Effects of sparsely and densely ionizing radiation on plants

One of the main purposes leading botanists to investigate the effects of ionizing radiations is to understand plant behaviour in space, where vegetal systems play an important role for nourishment, psychological support and functioning of life support systems. Ground-based experiments have been performed with particles of different charge and energy. Samples exposed to X- or γ-rays are often used as reference to derive the biological efficiency of different radiation qualities. Studies where biological samples are exposed directly to the space radiation environment have also been performed. The comparison of different studies has clarified how the effects observed after exposure are deeply influenced by several factors, some related to plant characteristics (e.g. species, cultivar, stage of development, tissue architecture and genome organization) and some related to radiation features (e.g. quality, dose, duration of exposure). In this review, we report main results from studies on the effect of ionizing radiations, including cosmic rays, on plants, focusing on genetic alterations, modifications of growth and reproduction and changes in biochemical pathways especially photosynthetic behaviour. Most of the data confirm what is known from animal studies: densely ionizing radiations are more efficient in inducing damages at several different levels, in comparison with sparsely ionizing radiation.