Physcomitrella patens has lipoxygenases for both eicosanoid and octadecanoid pathways

Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and eicosapentaenoic acid, in addition to the shorter chain C18 α-linolenic and linoleic acids, which are typical substrates of lipoxygenases in flowering plants. To identify the fatty acid substrates used by moss lipoxygenases, eight lipoxygenase genes from Physcomitrella patens were heterologously expressed in Escherichia coli, and then analyzed for lipoxygenase activity using linoleic, α-linolenic and arachidonic acids as substrates. Among the eight moss lipoxygenases, only seven were found to be enzymatically active in vitro, two of which selectively used arachidonic acid as the substrate, while the other five preferred α-linolenic acid. Based on enzyme assays using a Clark-type oxygen electrode, all of the active lipoxygenases had an optimum pH at 7.0, except for one with highest activity at pH 5.0. HPLC analyses indicated that the two arachidonic acid lipoxygenases form (12S)-hydroperoxy eicosatetraenoic acid as the main product, while the other five lipoxygenases produce mainly (13S)-hydroperoxy octadecatrienoic acid from α-linolenic acid. These results suggest that mosses may have both C20 and C18 based oxylipin pathways.     

Proteomic analysis identifies in vivo candidate matrix metalloproteinase‐9 substrates in the left ventricle post‐myocardial infarction

Matrix metalloproteinase‐9 (MMP‐9) deletion has been shown to improve remodeling of the left ventricle post‐myocardial infarction (MI), but the mechanisms to explain this improvement have not been fully elucidated. MMP‐9 has a broad range of in vitro substrates, but relevant in vivo substrates are incompletely defined. Accordingly, we evaluated the infarct regions of wild‐type (wt) and MMP‐9 null (null) mice using a proteomic strategy. Wt and null groups showed similar infarct sizes (48±3 in wt and 45±3% in null), indicating that both groups received an equal injury stimulus. Left ventricle infarct tissue was homogenized and analyzed by 2‐DE and MS. Of 31 spot intensity differences, the intensities of 9 spots were higher and 22 spots were lower in null mice compared to wt (all p<0.05). Several extracellular matrix proteins were identified in these spots by MS, including fibronectin, tenascin‐C, thrombospondin‐1, and laminin. Fibronectin was observed on the gels at a lower than expected molecular weight in the wt group, which suggested substrate cleavage, and the lower molecular weight spot was observed at lower intensity in the MMP‐9 null group, which suggested cleavage by MMP‐9. Immunoblotting confirmed the presence of fibronectin cleavage products in the wt samples and lower levels in the absence of MMP‐9. In conclusion, examining infarct tissue from wt and MMP‐9 null mice by proteomic analysis provides a powerful and unique method to identify in vivo candidate MMP substrates.     

Molecular Modeling of Calixarenes with Group I Metal Ions

Molecular mechanics calculations have been used to model the geometries of the complexes of Group I metal ions with calix[n]arenes (n = 4,5). A simple procedure in which the calixarene atoms are assigned partial charges on the basis of AM1 calculations and the metal ions are allowed to bind electrostatically to the calixarenes produces surprising good results when the resulting structures are compared to known crystallographic data on the complexes. Encapsulated solvent molecules and/or counterions can be included in the calculations and, indeed, are necessary to reproduce the X-ray data. Electronic Supplementary Material available.     

Ecosystem responses to changes in plant functional type composition: An example from the Patagonian steppe

Abstract. Grass cover along a grazing intensity gradient in Patagonia decreases, whereas bare soil and shrub cover increases. Our objective was to study the effect of a change in the dominant plant functional type on soil water balance, primary production, herbivore biomass, roughness, and albedo. Using a soil water balance model, we found increases in evaporation and deep drainage, and a decrease in total transpiration along the grazing intensity gradient. Above-ground primary production, estimated from transpiration, decreased along the grazing intensity gradient because shrubs did not fully compensate for the decrease in grass production. Using a statistical model, we calculated herbivore biomass from estimates of above-ground primary production. Estimated herbivore biomass was lowest in the shrub-dominated extreme of the grazing gradient. Roughness increased from the grass-dominated to the shrub-dominated community. Albedo had a maximum at an intermediate position along the gradient. Our results suggest that changes in plant functional type composition, independent of changes in biomass, affect ecosystem functioning and the exchange of energy and material with the atmosphere. Grasses and shrubs proved to be appropriate plant functional types to link structure and function of ecosystems.     

Silurian calcispheres (Calcitarcha) of Gotland (Sweden): Comparisons with calcareous dinoflagellates

Scanning electron microscope examinations of polished and etched surfaces of sediments from the Silurian carbonate platform of Gotland, Sweden, revealed the presence of numerous, morphologically diverse “calcispheres” (Calcitarcha). Some of these spherical calcareous microfossils display wall structures that are surprisingly similar to those of calcareous dinoflagellate cysts. In analogy to the interpretation of the biological affinities of Palaeozoic acritarchs as cysts of organisms that might have been the ancestors of organic-walled dinoflagellates, the Calcitarcha from Gotland can be compared and may possibly be related to organisms that may have been the ancestors of calcareous cyst-producing dinoflagellates that so far have not been observed before the Late Jurassic.     

Using evolutionary information and ancestral sequences to understand the sequence-function relationship in GLP-1 agonists

Glucagon-like peptide-1 (GLP-1) is an incretin hormone with therapeutic potential for type 2 diabetes. A variety of GLP-1 sequences are known from amphibian species, and some of these have been tested here and found to be able to bind and activate the human GLP-1 receptor. While little difference was observed for the in vitro potency for the human GLP-1 receptor, larger differences were found in the enzymatic stability of these peptides. Two peptides showed increased enzymatic stability, and they group together phylogenetically, though they originate from Amphibia and Reptilia. We have used ancestral sequence reconstruction to analyze the evolution of these GLP-1 molecules, including the synthesis of new peptides. We find that the increased stability could not be observed in the resurrected peptides from the common ancestor of frogs, even though they maintain the ability to activate the human GLP-1 receptor. Another method, using residue mapping on evolutionary branches yielded peptides that had maintained potency towards the receptor and also showed increased stability. This represents a new approach using evolutionary data in protein engineering.     

Compromised proteasome degradation elevates neuronal nitric oxide synthase levels and induces apoptotic cell death

The significance of impairment of proteasome activity in PC12 cells was examined in connection with nitrative/nitrosative stress and apoptotic cell death. Treatment of differentiated PC12 cells with MG132, a proteasome inhibitor, elicited a dose- and time-dependent increase in neuronal nitric oxide synthase (nNOS) protein levels, decreased cell viability, and increased cytotoxicity. Viability and cytotoxicity were ameliorated by L-NAME (a broad NOS inhibitor). Nitric oxide/peroxynitrite formation was increased upon treatment of PC12 cells with MG132 and decreased upon treatment with the combination of MG132 and 7-NI (a specific inhibitor of nNOS). The decreases in cell viability appeared to be effected by an activation of JNK and its effect on mitochondrial Bcl-x_L phosphorylation. These effects are strengthened by the activation of caspase-9 along with increased caspase-3 activity upon treatment of PC12 cells with MG132. These results suggest that impairment of proteasome activity and consequent increases in nNOS levels lead to a nitrative stress that involves the coordinated response of JNK cytosolic signaling and mitochondrion-driven apoptotic pathways.     

Intracellular transport and degradation of 125I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

Improving aquaporin Z expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> by fusion partners and subsequent condition optimization

Aquaporin Z (AqpZ), a typical orthodox aquaporin with six transmembrane domains, was expressed as a fusion protein with TrxA in E. coli in our previous work. In the present study, three fusion partners (DsbA, GST and MBP) were employed to improve the expression level of this channel protein in E. coli. The result showed that, compared with the expression level of TrxA-AqpZ, five- to 40-fold increase in the productivity of AqpZ with fusion proteins was achieved by employing these different fusion partners, and MBP was the most efficient fusion partner to increase the expression level. By using E. coli C43 (DE3)/pMAL-AqpZ, the effects of different expression conditions were investigated systematically to improve the expression level of MBP-AqpZ in E. coli. The high productivity of MBP-AqpZ (200 mg/l) was achieved under optimized conditions. The present work provides a novel approach to improve the expression level of membrane proteins in E. coli.     

Structural basis of the honey bee PBP pheromone and pH-induced conformational change

The behavior of insects and their perception of their surroundings are driven, in a large part, by odorants and pheromones. This is especially true for social insects, such as the honey bee, where the queen controls the development and the caste status of the other individuals. Pheromone perception is a complex phenomenon relying on a cascade of recognition events, initiated in antennae by pheromone recognition by a pheromone-binding protein and finishing with signal transduction at the axon membrane level. With to the objective of deciphering this initial step, we have determined the structures of the bee antennal pheromone-binding protein (ASP1) in the apo form and in complex with the main component of the queen mandibular pheromonal mixture, 9-keto-2(E)-decenoic acid (9-ODA) and with nonpheromonal components. In the apo protein, the C terminus obstructs the binding site. In contrast, ASP1 complexes have different open conformations, depending on the ligand shape, leading to different volumes of the binding cavity. The binding site integrity depends on the C terminus (111-119) conformation, which involves the interplay of two factors; i.e. the presence of a ligand and a low pH. Ligand binding to ASP1 is favored by low pH, opposite to what is observed with other pheromone-binding proteins, such as those of Bombyx mori and Anopheles gambiae.