Reduced food intake and body weight in mice deficient for the G protein-coupled receptor GPR82

G protein-coupled receptors (GPCR) are involved in the regulation of numerous physiological functions. Therefore, GPCR variants may have conferred important selective advantages during periods of human evolution. Indeed, several genomic loci with signatures of recent selection in humans contain GPCR genes among them the X-chromosomally located gene for GPR82. This gene encodes a so-called orphan GPCR with unknown function. To address the functional relevance of GPR82 gene-deficient mice were characterized. GPR82-deficient mice were viable, reproduced normally, and showed no gross anatomical abnormalities. However, GPR82-deficient mice have a reduced body weight and body fat content associated with a lower food intake. Moreover, GPR82-deficient mice showed decreased serum triacylglyceride levels, increased insulin sensitivity and glucose tolerance, most pronounced under Western diet. Because there were no differences in respiratory and metabolic rates between wild-type and GPR82-deficient mice our data suggest that GPR82 function influences food intake and, therefore, energy and body weight balance. GPR82 may represent a thrifty gene most probably representing an advantage during human expansion into new environments.     

Thermoregulation behaviour in codling moth larvae

Abstract. The thermoregulation behaviour of the codling moth, Cydia pomonella, is investigated in temperature gradient experiments with larvae feeding within apples, and with mature larvae searching for cocooning sites. Feeding larvae appear to prefer the apple hemisphere with a higher temperature (i.e. they build larger cavities in the radiated, warmer part of the fruit). The proportion of larval cavities in the warmer hemisphere is positively related to increasing apple temperature on that side, as well as to the temperature difference between the warm and the cool fruit hemisphere. The mechanism in feeding larvae can be termed as cryptic basking because, during microhabitat selection, the caterpillars exploit temperature differences that are caused explicitly by incident solar radiation. Fifth-instar larvae in search of cocooning sites show no temperature preference within the large gradient offered (9–29 °C), with no difference between males and females. During larval development, the insect changes its thermoregulation behaviour in response to a possible shift in benefits of an elevated body temperature with respect to environmental conditions. Both the thermoregulation behaviour and such a shift of behavioural response should be respected when simulating body temperatures of the species.     

The basis of gender assignment in disorders of somatosexual differentiation

Sex assignment of patients with disorders of somatosexual differentiation is a controversial topic. The aim is to enable the patient to develop a stable gender identity during childhood, adolescence and adulthood. Enormous advances have recently been made in our knowledge of the molecular mechanisms of sexual differentiation and it is understood that long-term outcome may depend on the underlying diagnosis. There is increasing evidence that genital development is dependent on the action of androgenic steroids; moreover, both androgens and oestrogens may have an impact on other developing organs including neuronal structures such as the brain. Long-term outcome studies on the various intersexuality disorders are desperately needed in order to establish a basis for evidence-based medicine regarding sex assignment and treatment options. Premature decisions leading to irreversible interventions before an accurate diagnosis has been established must be avoided.     

Characterization of the in vitro co-assembly process of the intermediate filament proteins vimentin and desmin: mixed polymers at all stages of assembly

We have investigated the co-assembly properties of the intermediate filament (IF) proteins vimentin and desmin. First, the soluble complexes formed by both proteins separately in 5 mM Tris-HCl, pH 8.4, were characterized by analytical ultracentrifugation. In both cases, s-values of around 5 S were obtained corresponding to the formation of tetramers. However, at pH 7.5 and in the presence of 1 mM EDTA, both proteins behaved quite differently; whereas vimentin sedimented at 7.2 S, desmin assembled into much larger complexes of about 13 S. A mixture of equimolar amounts of vimentin and desmin in 8 M urea yielded, after reconstitution into 5 mM Tris-HCl, pH 7.5, and 1 mM EDTA, complexes exhibiting a sharp peak at 10.9 S. This intermediate s-value indicated that co-assembly into a distinct new set of complexes had occurred. As judged by electron microscopy and viscometry, these mixtures assembled into IFs with characteristics similar to those of pure vimentin and desmin. Furthermore, when vimentin and desmin tetramers were mixed in 5 mM Tris-HCl, pH 8.4, and subsequently subjected to IF assembly conditions, again "hybrid" filaments were obtained. Most interestingly, after 10 min of assembly, mass-per-length (MPL) measurements by scanning transmission electron microscopy yielded IFs with an MPL-peak value of 36 +/- 5 kDa/nm, hence closer to that of vimentin IFs (33 +/- 4 kDa/nm) than to that of desmin IFs (48 +/- 8 kDa/nm). Finally, when unit length-filaments (ULF) of vimentin and desmin were mixed and assembled further, the diameters of individual mature IFs formed exhibited a significantly higher degree of width inhomogeneity along their length than vimentin and desmin IFs as might be expected for a modular mode of assembly. Last but not least, atomic force microscopy provided further direct evidence that desmin IFs are able to fuse end-to-end with vimentin IFs. In summary, we have shown that vimentin and desmin are able to co-assemble at the dimer, tetramer, ULF and even the mature IF level.     

Identification of N-acylhomoserine lactones in mucopurulent respiratory secretions from cystic fibrosis patients

Pseudomonas aeruginosa and species of the Burkholderia cepacia complex are the primary bacterial pathogens contributing to lung disease in patients with cystic fibrosis. Quorum sensing systems using N-acyl homoserine lactone (AHL) signal molecules are involved in the regulation of a number of virulence factors in these species. Extracts of mucopurulent respiratory secretions from 13 cystic fibrosis patients infected with P. aeruginosa and/or strains of the B. cepacia complex were fractionated using reverse-phase fast pressure liquid chromatography and analyzed for the presence of AHLs using a traI-luxCDABE-based reporter that responds to AHLs with acyl chains ranging between 4 and 12 carbons. Using this assay system, a broad range of AHLs were detected and identified despite being present at low concentrations in limited sample volumes. N-(3-oxo-dodecanoyl)-l-homoserine lactone, N-(3-oxo-decanoyl)-l-homoserine lactone and N-octanoyl-l-homoserine lactone (OHL) were the AHLs most frequently identified. OHL and N-decanoyl-l-homoserine lactone were detected in nanomolar concentrations compared to picomolar amounts of the 3-oxo-derivatives of the AHLs identified.     

Regulated expression of endothelial lipase by porcine brain capillary endothelial cells constituting the blood-brain barrier

Normal neurological function depends on a constant supply of polyunsaturated fatty acids to the brain. A considerable proportion of essential fatty acids originates from lipoprotein-associated lipids that undergo uptake and/or catabolism at the blood-brain barrier (BBB). This study aimed at identifying expression and regulation of endothelial lipase (EL) in brain capillary endothelial cells (BCEC), major constituents of the BBB. Our results revealed that BCEC are capable of EL synthesis and secretion. Overexpression of EL resulted in enhanced hydrolysis of extracellular high-density lipoprotein (HDL)-associated sn-2-labeled [(14)C]20 : 4 phosphatidylcholine. [(14)C]20 : 4 was recovered in cellular lipids, indicating re-uptake and intracellular re-esterification. To investigate local regulation of EL in the cerebrovasculature, BCEC were cultured in the presence of peroxisome-proliferator activated receptor (PPAR)- and liver X receptor (LXR)-agonists, known to regulate HDL levels. These experiments revealed that 24(S)OH-cholesterol (a LXR agonist), bezafibrate (a PPARalpha agonist), or pioglitazone (a PPARgamma agonist) resulted in down-regulation of EL mRNA and protein levels. Our findings implicate that EL could generate fatty acids at the BBB for transport to deeper regions of the brain as building blocks for membrane phospholipids. In addition PPAR and LXR agonists appear to contribute to HDL homeostasis at the BBB by regulating EL expression.     

Effects of Ebola virus glycoproteins on endothelial cell activation and barrier function

Ebola virus causes severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. Vascular instability and dysregulation are disease-decisive symptoms during severe infection. While the transmembrane glycoprotein GP(1,2) has been shown to cause endothelial cell destruction, the role of the soluble glycoproteins in pathogenesis is largely unknown; however, they are hypothesized to be of biological relevance in terms of target cell activation and/or increase of endothelial permeability. Here we show that virus-like particles (VLPs) consisting of the Ebola virus matrix protein VP40 and GP(1,2) were able to activate endothelial cells and induce a decrease in barrier function as determined by impedance spectroscopy and hydraulic conductivity measurements. In contrast, the soluble glycoproteins sGP and delta-peptide did not activate endothelial cells or change the endothelial barrier function. The VLP-induced decrease in barrier function was further enhanced by the cytokine tumor necrosis factor alpha (TNF-alpha), which is known to induce a long-lasting decrease in endothelial cell barrier function and is hypothesized to play a key role in Ebola virus pathogenesis. Surprisingly, sGP, but not delta-peptide, induced a recovery of endothelial barrier function following treatment with TNF-alpha. Our results demonstrate that Ebola virus GP(1,2) in its particle-associated form mediates endothelial cell activation and a decrease in endothelial cell barrier function. Furthermore, sGP, the major soluble glycoprotein of Ebola virus, seems to possess an anti-inflammatory role by protecting the endothelial cell barrier function.     

Ectopic expression of an amino acid transporter (VfAAP1) in seeds of Vicia narbonensis and pea increases storage proteins

Storage protein synthesis is dependent on available nitrogen in the seed, which may be controlled by amino acid import via specific transporters. To analyze their rate-limiting role for seed protein synthesis, a Vicia faba amino acid permease, VfAAP1, has been ectopically expressed in pea (Pisum sativum) and Vicia narbonensis seeds under the control of the legumin B4 promoter. In mature seeds, starch is unchanged but total nitrogen is 10% to 25% higher, which affects mainly globulin, vicilin, and legumin, rather than albumin synthesis. Transgenic seeds in vitro take up more [14C]-glutamine, indicating increased sink strength for amino acids. In addition, more [14C] is partitioned into proteins. Levels of total free amino acids in growing seeds are unchanged but with a shift toward higher relative abundance of asparagine, aspartate, glutamine, and glutamate. Hexoses are decreased, whereas metabolites of glycolysis and the tricarboxylic acid cycle are unchanged or slightly lower. Phosphoenolpyruvate carboxylase activity and the phosphoenolpyruvate carboxylase-to-pyruvate kinase ratios are higher in seeds of one and three lines, indicating increased anaplerotic fluxes. Increases of individual seed size by 20% to 30% and of vegetative biomass indicate growth responses probably due to improved nitrogen status. However, seed yield per plant was not altered. Root application of [15N] ammonia results in significantly higher label in transgenic seeds, as well as in stems and pods, and indicates stimulation of nitrogen root uptake. In summary, VfAAP1 expression increases seed sink strength for nitrogen, improves plant nitrogen status, and leads to higher seed protein. We conclude that seed protein synthesis is nitrogen limited and that seed uptake activity for nitrogen is rate limiting for storage protein synthesis.     

Role of Ebola virus secreted glycoproteins and virus-like particles in activation of human macrophages

Ebola virus, a member of the family Filoviridae, causes one of the most severe forms of viral hemorrhagic fever. In the terminal stages of disease, symptoms progress to hypotension, coagulation disorders, and hemorrhages, and there is prominent involvement of the mononuclear phagocytic and reticuloendothelial systems. Cells of the mononuclear phagocytic system are primary target cells and producers of inflammatory mediators. Ebola virus efficiently produces four soluble glycoproteins during infection: sGP, delta peptide (Delta-peptide), GP(1), and GP(1,2Delta). While the presence of these glycoproteins has been confirmed in blood (sGP) and in vitro systems, it is hypothesized that they are of biological relevance in pathogenesis, particularly target cell activation. To gain insight into their function, we expressed the four soluble glycoproteins in mammalian cells and purified and characterized them. The role of the transmembrane glycoprotein in the context of virus-like particles was also investigated. Primary human macrophages were treated with glycoproteins and virus-like particles and subsequently tested for activation by detection of several critical proinflammatory cytokines (tumor necrosis factor alpha, interleukin-6 [IL-6], and IL-1 beta) and the chemokine IL-8. The presentation of the glycoprotein was determined to be critical since virus-like particles, but not soluble glycoproteins, induced high levels of activation. We propose that the presentation of GP(1,2) in the rigid form such as that observed on the surface of particles is critical for initiating a sufficient signal for the activation of primary target cells. The secreted glycoproteins do not appear to play any role in exogenous activation of these cells during Ebola virus infection.     

Lethal effects of experimental warming approximating a future climate scenario on southern African quartz-field succulents: a pilot study

Here we examine the response of succulents in a global biodiversity hot spot to experimental warming consistent with a future African climate scenario. Passive daytime warming (averaging 5.5 degrees C above ambient) of the natural vegetation was achieved with 18 transparent hexagonal open-top chamber arrays randomized in three different quartz-field communities. After 4-months summer treatment, the specialized-dwarf and shrubby succulents displayed between 2.1 and 4.9 times greater plant and canopy mortalities in the open-top chambers than in the control plots. Those surviving in cooler ventilated areas and shaded refuges in the chambers had lower starch concentrations and water contents; the shrubby succulents also exhibited diminished chlorophyll concentrations. It is concluded that current thermal regimes are likely to be closely proximate to tolerable extremes for many endemic succulents in the region, and that anthropogenic warming could significantly exceed their thermal thresholds. Further investigation is required to elucidate the importance of associated moisture deficits in these warming experiments, a potential consequence of supplementary (fog and dew) precipitation interception by open-top chambers and higher evaporation therein, on plant mortalities.