A large disordered region confers a wide spanning volume to vertebrate Suppressor of Fused as shown in a trans-species solution study.

Hedgehog (Hh) pathway inhibition by the conserved protein Suppressor of Fused (SuFu) is crucial to vertebrate development. By constrast, SuFu loss-of-function mutant has little effect in drosophila. Previous publications showed that the crystal structures of human and drosophila SuFu consist of two ordered domains that are capable of breathing motions upon ligand binding. However, the crystal structure of human SuFu does not give information about twenty N-terminal residues (IDR1) and an eighty-residue-long region predicted as disordered (IDR2) in the C-terminus, whose function is important for the pathway repression. These two intrinsically disordered regions (IDRs) are species-dependent. To obtain information about the IDR regions, we studied full-length SuFu's structure in solution, both with circular dichroism and small angle X-ray scattering, comparing drosophila, zebrafish and human species, to better understand this considerable difference. Our studies show that, in spite of similar crystal structures restricted to ordered domains, drosophila and vertebrate SuFu have very different structures in solution. The IDR2 of vertebrates spans a large area, thus enabling it to reach for partners and be accessible for post-translational modifications. Furthermore, we show that the IDR2 region is highly conserved within phyla but varies in length and sequence, with insects having a shorter disordered region while that of vertebrates is broad and mobile. This major variation may explain the different phenotypes observed upon SuFu removal.

Microbial fermentation of flaxseed fibers modulates the transcriptome of GPR41-expressing enteroendocrine cells and protects mice against diet-induced obesity.

Dietary fibers, an integral part of the human diet, require the enzymatic activity of the gut microbiota for complete metabolism into short-chain fatty acids (SCFAs). SCFAs are important modulators of host metabolism and physiology and act in part as signaling molecules by activating G protein-coupled receptors (GPCRs), such as GPR41. Flaxseed fibers improve metabolism in rodents and mice, but their fermentation profiles, effects on enteroendocrine cells, and associated metabolic benefits are unknown. We fed GPR41-red fluorescent protein mice, an enteroendocrine reporter mouse strain, chow, high-fat diet (HFD), or HFD supplemented either with 10% nonfermentable fiber cellulose or fermentable flaxseed fibers for 12 wk to assess changes in cecal gut microbiota, enteroendocrine cell transcriptome in the ileum and colon, and physiological parameters. We observed that flaxseed fibers restructured the gut microbiota and promoted proliferation of the genera Bifidobacterium and Akkermansia compared with HFD. The shifts in cecal bacterial composition restored levels of the SCFAs butyrate similar to the chow diet, resulting in colonic but not ileal enteroendocrine cell transcriptional changes in genes related to cell cycle, mRNA, and protein transport compared with HFD. Consistent with the effects on enteroendocrine functions, flaxseed fibers also protected mice from diet-induced obesity, potentially by preventing a reduction in energy expenditure induced by an HFD. Our study shows that flaxseed fibers alter cecal microbial ecology, are fermented to SCFAs in the cecum, and modulate enteroendocrine cell transcriptome in the colon, which may contribute to their metabolically favorable phenotype.

Anxiolytic-Like Effects of Increased Ghrelin Receptor Signaling in the Amygdala.

BACKGROUND: Besides the well-known effects of ghrelin on adiposity and food intake regulation, the ghrelin system has been shown to regulate aspects of behavior including anxiety and stress. However, the effect of virus-mediated overexpression of the ghrelin receptor in the amygdala has not previously been addressed directly. METHODS: First, we examined the acute effect of peripheral ghrelin administration on anxiety- and depression-like behavior using the open field, elevated plus maze, forced swim, and tail suspension tests. Next, we examined the effect of peripheral ghrelin administration and ghrelin receptor deficiency on stress in a familiar and social environment using the Intellicage system. Importantly, we also used a novel approach to study ghrelin receptor signaling in the brain by overexpressing the ghrelin receptor in the amygdala. We examined the effect of ghrelin receptor overexpression on anxiety-related behavior before and after acute stress and measured the modulation of serotonin receptor expression. RESULTS: We found that ghrelin caused an anxiolytic-like effect in both the open field and elevated plus maze tests. Additionally, it attenuated air-puff-induced stress in the social environment, while the opposite was shown in ghrelin receptor deficient mice. Finally, we found that overexpression of the ghrelin receptor in the basolateral division of the amygdala caused an anxiolytic-like effect and decreased the 5HT1a receptor expression. CONCLUSIONS: Ghrelin administration and overexpression of the ghrelin receptor in the amygdala induces anxiolytic-like behavior. Since the ghrelin receptor has high constitutive activity, ligand-independent signaling in vivo may be important for the observed anxiolytic-like effects. The anxiolytic effects seem to be mediated independently from the HPA axis, potentially engaging the central serotonin system.

Dataset on the soils of medieval archaeological monuments in the forest-steppe zone of the East European plain.

One of the natural archives that can save information about the environmental conditions of the past is soils buried under embankments of burial complexes. Due to isolation from external environmental factors soils retain information about the features of the natural environment at the time of its burial. In this work we present a dataset on soils buried under four mounds in the Middle Ages. The soils were buried under mounds in a short time interval - 25-50 years. For comparison, the data on the surface soil located near the barrows are also presented. Obtained dataset includes detailed morphological field description of the soils and their physico-chemical analysis, such as granulometry, elemental analysis, fractions of iron and selected chemical data. Obtained data can be used to identify the dynamics of forest-steppe landscapes in the XIth century. The Medieval Warm Period and the subsequent humidisation of the climate over a short time interval had a significant impact on natural conditions and the migration of the population of the steppes of Eurasia. A comparative analysis of the properties of soils buried under archaeological sites of different ages allows examining in details the changes in the natural environment and its components over time. Moreover, soils are capable of storing a whole range of additional features of non-pedogenic origin that can be used for a more detailed reconstruction of the natural environment. The data on spores, pollen and non-pollen palynomorphs of the soil profiles are also presented in this article.

Ghrelin-mediated improvements in the metabolic phenotype in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is a heritable neurodegenerative disorder, characterised by metabolic disturbances, along with cognitive and psychiatric impairments. Targeting metabolic HD dysfunction via the maintenance of body weight and fat mass and restoration of peripheral energy metabolism can improve the progression of neurological symptoms. In this respect, we focused on the therapeutic potential of the orexigenic peptide hormone ghrelin, which plays an important role in promoting a positive energy balance. In the present study, we found a significant disruption of circadian metabolic regulation in a R6/2 mouse HD model in the late stage of disease. Daily circadian rhythms of activity, energy expenditure, respiratory exchange ratio and feeding were strongly attenuated in R6/2 mice. During the rest phase, R6/2 mice had a higher total activity, elevated energy expenditure and excessive water consumption compared to control mice. We also found that, in the late stage of disease, R6/2 mice had ghrelin axis deficiency as a result of low circulating ghrelin levels, in addition to down-regulation of the ghrelin receptor and several key signalling molecules in the hypothalamus, as well as a reduced responsiveness to exogenous peripheral ghrelin. We demonstrated that, in pre-symptomatic mice, responsiveness to ghrelin is preserved. Chronic ghrelin treatment efficiently increased lean body mass and decreased the energy expenditure and fat utilisation of R6/2 mice in the early stage of disease. In addition, ghrelin treatment was also effective in the normalisation of drinking behaviour and the rest activity of these mice. Ghrelin treatment could provide a novel therapeutic possibility for delaying disease progression; however, deficiency in ghrelin receptor expression could limit its therapeutic potential in the late stage of disease.

The aromatic amino acid sensor GPR142 controls metabolism through balanced regulation of pancreatic and gut hormones.

OBJECTIVES: GPR142, which is highly expressed in pancreatic islets, has recently been deorphanized as a receptor for aromatic amino acids; however, its physiological role and pharmacological potential is unclear. METHODS AND RESULTS: We find that GPR142 is expressed not only in ß- but also in α-cells of the islets as well as in enteroendocrine cells, and we confirm that GPR142 is a highly selective sensor of essential aromatic amino acids, in particular Trp and oligopeptides with N-terminal Trp. GPR142 knock-out mice displayed a very limited metabolic phenotype but demonstrated that L-Trp induced secretion of pancreatic and gut hormones is mediated through GPR142 but that the receptor is not required for protein-induced hormone secretion. A synthetic GPR142 agonist stimulated insulin and glucagon as well as GIP, CCK, and GLP-1 secretion. In particular, GIP secretion was sensitive to oral administration of the GPR142 agonist an effect which in contrast to the other hormones was blocked by protein load. Oral administration of the GPR142 agonist increased [3H]-2-deoxyglucose uptake in muscle and fat depots mediated through insulin action while it lowered liver glycogen conceivably mediated through glucagon, and, consequently, it did not lower total blood glucose. Nevertheless, acute administration of the GPR142 agonist strongly improved oral glucose tolerance in both lean and obese mice as well as Zucker fatty rat. Six weeks in-feed chronic treatment with the GPR142 agonist did not affect body weight in DIO mice, but increased energy expenditure and carbohydrate utilization, lowered basal glucose, and improved insulin sensitivity. CONCLUSIONS: GPR142 functions as a sensor of aromatic amino acids, controlling GIP but also CCK and GLP-1 as well as insulin and glucagon in the pancreas. GPR142 agonists could have novel interesting potential in modifying metabolism through a balanced action of gut hormones as well as both insulin and glucagon.

GPCR-Mediated Signaling of Metabolites.

In addition to their bioenergetic intracellular function, several classical metabolites act as extracellular signaling molecules activating cell-surface G-protein-coupled receptors (GPCRs), similar to hormones and neurotransmitters. "Signaling metabolites" generated from nutrients or by gut microbiota target primarily enteroendocrine, neuronal, and immune cells in the lamina propria of the gut mucosa and the liver and, through these tissues, the rest of the body. In contrast, metabolites from the intermediary metabolism act mainly as metabolic stress-induced autocrine and paracrine signals in adipose tissue, the liver, and the endocrine pancreas. Importantly, distinct metabolite GPCRs act as efficient pro- and anti-inflammatory regulators of key immune cells, and signaling metabolites may thus function as important drivers of the low-grade inflammation associated with insulin resistance and obesity. The concept of key metabolites as ligands for specific GPCRs has broadened our understanding of metabolic signaling significantly and provides a number of novel potential drug targets.

Effects of FGF receptor peptide agonists on animal behavior under normal and pathological conditions.

Hexafins are recently identified low-molecular-weight peptide agonists of the fibroblast growth factor receptor (FGFR), derived from the beta6-beta7 loop region of various FGFs. Synthetic hexafin peptides have been shown to bind to and induce tyrosine phosphorylation of FGFR1, stimulate neurite outgrowth, and promote neuronal survival in vitro. Thus, the pronounced biological activities of hexafins in vitro make them attractive compounds for pharmacological studies in vivo. The present study investigated the effects of subcutaneous administration of hexafin1 and hexafin2 (peptides derived from FGF1 and FGF2, respectively) on social memory, exploratory activity, and anxiety-like behavior in adult rats. Treatment with hexafin1 and hexafin2 resulted in prolonged retention of social memory. Furthermore, rats treated with hexafin2 exhibited decreased anxiety-like behavior in the elevated plus maze. Employing an R6/2 mouse model of Huntington's disease (HD), we found that although hexafin2 did not affect the progression of motor symptoms, it alleviated deficits in activity related to social behavior, including sociability and social novelty. Thus, hexafin2 may have therapeutic potential for the treatment of HD.

Detection of early behavioral markers of Huntington's disease in R6/2 mice employing an automated social home cage.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder, for which no known cure or effective treatment exists. To facilitate the search for new potential treatments of HD, an automated system for analyzing the behavior of transgenic HD mice is urgently needed. A recently developed behavior screening system, the IntelliCage, allows automated testing of mouse behavior in the home cage employing individual recognition of animals living in social groups. The present study validates the ability of the IntelliCage system to detect behavioral and cognitive dysfunction in R6/2 mice, an established transgenic model of HD. The results indicate that the IntelliCage is a reliable system for recording exploratory activity, drinking behavior, circadian rhythm, spatial preference, and cognition in mice during prolonged periods of assessment. The system detected early dysfunctional behaviors in R6/2 mice, such as decrease in exploratory activity, sleep disturbances, increased drinking, and repetitive behavior. Additionally, the use of various learning tasks, such as spatial avoidance and spatial patrolling, revealed early cognitive changes in R6/2 mice. The simple learning tasks may be used at both early and late stages of the disease.

An automatic procedure for evaluation of single cell motility.

BACKGROUND: Cell motility is vital in many physiological and pathological processes, such as embryogenesis, inflammation, wound healing, and metastasis. However, the time-consuming step in the evaluation of individual cell motility is the analysis of hundreds of recorded images of cell cultures in general consisting of retrieving images, one at a time, and marking the positions of individual cells by a computer mouse. Therefore, the aim of the present study was to develop a novel automatic procedure for the evaluation of cell motility. MATERIALS AND METHODS: The procedure was tested on fibroblasts and glioma and adenocarcinoma cells engineered to express the green fluorescent protein by either transient transfection or adenovirus transduction, allowing automatic recognition of cell coordinates on retrieved images. RESULTS: The effects of serum growth factors, teratogenic compounds, and overexpression of transcription factors on the motile behavior of cultured cells were determined. Cell motility was estimated by both manual and automatic marking of cell position and subsequently motility parameters were computed. The results obtained by the two procedures were found to correlate significantly. CONCLUSIONS: We developed a procedure allowing automatic video recording of sparsely seeded cells transfected with a plasmid or tranduced with a recombinant virus expressing the enhanced green fluorescent protein (EGFP).

Triple effect of mimetic peptides interfering with neural cell adhesion molecule homophilic cis interactions.

The neural cell adhesion molecule (NCAM) is pivotal in neural development, regeneration, and learning. Here we characterize two peptides, termed P1-B and P2, derived from the homophilic binding sites in the first two N-terminal immunoglobulin (Ig) modules of NCAM, with regard to their effects on neurite extension and adhesion. To evaluate how interference of these mimetic peptides with NCAM homophilic interactions in cis influences NCAM binding in trans, we employed a coculture system in which PC12-E2 cells were grown on monolayers of fibroblasts with or without NCAM expression and the rate of neurite outgrowth subsequently was analyzed. P2, but not P1-B, induced neurite outgrowth in the absence of NCAM binding in trans. When PC12-E2 cells were grown on monolayers of NCAM-expressing fibroblasts, the effect of both P1-B and P2 on neurite outgrowth was dependent on peptide concentrations. P1-B and P2 acted as conventional antagonists, agonists, and reverse agonists of NCAM at low, intermediate, and high peptide concentrations, respectively. The demonstrated in vitro triple pharmacological effect of mimetic peptides interfering with the NCAM homophilic cis binding will be valuable for the understanding of the actions of these mimetics in vivo.

Modulation of the homophilic interaction between the first and second Ig modules of neural cell adhesion molecule by heparin.

The second Ig module (IgII) of the neural cell adhesion molecule (NCAM) is known to bind to the first Ig module (IgI) of NCAM (so-called homophilic binding) and to interact with heparan sulfate and chondroitin sulfate glycoconjugates. We here show by NMR that the heparin and chondroitin sulfate-binding sites (HBS and CBS, respectively) in IgII coincide, and that this site overlaps with the homophilic binding site. Using NMR and surface plasmon resonance (SPR) analyses we demonstrate that interaction between IgII and heparin indeed interferes with the homophilic interaction between IgI and IgII. Accordingly, we show that treatment of cerebellar granule neurons (CGNs) with heparin inhibits NCAM-mediated outgrowth. In contrast, treatment with heparinase III or chondroitinase ABC abrogates NCAM-mediated neurite outgrowth in CGNs emphasizing the importance of the presence of heparan/chondroitin sulfates for proper NCAM function. Finally, a peptide encompassing HBS in IgII, termed the heparin-binding peptide (HBP), is shown to promote neurite outgrowth in CGNs. These observations indicate that neuronal differentiation induced by homophilic NCAM interaction is modulated by interactions with heparan/chondroitin sulfates.