Deficiency of PTP1B in POMC neurons leads to alterations in energy balance and homeostatic response to cold exposure

The adipose tissue-derived hormone leptin regulates energy balance through catabolic effects on central circuits, including proopiomelanocortin (POMC) neurons. Leptin activation of POMC neurons increases thermogenesis and locomotor activity. Protein tyrosine phosphatase 1B (PTP1B) is an important negative regulator of leptin signaling. POMC neuron-specific deletion of PTP1B in mice results in reduced high-fat diet-induced body weight and adiposity gain due to increased energy expenditure and greater leptin sensitivity. Mice lacking the leptin gene (ob/ob mice) are hypothermic and cold intolerant, whereas leptin delivery to ob/ob mice induces thermogenesis via increased sympathetic activity to brown adipose tissue (BAT). Here, we examined whether POMC PTP1B mediates the thermoregulatory response of CNS leptin signaling by evaluating food intake, body weight, core temperature (T(C)), and spontaneous physical activity (SPA) in response to either exogenous leptin or 4-day cold exposure (4°C) in male POMC-Ptp1b-deficient mice compared with wild-type controls. POMC-Ptp1b(-/-) mice were hypersensitive to leptin-induced food intake and body weight suppression compared with wild types, yet they displayed similar leptin-induced increases in T(C). Interestingly, POMC-Ptp1b(-/-) mice had increased BAT weight and elevated plasma triiodothyronine (T(3)) levels in response to a 4-day cold challenge, as well as reduced SPA 24 h after cold exposure, relative to controls. These data show that PTP1B in POMC neurons plays a role in short-term cold-induced reduction of SPA and may influence cold-induced thermogenesis via enhanced activation of the thyroid axis.     

Ghrelin and food reward: the story of potential underlying substrates

The incidence of obesity is increasing at an alarming rate and this worldwide epidemic represents a significant decrease in life span and quality of life of a large part of the affected population. Therefore an understanding of mechanisms underlying food overconsumption and obesity development is urgent and essential to find potential treatments. Research investigating mechanisms underlying obesity and the control of food intake has recently experienced a major shift in focus, from the brain's hypothalamus to additional important neural circuits controlling emotion, cognition and motivated behavior. Among them, the mesolimbic system, and the changes in reward and motivated behavior for food, emerge as new promising treatment targets. Furthermore, there is also growing appreciation of the impact of peripheral hormones that signal nutrition status to the mesolimbic areas, and especially the only known circulating orexigenic hormone, ghrelin. This review article provides a synthesis of recent evidence concerning the impact of manipulation of ghrelin and its receptor on models of food reward/food motivation behavior and the mesolimbic circuitry. Particular attention is given to the potential neurocircuitry and neurotransmitter systems downstream of ghrelin's effects on food reward.     

Insights into xanthine riboswitch structure and metal ion-mediated ligand recognition

Riboswitches are conserved functional domains in mRNA that mostly exist in bacteria. They regulate gene expression in response to varying concentrations of metabolites or metal ions. Recently, the NMT1 RNA motif has been identified to selectively bind xanthine and uric acid, respectively, both are involved in the metabolic pathway of purine degradation. Here, we report a crystal structure of this RNA bound to xanthine. Overall, the riboswitch exhibits a rod-like, continuously stacked fold composed of three stems and two internal junctions. The binding-pocket is determined by the highly conserved junctional sequence J1 between stem P1 and P2a, and engages a long-distance Watson–Crick base pair to junction J2. Xanthine inserts between a G–U pair from the major groove side and is sandwiched between base triples. Strikingly, a Mg²⁺ ion is inner-sphere coordinated to O6 of xanthine and a non-bridging oxygen of a backbone phosphate. Two further hydrated Mg²⁺ ions participate in extensive interactions between xanthine and the pocket. Our structure model is verified by ligand binding analysis to selected riboswitch mutants using isothermal titration calorimetry, and by fluorescence spectroscopic analysis of RNA folding using 2-aminopurine-modified variants. Together, our study highlights the principles of metal ion-mediated ligand recognition by the xanthine riboswitch.     

Different temperature sensitivity and kinetics of soil enzymes indicate seasonal shifts in C,N and P nutrient stoichiometry in acid forest soil

Acid forest soils in the Bohemian Forest in Central Europe are biogeochemically imbalanced in organic C, N and P processing. We hypothesized that these imbalances can be due to different temperature sensitivities of soil enzyme activities and their affinities to substrate in litter and organic soil horizons. We measured potential activities of five main soil enzymes (β-glucosidase, cellobiohydrolase, Leu-aminopeptidase, Ala-aminopeptidase, and phosphatase) responsible for organic carbon, nitrogen and phosphorus acquisition. We also modeled potential in situ enzyme activities and nutrient release based on continuous in situ temperature measurements. We determined basic kinetic parameters (K_m, V_max), enzyme efficiencies (k_cat) and temperature sensitivities (E_a and Q₁₀) according to Michaelis–Menten kinetic and modified Arrhenius models. Our results showed significant differences in substrate affinities between the litter and organic soil horizons. Higher aminopeptidase affinity (lower K_m) in the litter soil horizon can lead to leaching of peptidic compounds to lower soil horizons. β-Glucosidase and phosphatase showed high temperature response following the Arrhenius model. However, both aminopeptidases showed no or even decreased activity with increasing temperature. The aminopeptidase temperature insensitivity means that peptidic compounds are degraded at the same or even lower rate in warmer and colder periods of the year in acid forest soils. This imbalance results in different release of available nutrients from plant litter and soil organic matter which may affect bacterial and fungal community composition and nutrient leaching from these ecosystems.     

Double trouble—Buffer selection and His-tag presence may be responsible for nonreproducibility of biomedical experiments

The availability of purified and active protein is the starting point for the majority of in vitro biomedical, biochemical, and drug discovery experiments. The use of polyhistidine affinity tags has resulted in great increases of the efficiency of the protein purification process, but can negatively affect structure and/or activity measurements. Similarly, buffer molecules may perturb the conformational stability of a protein or its activity. During the determination of the structure of a Gcn5-related N-acetyltransferase (GNAT) from Pseudomonas aeruginosa (PA4794), we found that both HEPES and the polyhistidine affinity tag bind (separately) in the substrate-binding site. In the case of HEPES, the molecule induces conformational changes in the active site, but does not significantly affect enzyme activity. In contrast, the uncleaved His-tag does not induce major conformational changes but acts as a weak competitive inhibitor of peptide substrate. In two other GNAT enzymes, we observed that the presence of the His-tag had a strong influence on the activity of these proteins. The influence of protein preparation on functional studies may affect the reproducibility of experiments in other laboratories, even when changes between protocols seem at first glance to be insignificant. Moreover, the results presented here show how critical it is to adjust the experimental conditions for each protein or family of proteins, and investigate the influence of these factors on protein activity and structure, as they may significantly alter the effectiveness of functional characterization and screening methods. Thus, we show that a polyhistidine tag and the buffer molecule HEPES bind in the substrate-binding site and influence the conformation of the active site and the activity of GNAT acetyltransferases. We believe that such discrepancies can influence the reproducibility of some experiments and therefore could have a significant “ripple effect” on subsequent studies.     

Distance between south-European and south-west Asiatic refugial areas involved morphological differentiation: Pinus sylvestris case study

The phenotypic differentiation of relic P. sylvestris in southern Europe and southwestern Asia was verified using thirty-two populations sampled from the Iberian Peninsula, Massif Central, Balkan Peninsula, Crimea and Anatolia. Twenty-one morphological and anatomical needle traits and 18 cone morphological characteristics were examined to describe the population diversity and differentiation. The needle characters were not correlated to those of cone. The differences between regions were significant based on 12 needle and 9 cone characteristics, suggesting spatial isolation. The differentiation between the Iberian and Anatolian populations was the highest, which indicates the isolation by distance. The high level of morphological differentiation was also found among Iberian populations, supporting the already known complex history of the species in that region. Populations within other regions were differentiated at lower levels; however, the West Anatolian populations differed morphologically from the eastern ones. The described pattern of morphological differentiation supports the idea of the long-lasting existence of P. sylvestris in the south-European and Anatolian mountain regions. To conserve this variation, seed transfer between regions in the forest economy should be restricted.     

Comparison of cerebral base arteries in antelopes of Tragelaphus,Taurotragus and Boselaphus genera

Studies were conducted on preparations of head arteries, including cerebral base arteries of sitatunga (n = 14), nyala n = 16) and Greater kudu n = 4) of Tragelaphus genus, Common eland n = 7) from the tribe of Tragelaphini and the largest Asiatic Nilgai antelope n = 9) from Boselaphini tribe. Blood vessels of the cerebral arterial circle in studied antelopes were found to arise from terminal division of intracranial segments of internal carotid arteries, which emerge from the pairwise rostral epidural rete mirabile. Due to obliteration of the extracranial segment of internal carotid artery, the cerebral arterial circle of studied antelopes is supplied with blood mainly through maxillary artery, with mediation of blood vessels of rostral epidural rete mirabile. The system of cerebral base arteries in selected representatives of Tragelaphus, Taurotragus and Boselaphus, in contrast to other species of Bovinae subfamily, manifests the absence of caudal epidural rete mirabile. The pattern of cerebral base arteries in studied antelopes is consistent with position of the species in taxonomy worked out by Simpson (Bull Am Mus Nat Hist 85:1–350, 1945) and McKenna and Bell (Classification of mammals above the species level. Columbia University Press, New York, pp I–XII, 1997).