Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PP_i). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP_i product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.     

Effects of Acute Exposure to Moderate Altitude on Vascular Function,Metabolism and Systemic Inflammation

Background

Travel to mountain areas is popular. However, the effects of acute exposure to moderate altitude on the cardiovascular system and metabolism are largely unknown.

Objectives

To investigate the effects of acute exposure to moderate altitude on vascular function, metabolism and systemic inflammation.

Methods

In 51 healthy male subjects with a mean (SD) age of 26.9 (9.3) years, oxygen saturation, blood pressure, heart rate, arterial stiffness, lipid profiles, low density lipoprotein (LDL) particle size, insulin resistance (HOMA-index), highly-sensitive C-reactive protein and pro-inflammatory cytokines were measured at 490 m (Zurich) and during two days at 2590 m, (Davos Jakobshorn, Switzerland) in randomized order. The largest differences in outcomes between the two altitudes are reported.

Results

Mean (SD) oxygen saturation was significantly lower at 2590 m, 91.0 (2.0)%, compared to 490 m, 96.0 (1.0)%, p<0.001. Mean blood pressure (mean difference +4.8 mmHg, p<0.001) and heart rate (mean difference +3.3 bpm, p<0.001) were significantly higher at 2590 m, compared to 490 m, but this was not associated with increased arterial stiffness. At 2590 m, lipid profiles improved (median difference triglycerides −0.14 mmol/l, p = 0.012, HDL +0.08 mmol/l, p<0.001, total cholesterol/HDL-ratio −0.25, p = 0.001), LDL particle size increased (median difference +0.45 nm, p = 0.048) and hsCRP decreased (median difference −0.18 mg/l, p = 0.024) compared to 490 m. No significant change in pro-inflammatory cytokines or insulin resistance was observed upon ascent to 2590 m.

Conclusions

Short-term stay at moderate altitude is associated with increased blood pressure and heart rate likely due to augmented sympathetic activity. Exposure to moderate altitude improves the lipid profile and systemic inflammation, but seems to have no significant effect on glucose metabolism.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01130948","term_id":"NCT01130948"}}NCT01130948     

Evolution and Allometry of Calcaneal Elongation in Living and Extinct Primates

Specialized acrobatic leaping has been recognized as a key adaptive trait tied to the origin and subsequent radiation of euprimates based on its observed frequency in extant primates and inferred frequency in extinct early euprimates. Hypothesized skeletal correlates include elongated tarsal elements, which would be expected to aid leaping by allowing for increased rates and durations of propulsive acceleration at takeoff. Alternatively, authors of a recent study argued that pronounced distal calcaneal elongation of euprimates (compared to other mammalian taxa) was related primarily to specialized pedal grasping. Testing for correlations between calcaneal elongation and leaping versus grasping is complicated by body size differences and associated allometric affects. We re-assess allometric constraints on, and the functional significance of, calcaneal elongation using phylogenetic comparative methods, and present an evolutionary hypothesis for the evolution of calcaneal elongation in primates using a Bayesian approach to ancestral state reconstruction (ASR). Results show that among all primates, logged ratios of distal calcaneal length to total calcaneal length are inversely correlated with logged body mass proxies derived from the area of the calcaneal facet for the cuboid. Results from phylogenetic ANOVA on residuals from this allometric line suggest that deviations are explained by degree of leaping specialization in prosimians, but not anthropoids. Results from ASR suggest that non-allometric increases in calcaneal elongation began in the primate stem lineage and continued independently in haplorhines and strepsirrhines. Anthropoid and lorisid lineages show stasis and decreasing elongation, respectively. Initial increases in calcaneal elongation in primate evolution may be related to either development of hallucal-grasping or a combination of grasping and more specialized leaping behaviors. As has been previously suggested, subsequent increases in calcaneal elongation are likely adaptations for more effective acrobatic leaping, highlighting the importance of this behavior in early euprimate evolution.     

A new blunt-snouted dyrosaurid,Anthracosuchus balrogus gen. et sp. nov. (Crocodylomorpha,Mesoeucrocodylia), from the Palaeocene of Colombia

A new exceptionally brevirostrine dyrosaurid is described from the middle Palaeocene (58–60 million years ago) Cerrejón Formation, northeastern Colombia, based on four partial skulls and associated postcrania. This taxon is unique among dyrosaurids not only in skull shape, but also in having orbital tuberosities, and osteoderms that are dorsoventrally thick and unpitted, a trait otherwise unknown in Crocodylomorpha. Results from a cladistic analysis of Dyrosauridae suggest that the new taxon, together with Cretaceous–Palaeocene Chenanisuchus lateroculi from Africa and Cerrejonisuchus improcerus also from the Cerrejón Formation, are the most basal members of the family. Results from a biogeographic analysis indicate at least three independent dispersals of dyrosaurids from Africa to the New World occurred in the Late Cretaceous or early Palaeocene. Widely set orbits in the new taxon indicate a deviation from surface-based predation, characteristic of other dyrosaurids, to sub-surface predation, as in modern Gavialis. Tooth impressions found on turtle shells recovered from the same locality match well with teeth of the new taxon indicating possible predation.http://www.zoobank.org/urn:lsid:zoobank.org:pub:AB2B24A5-27CC-4D3F-B580-F11F17851CE6     

Daily Behavioral Rhythmicity and Organization of the Suprachiasmatic Nuclei in the Diurnal Rodent,Lemniscomys barbarus

Wheel‐running activity was recorded in Lemniscomys barbarus exposed to different lighting conditions. This rodent shows rhythmic locomotor activity under natural twilight‐light/dark (LD) as well as squared‐LD cycles. A mean of 77% of the activity occurred during the light phase. Under different controlled photoperiods, the quantity of daily locomotor activity was relatively stable except for a lower level in the shortest photoperiod tested (LD 06∶18). The duration of the active phase tended to increase with the duration of the light phase, especially in the longer photoperiods. Whatever the lighting conditions, Lemniscomys barbarus started running before lights‐on and stopped after lights‐off. The phase angle of activity offset relative to lights‐off was stable in each squared‐photoperiod, whereas the phase angle of activity onset relative to lights‐on was significantly the highest under the shortest photoperiods. Recording of activity under constant lighting conditions showed that the daily rhythm of locomotor activity is fundamentally circadian. The endogenous period was slightly<24 h (mean=23.8 h) in permanent darkness and>24 h (mean=24.5 h) in continuous light. Re‐entrainment of the locomotor activity rhythm after a 6 h phase advance or delay requires only four days on average. Moreover, the phase‐responses curve to a 30 min light pulse (200 lux) in Lemniscomys barbarus kept in constant dark reveals large phase shifts according to circadian times (CT). With CT0 being defined as the onset of daily activity, maximum phase delay and advance shifts were observed at CT11 (Δ Ψ=‐5.7 h±2.3 h) and CT21 (Δ Ψ =4.9±1.2 h), respectively. Interestingly, the phase‐response curve to light did not show any dead zone. Immunohistochemical staining of the suprachiasmatic nuclei indicates that arginine vasopressin‐immunoreactive cell bodies and fibers delimited a dorsal subregion that extends laterally and medially. The ventral subregion is rich in vasoactive intestinal peptide‐immunoreactive neurones overlapping a smaller area containing gastrin‐releasing peptide‐expressing cells and receives numerous fibers labeled with neuropeptide Y antibody. The results of this study clearly demonstrate that Lemniscomys barbarus is a diurnal species highly sensitive to the shifting effects of light. Overall, this rodent can be considered a new and interesting model for circadian rhythm neurobiology.     

Dufour’s gland secretion,sterility and foraging behavior: Correlated behavior traits in bumblebee workers

Bombus terrestris colonies go through two major phases: the “pre-competition phase” in which the queen is the sole reproducer and aggression is rare, and the “competition phase” in which workers aggressively compete over reproduction. Conflicts over reproduction are partially regulated by a group of octyl esters that are produced in Dufour’s gland of reproductively subordinate workers and protect them from being aggressed. However, workers possess octyl esters even before overt aggression occurs, raising the question of why produce the ester-signal before it is functionally necessary?In most insect societies, foragers show reduced aggression and low dominance rank. We hypothesize that ester production in B. terrestris is not only correlated with sterility but also with foraging, signaling cooperative behavior by subordinate workers. Such a signal helps to maintain social organization, reduce the cost of fights between reproductives and helpers, and increase colony productivity, enabling subordinates to gain greater inclusive fitness. We demonstrate that foragers produce larger amounts of esters compared to non-foragers, and that their amounts positively correlate with foraging efforts. We further suggest that task performance, potential fecundity, and aggression are interlinked, and that worker–worker interactions are involved in regulating foraging behavior.B. terrestris, being an intermediate phase between primitive and derived eusocial insects, provides an excellent model for understanding the evolution of early phases of eusociality. Our results, combined with those in primitively eusocial wasps, suggest that at early stages of social evolution, reproduction was regulated by a “primordial division of labor”, that comprised foragers and reproducers, which further evolved to a more complex division of labor, a hallmark of eusociality.     

VKORC1L1, an Enzyme Rescuing the Vitamin K 2,3-Epoxide Reductase Activity in Some Extrahepatic Tissues during Anticoagulation Therapy

Vitamin K is involved in the γ-carboxylation of the vitamin K-dependent proteins, and vitamin K epoxide is a by-product of this reaction. Due to the limited intake of vitamin K, its regeneration is necessary and involves vitamin K 2,3-epoxide reductase (VKOR) activity. This activity is known to be supported by VKORC1 protein, but recently a second gene, VKORC1L1, appears to be able to support this activity when the encoded protein is expressed in HEK293T cells. Nevertheless, this protein was described as being responsible for driving the vitamin K-mediated antioxidation pathways. In this paper we precisely analyzed the catalytic properties of VKORC1L1 when expressed in Pichia pastoris and more particularly its susceptibility to vitamin K antagonists. Vitamin K antagonists are also inhibitors of VKORC1L1, but this enzyme appears to be 50-fold more resistant to vitamin K antagonists than VKORC1. The expression of Vkorc1l1 mRNA was observed in all tissues assayed, i.e. in C57BL/6 wild type and VKORC1-deficient mouse liver, lung, and testis and rat liver, lung, brain, kidney, testis, and osteoblastic cells. The characterization of VKOR activity in extrahepatic tissues demonstrated that a part of the VKOR activity, more or less important according to the tissue, may be supported by VKORC1L1 enzyme especially in testis, lung, and osteoblasts. Therefore, the involvement of VKORC1L1 in VKOR activity partly explains the low susceptibility of some extrahepatic tissues to vitamin K antagonists and the lack of effects of vitamin K antagonists on the functionality of the vitamin K-dependent protein produced by extrahepatic tissues such as matrix Gla protein or osteocalcin.     

Synthesis,characterization, and biological assessment of the four stereoisomers of the H3 receptor antagonist 5-fluoro-2-methyl-N-[2-methyl-4-(2-methyl[1,3′]bipyrrolidinyl-1′-yl)phenyl]benzamide

This Letter describes the asymmetric synthesis of the four stereoisomers (8a–8d) of a potent and highly selective histamine H₃ receptor (H₃R) antagonist, 5-fluoro-2-methyl-N-[2-methyl-4-(2-methyl[1,3′]bipyrrolidinyl-1′-yl) phenyl]benzamide (1). The physico-chemical properties, in vitro H₃R affinities and ADME of 8a–8d were determined. Stereoisomer 8c (2S,3′S) displayed superior in vitro H₃R affinity over other three stereoisomers and was selected for further profiling in in vivo PK and drug safety. Compound 8c exhibited excellent PK properties with high exposure, desired brain to plasma ratio and reasonable brain half life. However, all stereoisomers showed similar unwanted hERG affinities.     

Discovery of aryl ureas and aryl amides as potent and selective histamine H3 receptor antagonists for the treatment of obesity (Part I)

A series of structurally novel aryl ureas was derived from optimization of the HTS lead as selective histamine H₃ receptor (H₃R) antagonists. The SAR was explored and the data obtained set up the starting point and foundation for further optimization. The most potent tool compounds, as exemplified by compounds 2l, 5b, 5d, and 5e, displayed antagonism potencies in the subnanomolar range in in vitro human-H₃R FLIPR assays and rhesus monkey H₃R binding assays.