Characterization of the Catalytically Active Mn(II)-loaded \u3cem\u3eargE\u3c/em\u3e-encoded \u3cem\u3eN\u3c/em\u3e-acetyl-L-ornithine Deacetylase from \u3cem\u3eEscherichia coli\u3c/em\u3e

The catalytically competent Mn(II)-loaded form of the argE-encoded N-acetyl-l-ornithine deacetylase from Escherichia coli (ArgE) was characterized by kinetic, thermodynamic, and spectroscopic methods. Maximum N-acetyl-l-ornithine (NAO) hydrolytic activity was observed in the presence of one Mn(II) ion with k cat and K m values of 550 s−1 and 0.8 mM, respectively, providing a catalytic efficiency (k cat/K m) of 6.9 × 105 M−1 s−1. The ArgE dissociation constant (K d) for Mn(II) was determined to be 0.18 μM, correlating well with a value obtained by isothermal titration calorimetry of 0.30 μM for the first metal binding event and 5.3 μM for the second. An Arrhenius plot of the NAO hydrolysis for Mn(II)-loaded ArgE was linear from 15 to 55 °C, suggesting the rate-limiting step does not change as a function of temperature over this range. The activation energy, determined from the slope of this plot, was 50.3 kJ mol−1. Other thermodynamic parameters were ΔG ‡ = 58.1 kJ mol−1, ΔH ‡ = 47.7 kJ mol−1, and ΔS ‡ = –34.5 J mol−1 K−1. Similarly, plots of lnK m versus 1/T were linear, suggesting substrate binding is controlled by a single step. The natural product, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]leucine (bestatin), was found to be a competitive inhibitor of ArgE with a K i value of 67 μM. Electron paramagnetic resonance (EPR) data recorded for both [Mn(II)_(ArgE)] and [Mn(II)Mn(II)(ArgE)] indicate that the two Mn(II) ions form a dinuclear site. Moreover, the EPR spectrum of [Mn(II)Mn(II)(ArgE)] in the presence of bestatin indicates that bestatin binds to ArgE but does not form a µ-alkoxide bridge between the two metal ions

\u3cem\u3eargE\u3c/em\u3e-Encoded \u3cem\u3eN\u3c/em\u3e-Acetyl-l-Ornithine Deacetylase from \u3cem\u3eEscherichia coli\u3c/em\u3e Contains a Dinuclear Metalloactive Site

The catalytic and structural properties of the argE-encoded N-acetyl-l-ornithine deacetylase (ArgE) from Escherichia coli were investigated. On the basis of kinetic and ITC (isothermal titration calorimetry) data, Zn(II) binds to ArgE with Kd values that differ by ∼20 times. Moreover, ArgE exhibits ∼90% of its full catalytic activity upon addition of one metal ion. Therefore, ArgE behaves similarly to the aminopeptidase from Aeromonas proteolytica (AAP) in that one metal ion is the catalytic metal ion while the second likely plays a structural role. The N-acetyl-l-ornithine (NAO) deacetylase activity of ArgE showed a linear temperature dependence from 20 to 45 °C, indicating that the rate-limiting step does not change over this temperature range. The activation energy for NAO hydrolysis by ArgE was 25.6 kJ/mol when loaded with Zn(II) and 34.3 kJ/mol when loaded with Co(II). Electronic absorption and EPR (electron paramagnetic resonance) spectra of [Co·(ArgE)] and [CoCo(ArgE)] indicate that both divalent metal binding sites are five coordinate. In addition, EPR data show clear evidence of spin−spin coupling between the Co(II) ions in the active site but only after addition of a second equivalent of Co(II). Combination of these data provides the first physical evidence that the ArgE from E. coli contains a dinuclear Zn(II) active site, similar to AAP and the carboxypeptidase G2 from Pseudomonas sp. strain RS-16 (CPG2)

Identification of a Histidine Metal Ligand in the \u3cem\u3eargE\u3c/em\u3e-Encoded \u3cem\u3eN\u3c/em\u3e-Acetyl-L-Ornithine Deacetylase from \u3cem\u3eEscherichia coli\u3c/em\u3e

The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, however, only the H355A enzyme was found to be soluble. Kinetic analysis of the Co(II)-loaded H355A exhibited activity levels that were 380-fold less than Co(II)-loaded WT ArgE. Electronic absorption spectra of Co(II)-loaded H355A-ArgE indicate that the bound Co(II) ion resides in a distorted, five-coordinate environment and Isothermal Titration Calorimetry (ITC) data for Zn(II) binding to the H355A enzyme provided a dissociation constant (Kd) of 39 μM. A three-dimensional homology model of ArgE was generated using the X-ray crystal structure of the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae confirming the assignment of H355 as well as H80 as active site ligands

Structural Characterization of Zn(II)-, Co(II)-, and Mn(II)-loaded Forms of the argE-encoded \u3cem\u3eN\u3c/em\u3e-acetyl-L-ornithine Deacetylase from \u3cem\u3eEscherichia coli\u3c/em\u3e

The Zn, Co, and Mn K-edge extended X-ray absorption fine structure (EXAFS) spectra of the N-acetyl-l-ornithine deacetylase (ArgE) from Escherichia coli, loaded with one or two equivalents of divalent metal ions (i.e., [Zn(II)_(ArgE)], [Zn(II)Zn(II)(ArgE)], [Co(II)_(ArgE)], [Co(II)Co(II)(ArgE)], [Mn(II)_(ArgE)], and [Mn(II)Mn(II)(ArgE)]), were recorded. The Fourier transformed data (FT) for [Zn(II)_(ArgE)], [Zn(II)Zn(II)(ArgE)], [Co(II)_(ArgE)] and [Co(II)Co(II)(ArgE)] are dominated by a peak at 2.05 Å, that can be fit assuming five or six light atom (N,O) scatterers. Inclusion of multiple-scattering contributions from the outer-shell atoms of a histidine-imidazole ring resulted in reasonable Debye–Waller factors for these contributions and a slight reduction in the goodness-of-fit value (f′). Furthermore, the data best fit a model that included a M–M vector at 3.3 and 3.4 Å for Zn(II) and Co(II), respectively, suggesting the formation of a dinuclear site. Multiple scattering contributions from the outer-shell atoms of a histidine-imidazole rings are observed at ~ 3 and 4 Å for Zn(II)- and Co(II)-loaded ArgE suggesting at least one histidine ligand at each metal binding site. Likewise, EXAFS data for Mn(II)-loaded ArgE are dominated by a peak at 2.19 Å that was best fit assuming six light atom (N,O) scatterers. Due to poor signal to noise ratios for the Mn EXAFS spectra, no Mn–Mn vector could be modeled. Peak intensities for [M(II)_(ArgE)] vs. [M(II)M(II)(ArgE)] suggest the Zn(II), Co(II), and Mn(II) bind to ArgE in a cooperative manner. Since no structural data has been reported for any ArgE enzyme, the EXAFS data reported herein represent the first structural glimpse for ArgE enzymes. These data also provide a structural foundation for the future design of small molecules that function as inhibitors of ArgE and may potentially function as a new class of antibiotics

The Black Hole Mass of NGC 4151. II. Stellar Dynamical Measurement from Near-Infrared Integral Field Spectroscopy

We present a revised measurement of the mass of the central black hole (Mbh) in the Seyfert 1 galaxy NGC 4151. The new stellar dynamical mass measurement is derived by applying an axisymmetric orbit-superposition code to near-infrared integral field data obtained using adaptive optics with the Gemini NIFS spectrograph. When our models attempt to fit both the NIFS kinematics and additional low spatial resolution kinematics, our results depend sensitively on how chi-squared is computed--probably a consequence of complex bar kinematics that manifest immediately outside the nuclear region. The most robust results are obtained when only the high spatial resolution kinematic constraints in the nuclear region are included in the fit. Our best estimates for the BH mass and H-band mass-to-light ratio are Mbh~(3.76+/-1.15)E7 Msun (1-sigma error) and M/L(H-band)~0.34+/-0.03 Msun/Lsun (3-sigma error), respectively (the quoted errors reflect the model uncertainties). Our BH mass measurement is consistent with estimates from both reverberation mapping (3.57[+0.45/-0.37]E7 Msun) and gas kinematics (3.0[+0.75/-2.2]E7 Msun; 1-sigma errors), and our best-fit mass-to-light ratio is consistent with the photometric estimate of M/L(H-band)=0.4+/-0.2 Msun/Lsun. The NIFS kinematics give a central bulge velocity dispersion sigma_c=116+/-3 km/s, bringing this object slightly closer to the M-sigma relation for quiescent galaxies. Although NGC 4151 is one of only a few Seyfert 1 galaxies in which it is possible to obtain a direct dynamical BH mass measurement--and thus, an independent calibration of the reverberation mapping mass scale--the complex bar kinematics makes it less than ideally suited for this purpose.Comment: 21 pages, 15 figures. Accepted for publication in Ap

CUMYL-4CN-BINACA Is an Efficacious and Potent Pro-Convulsant Synthetic Cannabinoid Receptor Agonist

Synthetic cannabinoid receptor agonists (SCRAs) are the largest class of new psychoactive substances (NPS). New examples are detected constantly, and some are associated with a series of adverse effects, including seizures. CUMYL-4CN-BINACA (1-(4-cyanobutyl)-N-(2-phenylpropan-2-yl)indazole-3-carboxamide) is structurally related to potent, cumylamine-derived SCRAs such as 5F-CUMYL-PINACA, but is unusual due to a terminal aliphatic nitrile group not frequently encountered in SCRAs or pharmaceuticals. We report here that CUMYL-4CN-BINACA is a potent CB1 receptor agonist (Ki = 2.6 nM; EC50 = 0.58 nM) that produces pro-convulsant effects in mice at a lower dose than reported for any SCRA to date (0.3 mg/kg, i.p). Hypothermic and pro-convulsant effects in mice could be reduced or blocked, respectively, by pretreatment with CB1 receptor antagonist SR141716, pointing to at least partial involvement of CB1 receptors in vivo. Pretreatment with CB2 receptor antagonist AM-630 had no effect on pro-convulsant activity. The pro-convulsant properties and potency of CUMYL-4CN-BINACA may underpin the toxicity associated with this compound in humans

Parameters That Affect Fear Responses in Rodents and How to Use Them for Management

The strong innate fear response shown by laboratory rodents to predator cues could provide powerful and innovative tools for pest management. Predator cues are routinely used to induce fear and anxiety in laboratory rodents for pharmacological studies. However, research on the fear response induced by predator cues in different species of rodents in the wild has been inconclusive with results often contradictory to laboratory experiments. Potential explanations for this inconsistency include the prey's: (i) physiological state; (ii) parasite load; (iii) differential intensity of perceived threats; (iv) fear learning and habituation; and (v) information gathering. In this review, we first explore current knowledge on the sensory mechanisms and capabilities of rodents, followed by the discussion of each of these explanations within the context of their implications for the use of antipredator response as a pest rodent management tool. Finally, we make recommendations on potential solutions and strategies to resolve issues in rodent management related to these hypotheses

Effects of Cannabidiol on exercise physiology and bioenergetics : a randomised controlled pilot trial

Background: Cannabidiol (CBD) has demonstrated anti-inflammatory, analgesic, anxiolytic and neuroprotective effects that have the potential to benefit athletes. This pilot study investigated the effects of acute, oral CBD treatment on physiological and psychological responses to aerobic exercise to determine its practical utility within the sporting context. Methods: On two occasions, nine endurance-trained males (mean±SD V̇O2max: 57.4±4.0 mL·min−1 ·kg−1 ) ran for 60 min at a fixed intensity (70% V̇O2max) (RUN 1) before completing an incremental run to exhaustion (RUN 2). Participants received CBD (300 mg; oral) or placebo 1.5 h before exercise in a randomised, double-blind design. Respiratory gases (V̇O2), respiratory exchange ratio (RER), heart rate (HR), blood glucose (BG) and lactate (BL) concentrations, and ratings of perceived exertion (RPE) and pleasure–displeasure were measured at three timepoints (T1–3) during RUN 1. V̇O2max, RERmax, HRmax and time to exhaustion (TTE) were recorded during RUN 2. Venous blood was drawn at Baseline, Pre- and Post-RUN 1, Post-RUN 2 and 1 h Post-RUN 2. Data were synthesised using Cohen’s dz effect sizes and 85% confidence intervals (CIs). Effects were considered worthy of further investigation if the 85% CI included±0.5 but not zero. Results: CBD appeared to increase V̇O2 (T2:+38±48 mL·min−1, dz: 0.25–1.35), ratings of pleasure (T1:+0.7±0.9, dz: 0.22–1.32; T2:+0.8±1.1, dz: 0.17–1.25) and BL (T2:+3.3±6.4 mmol·L−1, dz:>0.00–1.03) during RUN 1 compared to placebo. No differences in HR, RPE, BG or RER were observed between treatments. CBD appeared to increase V̇O2max (+119±206 mL·min−1, dz: 0.06–1.10) and RERmax (+0.04±0.05 dz: 0.24–1.34) during RUN 2 compared to placebo. No differences in TTE or HRmax were observed between treatments. Exercise increased serum interleukin (IL)-6, IL-1β, tumour necrosis factor-α, lipopolysaccharide and myoglobin concentrations (i.e. Baseline vs. Post-RUN 1, Post-RUN 2 and/or 1-h Post-RUN 2, p’s<0.05). However, the changes were small, making it difficult to reliably evaluate the effect of CBD, where an effect appeared to be present. Plasma concentrations of the endogenous cannabinoid, anandamide (AEA), increased Post-RUN 1 and Post-RUN 2, relative to Baseline and Pre-RUN 1 (p’s<0.05). CBD appeared to reduce AEA concentrations Post-RUN 2, compared to placebo (−0.95±0.64 pmol·mL−1, dz: −2.19, −0.79). Conclusion: CBD appears to alter some key physiological and psychological responses to aerobic exercise without impairing performance. Larger studies are required to confirm and better understand these preliminary findings

Inland Pacific Northwest pasture calendar

The Inland Pacific Northwest (PNW) region, historically referred to as the 'Inland Empire', extends from the Cascade Mountains in the west to former Glacial Lake Missoula in the Rockies in the east and from the Canadian border in the north past the Snake River Canyon in the south. The greatest agricultural enterprises are hay (from various forage species), irrigated and non-irrigated grassland pastures, rangelands, and livestock production. It is also home to numerous wildlife species and flyways for migrating birds. The Inland PNW Pasture Calendar is designed to be a comprehensive guide for improved grassland management in support of forage-livestock systems. Applying the principles described will lead to improved regional economic, ecological, and social sustainability. This publication describes the opportunities and challenges of sustainably raising harvested and grazed forages and developing forage-livestock systems in this incredibly diverse environment. Appendix Chapters provide key information on grass / legume / forb species, how pasture species grow and regrow, soil nutrient management and organic matter, grazing philosophies and systems, matching grazing needs for pasture production, and forage-related animal health issues. Learning these key principles and following best management practices described will lead to improved sustainability of forage-livestock systems