Mutagenesis and computer modeling studies of a GPCR conserved residue W5.43(194) in ligand recognition and signal transduction for CB2 receptor

W5.43(194), a conserved tryptophan residue among G-protein coupled receptors (GPCRs) and cannabinoid receptors (CB), was examined in the present report for its significance in CB2 receptor ligand binding and adenylyl cyclase (AC) activity. Computer modeling postulates that this site in CB2 may be involved in the affinity of WIN55212-2 and SR144528 through aromatic contacts. In the present study, we reported that a CB2 receptor mutant, W5.43(194)Y, which had a tyrosine (Y) substitution for tryptophan (W), retained the binding affinity for CB agonist CP55940, but reduced binding affinity for CB2 agonist WIN55212-2 and inverse agonist SR144528 by 8-fold and 5-fold, respectively; the CB2 W5.43(194)F and W5.43(194)A mutations significantly affect the binding activities of CP55940, WIN55212-2 and SR144528. Furthermore, we found that agonist-mediated inhibition of the forskolin-induced cAMP production was dramatically diminished in the CB2 mutant W5.43(194)Y, whereas W5.43(194)F and W5.43(194)A mutants resulted in complete elimination of downstream signaling, suggesting that W5.43(194) was essential for the full activation of CB2. These results indicate that both aromatic interaction and hydrogen bonding are involved in ligand binding for the residue W5.43(194), and the mutations of this tryptophan site may affect the conformation of the ligand binding pocket and therefore control the active conformation of the wild type CB2 receptor. W5.43(194)Y/F/A mutations also displayed noticeable enhancement of the constitutive activation probably attributed to the receptor conformational changes resulted from the mutations.

Two enzymes responsible for the formation of herbivore-induced volatiles of maize, the methyltransferase AAMT1 and the terpene synthase TPS23, are regulated by a similar signal transduction pathway

Herbivore-induced volatiles play an important role in the indirect defense of plants. After herbivore damage, volatiles are released from the plant and can attract herbivore enemies that protect the plant from additional damage. The herbivore-induced volatile blend is complex and usually consists of mono- and sesquiterpenes, aromatic compounds, and indole. Although these classes of compounds are generally produced at different times after herbivore damage, the release of the terpene (E)-β-caryophyllene and the aromatic ester methyl anthranilate appear to be tightly coordinated. We have studied the herbivore induction patterns of two terpene synthases from Zea mays L. (Poaceae), TPS23 and TPS10, as well as S-adenosyl-L-methionine:anthranilic acid carboxyl methyltransferases (AAMT1), which are critical for the production of terpenes and anthranilate compounds, respectively. The transcript levels of tps23 and aamt1 displayed the same kinetics after damage by the larvae of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), and showed the same organ-specific and haplotype-specific expression patterns. Despite its close functional relation to TPS23, the terpene synthase TPS10 is not expressed in roots and does not display the haplotype-specific expression pattern. The results indicate that the same JA-mediated signaling cascade maycontrol the production of both the terpene (E)-β-caryophyllene and aromatic ester methyl anthranilate.

CE provides evidence of the stereoselective hydroxylation of norketamine in equines

CE with multiple isomer sulfated-CD as selector was used for the simultaneous analysis of the stereoisomers of ketamine, norketamine, 5,6-dehydronorketamine and hydroxylated metabolites of norketamine in liquid/liquid extracts of (i) in vitro incubations with ketamine or norketamine and equine liver microsomes and (ii) plasma and urine of ponies receiving a target-controlled infusion of ketamine under isoflurane anesthesia. Hydroxynorketamine metabolites with the hydroxy group at the cyclohexanone ring could be shown to be formed stereoselectively both in vitro and in vivo. Due to the lack of standard compounds, urinary extracts were fractionated by HPLC followed by characterization of the collected fractions with CE and LC-MS(n) with 0.7 mmu mass discrimination. Comparison of LC-MS(n) data obtained with the fractions, an in vitro microsomal sample, and both pony urine and hydrolyzed pony urine led to the identification of four hydroxylated norketamine metabolites with hydroxylation at the cyclohexanone ring, two with hydroxylation at the aromatic ring and four hydroxylated metabolites of ketamine. Due to the lower detection sensitivity, only the four hydroxynorketamine metabolites with hydroxylation at the cyclohexanone ring were observed by CE. The data suggest that demethylation of ketamine followed by hydroxylation of norketamine at the cyclohexanone ring is the major metabolic pathway in equine species and that the ketamine metabolism is highly stereoselective.

Luminescence Enhancement after Adding Stoppers to Europium(III) Nanozeolite L

Stopper molecules attached to nanozeolite L (NZL) boost the luminescence of confined Eu3+-β-diketonate complexes. The mechanism that is responsible was elucidated by comparing two diketonate ligands of different pKa and two aromatic imines, and by applying stationary and time resolved spectroscopy. The result is that the presence of the imidazolium based stopper is favorable to the sustainable formation of Eu3+-β-diketonate complexes with high coordination by decreasing the proton strength inside the channels of NZL. A consequence is that strongly luminescent transparent films can be prepared using aqueous suspension of the stopper modified composites.

Diagenesis of a light, tight-oil chert reservoir at the Ediacaran/Cambrian boundary, Sultanate of Oman

The Al Shomou Silicilyte Member (Athel Formation) in the South Oman Salt Basin shares many of the characteristics of a light, tight-oil (LTO) reservoir: it is a prolifi c source rock mature for light oil, it produces light oil from a very tight matrix and reservoir, and hydraulic fracking technology is required to produce the oil. What is intriguing about the Al Shomou Silicilyte, and different from other LTO reservoirs, is its position related to the Precambrian/Cambrian Boundary (PCB) and the fact that it is a ‘laminated chert‘ rather than a shale. In an integrated diagenetic study we applied microstructural analyses (SEM, BSE) combined with state-of-the-art stable isotope and trace element analysis of the silicilyte matrix and fractures. Fluid inclusion microthermometry was applied to record the salinity and minimum trapping temperatures. The microstructural investigations reveal a fi ne lamination of the silicilyte matrix with a mean lamina thickness of ca. 20 μm consisting of predominantly organic matter-rich and fi nely crystalline quartz-rich layers, respectively. Authigenic, micron-sized idiomorphic quartz crystals are the main matrix components of the silicilyte. Other diagenetic phases are pyrite, apatite, dolomite, magnesite and barite cements. Porosity values based on neutron density logs and core plug data indicate porosity in the silicilyte ranges from less than 2% to almost to 40%. The majority of the pore space in the silicilyte is related to (primary) inter-crystalline pores, with locally important oversized secondary pores. Pore casts of the silica matrix show that pores are extremely irregular in three dimensions, and are generally interconnected by a complex web or meshwork of fi ne elongate pore throats. Mercury injection capillary data are in line with the microstructural observations suggesting two populations of pore throats, with an effective average modal diameter of 0.4 μm. The acquired geochemical data support the interpretation that the primary source of the silica is the ambient seawater rather than hydrothermal or biogenic. A maximum temperature of ca. 45°C for the formation of microcrystalline quartz in the silicilyte is good evidence that the lithifi cation and crystallization of quartz occurred in the fi rst 5 Ma after deposition. Several phases of brittle fracturing and mineralization occurred in response to salt tectonics during burial. The sequences of fracture-fi lling mineral phases (dolomite - layered chalcedony – quartz – apatite - magnesite I+II - barite – halite) indicates a complex fl uid evolution after silicilyte lithifi cation. Primary, all-liquid fl uid inclusions in the fracturefi lling quartz are good evidence of growth beginning at low temperatures, i.e. ≤ 50ºC. Continuous precipitation during increasing temperature and burial is documented by primary two-phase fl uid inclusions in quartz cements that show brines at 50°C and fi rst hydrocarbons at ca. 70°C. The absolute timing of each mineral phase can be constrained based on U-Pb geochronometry, and basin modelling. Secondary fl uid inclusions in quartz, magnesite and barite indicate reactivation of the fracture system after peak burial temperature during the major cooling event, i.e. uplift, between 450 and 310 Ma. A number of fi rst-order trends in porosity and reservoir-quality distribution are observed which are strongly related to the diagenetic and fl uid history of the reservoir: the early in-situ generation of hydrocarbons and overpressure development arrests diagenesis and preserves matrix porosity. Chemical compaction by pressure dissolution in the fl ank areas could be a valid hypothesis to explain the porosity variations in the silicilitye slabs resulting in lower porosity and poorer connectivity on the fl anks of the reservoir. Most of the hydrocarbon storage and production comes from intervals characterized by Amthor et al. 114488 preserved micropores, not hydrocarbon storage in a fracture system. The absence of oil expulsion results in present-day high oil saturations. The main diagenetic modifi cations of the silicilyte occurred and were completed relatively early in its history, i.e. before 300 Ma. An instrumental factor for preserving matrix porosity is the diffi culty for a given slab to evacuate all the fl uids (water and hydrocarbons), or in other words, the very good sealing capacity of the salt embedding the slab.

Prototype of the gas chromatograph-mass spectrometer to investigate volatile species in the lunar soil for the Luna-Resurs mission

In preparation for the Russian Luna-Resurs mission we combined our compact time-of-flight mass spectrometer (TOF-MS) with a chemical pre-separation of the species by gas chromatography (GC). Coupled measurements with both instruments were successfully performed with the prototype of the mass spectrometer and a flight-like gas chromatograph. The system was tested with two test gas mixtures, a mixture of hydrocarbons and a mixture of noble gases. Due to its capability to record mass spectra over the full mass range at once with high sensitivity and a dynamic range of up to 10(6) within 1 s, the TOF-MS system is a valuable extension of the GC analytical system. Based on the measurements with calibration gases performed with the combined GC-MS prototype and under assumption of mean characteristics for the Moon's regolith, the detection limit for volatile species in a soil sample is estimated to 2.10(-10) by mass for hydrocarbons and 2.10(-9) by mass for noble gases. (C) 2015 Elsevier Ltd. All rights reserved.