49 research outputs found
Comprehensive review of the detection methods for synthetic cannabinoids and cathinones
Effects of Methylprednisolone and Its Liver-Targeted Dextran Prodrug on Ischemia–Reperfusion Injury in a Rat Liver Transplantation Model
Quantifying the role of online news in linking conservation research to Facebook and Twitter
Conservation Biology293825-83
Wicked conflict: using wicked problem thinking for holistic management of conservation conflict
Qualitative Confirmation of 9 Synthetic Cannabinoids and 20 Metabolites in Human Urine Using LC–MS/MS and Library Search
Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane
Lipopolysaccharide (LPS) is the major glycolipid that is present in the outer membranes (OMs) of most Gram-negative bacteria. LPS molecules are assembled with divalent metal cations in the outer leaflet of the OM to form an impervious layer that prevents toxic compounds from entering the cell. For most Gram-negative bacteria, LPS is essential for growth. In Escherichia coli, eight essential proteins have been identified to function in the proper assembly of LPS following its biosynthesis. This assembly process involves release of LPS from the inner membrane (IM), transport across the periplasm, and insertion into the outer leaflet of the OM. Here, we describe the biochemical characterization of the two-protein complex consisting of LptD and LptE that is responsible for the assembly of LPS at the cell surface. We can overexpress and purify LptD and LptE as a stable complex in a 1∶1 stoichiometry. LptD contains a soluble N-terminal domain and a C-terminal transmembrane domain. LptE stabilizes LptD by interacting strongly with the C-terminal domain of LptD. We also demonstrate that LptE binds LPS specifically and may serve as a substrate recognition site at the OM
Lipoprotein LptE is required for the assembly of LptD by the β-barrel assembly machine in the outer membrane of Escherichia coli
Most Gram-negative bacteria contain lipopolysaccharide (LPS), a glucosamine-based phospholipid, in the outer leaflet of the outer membrane (OM). LPS is unique to the bacterial OM and, in most cases, essential for cell viability. Transport of LPS from its site of synthesis to the cell surface requires eight essential proteins, MsbA and LptABCDEFG. Although the key players have been identified, the mechanism of LPS transport and assembly is not clear. The stable LptD/E complex is present at the OM and functions in the final stages of LPS assembly. Here, we have identified the mutant allele lptE6, which causes a two–amino-acid deletion in the lipoprotein LptE that affects its interaction with LptD. Highly specific suppressor mutations were isolated not only in lptD but also in bamA, which encodes the central component of the β-barrel assembly machine. We show that lptE6 and both suppressor mutations affect the assembly of the LptD/E complex and suggest that the lipoprotein LptE interacts with LptD while this protein is being assembled by the β-barrel assembly machine
Targeted Metabolomic Approach for Assessing Human Synthetic Cannabinoid Exposure and Pharmacology
Designer
synthetic cannabinoids like JWH-018 and AM2201 have unique
clinical toxicity. Cytochrome-P450-mediated metabolism of each leads
to the generation of pharmacologically active (ω)- and (ω-1)-monohydroxyl
metabolites that retain high affinity for cannabinoid type-1 receptors,
exhibit Δ<sup>9</sup>-THC-like effects in rodents, and are conjugated
with glucuronic acid prior to excretion in human urine. Previous studies
have not measured the contribution of the specific (ω-1)-monohydroxyl
enantiomers in human metabolism and toxicity. This study uses a chiral
liquid chromatography–tandem mass spectroscopy approach (LC–MS/MS)
to quantify each specific enantiomer and other nonchiral, human metabolites
of JWH-018 and AM2201 in human urine. The accuracy (average % RE =
18.6) and reproducibility (average CV = 15.8%) of the method resulted
in low-level quantification (average LLQ = 0.99 ng/mL) of each metabolite.
Comparisons with a previously validated nonchiral method showed strong
correlation between the two approaches (average <i>r</i><sup>2</sup> = 0.89). Pilot data from human urine samples demonstrate
enantiospecific excretion patterns. The (<i>S</i>)-isomer
of the JWH-018-(ω-1)-monohydroxyl metabolite was predominantly
excreted (>87%) in human urine as the glucuronic acid conjugate,
whereas
the relative abundance of the corresponding AM2201-(ω-1)-metabolite
was low (<5%) and did not demonstrate enantiospecificity (approximate
50:50 ratio of each enantiomer). The new chiral method provides a
comprehensive, targeted metabolomic approach for studying the human
metabolism of JWH-018 and AM2201. Preliminary evaluations of specific
enantiomeric contributions support the use of this approach in future
studies designed to understand the pharmacokinetic properties of JWH-018
and/or AM2201