Oxidation of lysergic acid diethylamide (LSD) by peroxidases: a new metabolic pathway

Lysergic acid diethylamide (LSD) is a potent hallucinogen that is primarily metabolized to 2-oxo-3-hydroxy-LSD (O-H-LSD) and N-desmethyl-LSD (nor-LSD) by cytochrome P450 complex liver enzymes. Due to its extensive metabolism, there still is an interest in the identification of new metabolites and new routes of its metabolism in humans. In the present study, we investigated whether LSD could be a substrate for horseradish peroxidase or myeloperoxidase (MPO). Using liquid chromatography coupled to UV detection and electrospray ionization mass spectrometry (LC-UV-ESI-MS), we found that both peroxidases were capable of metabolizing LSD to the same compounds that have been observed in vivo (i.e., O-H-LSD and nor-LSD). In addition, we found another major metabolite, N,N-diethyl-7-formamido-4-methyl-6-oxo-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carboxamide (FOMBK), which is an opened indolic ring compound. Hydrolysis of FOMBK led to the deformylated compound 7-amino-N,N-diethyl-4-methyl-6-oxo-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carboxamide. The reactions of LSD with the peroxidases were chemiluminescent and sensitive to inhibition by reactive oxygen scavengers, which indicated that the classic peroxidase cycle is involved in this new alternative metabolic pathway. Considering that MPO is abundant in immune cells and also present in the central nervous system, the degradation pathway described in this study suggests a possible route of LSD metabolism that may occur concurrently with the in vivo reaction catalyzed by the cytochrome P450 system.

Selectivity and Mechanisms Driven by Reaction Dynamics: The Case of the Gas-Phase OH-+CH3ONO2 Reaction

Well-established statistical approaches such as transition-state theory based on high-level calculated potential energy profiles are unable to account for the selectivity observed in the gas-phase OH- + CH3ONO2 reaction. This reaction can undergo bimolecular nucleophilic displacement at either the carbon center (S(N)2@C) or the nitrogen center (S(N)2@N) as well as a proton abstraction followed by dissociation (E(CO)2) pathway. Direct dynamics simulations yield an S(N)2:E(CO)2 product ratio in close agreement with experiment and show that the lack of reactivity at the nitrogen atom is due to the highly negative electrostatic potential generated by the oxygen atoms in the ONO2 group that scatters the incoming OH-. In addition to these dynamical effects, the nonstatistical behavior of these reactions is attributed to the absence of equilibrated reactant complexes and to the large number of recrossings, which might be present in several ion-molecule gas-phase reactions.

The regulation of NHE₁ and NHE₃ activity by angiotensin II is mediated by the activation of the angiotensin II type I receptor/phospholipase C/calcium/calmodulin pathway in distal nephron cells

Angiotensin II (Ang II), acting via the AT1 receptor, induces an increase in intracellular calcium [Ca(2+)]i that then interacts with calmodulin (CaM). The Ca(2+)/CaM complex directly or indirectly activates sodium hydrogen exchanger 1 (NHE1) and phosphorylates calmodulin kinase II (CaMKII), which then regulates sodium hydrogen exchanger 3 (NHE3) activity. In this study, we investigated the cellular signaling pathways responsible for Ang II-mediated regulation of NHE1 and NHE3 in Madin-Darby canine kidney (MDCK) cells. The NHE1- and NHE3-dependent pHi recovery rates were evaluated by fluorescence microscopy using the fluorescent probe BCECF/AM, messenger RNA was evaluated with the reverse transcription polymerase chain reaction (RT-PCR), and protein expression was evaluated by immunoblot. We demonstrated that treatment with Ang II (1pM or 1 nM) for 30 min induced, via the AT1 but not the AT2 receptor, an equal increase in NHE1 and NHE3 activity that was reduced by the specific inhibitors HOE 694 and S3226, respectively. Ang II (1 nM) did not change the total expression of NHE1, NHE3 or calmodulin, but it induced CaMKII, cRaf-1, Erk1/2 and p90(RSK) phosphorylation. The stimulatory effects of Ang II (1 nM) on NHE1 or NHE3 activity or protein abundance was reduced by ophiobolin-A (CaM inhibitor), KN93 (CaMKII inhibitor) or PD98059 (Mek inhibitor). These results indicate that after 30 min, Ang II treatment may activate G protein-dependent pathways, including the AT1/PLC/Ca(2+)/CaM pathway, which induces CaMKII phosphorylation to stimulate NHE3 and induces cRaf-1/Mek/Erk1/2/p90(RSK) activity to stimulate NHE1

Phosphatidylethanolamine in Trypanosoma brucei is organized in two separate pools and is synthesized exclusively by the Kennedy pathway

Phosphatidylethanolamine is a major phospholipid class of all eukaryotic cells. It can be synthesized via the CDP-ethanolamine branch of the Kennedy pathway, by decarboxylation of phosphatidylserine, or by base exchange with phosphatidylserine. The contributions of these pathways to total phosphatidylethanolamine synthesis have remained unclear. Although Trypanosoma brucei, the causative agent of human and animal trypanosomiasis, has served as a model organism to elucidate the entire reaction sequence for glycosylphosphatidylinositol biosynthesis, the pathways for the synthesis of the major phospholipid classes have received little attention. We now show that disruption of the CDP-ethanolamine branch of the Kennedy pathway using RNA interference results in dramatic changes in phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. By targeting individual enzymes of the pathway, we demonstrate that de novo phosphatidylethanolamine synthesis in T. brucei procyclic forms is strictly dependent on the CDP-ethanolamine route. Interestingly, the last step in the Kennedy pathway can be mediated by two separate activities leading to two distinct pools of phosphatidylethanolamine, consisting of predominantly alk-1-enyl-acyl- or diacyl-type molecular species. In addition, we show that phosphatidylserine in T. brucei procyclic forms is synthesized exclusively by base exchange with phosphatidylethanolamine.

Statins induce regulatory T cell recruitment via a CCL1 dependent pathway

The statins, a group of inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase, are reported to influence a variety of immune system activities through 3-hydroxy-3-methylglutaryl coenzyme A reductase-dependent and -independent mechanisms. How statin treatment regulates immune system function in vivo nonetheless remains to be fully defined. We analyzed the immunomodulatory effects of lovastatin in a Candida albicans-induced delayed-type hypersensitivity reaction in mice. In this model, lovastatin administration reduced the acute inflammatory response elicited by C. albicans challenge. This anti-inflammatory activity of lovastatin was associated with a shift from a Th1 to a Th2 immune response, as well as an increase in the percentage of regulatory T cells at the inflammation site and in the regional draining lymph node. The lovastatin-induced increase in regulatory T cells in the inflamed skin was dependent on expression of CCL1, a chemokine that is locally up-regulated by statin administration. The anti-inflammatory effect of lovastatin was abrogated in CCL1-deficient mice. These results suggest that local regulation of chemokine expression may be an important process in statin-induced modulation of the immune system.

Role of acetyl-phosphate in activation of the Rrp2-RpoN-RpoS pathway in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease spirochete, dramatically alters its transcriptome and proteome as it cycles between the arthropod vector and mammalian host. During this enzootic cycle, a novel regulatory network, the Rrp2-RpoN-RpoS pathway (also known as the σ(54)-σ(S) sigma factor cascade), plays a central role in modulating the differential expression of more than 10% of all B. burgdorferi genes, including the major virulence genes ospA and ospC. However, the mechanism(s) by which the upstream activator and response regulator Rrp2 is activated remains unclear. Here, we show that none of the histidine kinases present in the B. burgdorferi genome are required for the activation of Rrp2. Instead, we present biochemical and genetic evidence that supports the hypothesis that activation of the Rrp2-RpoN-RpoS pathway occurs via the small, high-energy, phosphoryl-donor acetyl phosphate (acetyl∼P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) pathway that converts acetate to acetyl-CoA. Supplementation of the growth medium with acetate induced activation of the Rrp2-RpoN-RpoS pathway in a dose-dependent manner. Conversely, the overexpression of Pta virtually abolished acetate-induced activation of this pathway, suggesting that acetate works through acetyl∼P. Overexpression of Pta also greatly inhibited temperature and cell density-induced activation of RpoS and OspC, suggesting that these environmental cues affect the Rrp2-RpoN-RpoS pathway by influencing acetyl∼P. Finally, overexpression of Pta partially reduced infectivity of B. burgdorferi in mice. Taken together, these findings suggest that acetyl∼P is one of the key activating molecule for the activation of the Rrp2-RpoN-RpoS pathway and support the emerging concept that acetyl∼P can serve as a global signal in bacterial pathogenesis.

The kynurenine pathway is involved in bacterial meningitis.

BackgroundBacterial meningitis (BM) is characterized by an intense host inflammatory reaction, which contributes to the development of brain damage and neuronal sequelae. Activation of the kynurenine (KYN) pathway (KP) has been reported in various neurological diseases as a consequence of inflammation. Previously, the KP was shown to be activated in animal models of BM, and the association of the SNP AADAT¿+¿401C/T (kynurenine aminotransferase II - KAT II) with the host immune response to BM has been described. The aim of this study was to investigate the involvement of the KP during BM in humans by assessing the concentrations of KYN metabolites in the cerebrospinal fluid (CSF) of BM patients and their relationship with the inflammatory response compared to aseptic meningitis (AM) and non-meningitis (NM) groups.MethodsThe concentrations of tryptophan (TRP), KYN, kynurenic acid (KYNA) and anthranilic acid (AA) were assessed by HPLC from CSF samples of patients hospitalized in the Giselda Trigueiro Hospital in Natal (Rio Grande do Norte, Brazil). The KYN/TRP ratio was used as an index of indoleamine 2,3-dioxygenase (IDO) activity, and cytokines were measured using a multiplex cytokine assay. The KYNA level was also analyzed in relation to AADAT¿+¿401C/T genotypes.ResultsIn CSF from patients with BM, elevated levels of KYN, KYNA, AA, IDO activity and cytokines were observed. The cytokines INF-¿ and IL-1Ra showed a positive correlation with IDO activity, and TNF-¿ and IL-10 were positively correlated with KYN and KYNA, respectively. Furthermore, the highest levels of KYNA were associated with the AADAT¿+¿401 C/T variant allele.ConclusionThis study suggests a downward modulatory effect of the KP on CSF inflammation during BM.

A copper-induced quinone degradation pathway provides protection against combined copper/quinone stress in Lactococcus lactis IL1403.

Quinones are ubiquitous in the environment. They occur naturally but are also in widespread use in human and industrial activities. Quinones alone are relatively benign to bacteria, but in combination with copper, they become toxic by a mechanism that leads to intracellular thiol depletion. Here, it was shown that the yahCD-yaiAB operon of Lactococcus lactis IL1403 provides resistance to combined copper/quinone stress. The operon is under the control of CopR, which also regulates expression of the copRZA copper resistance operon as well as other L. lactis genes. Expression of the yahCD-yaiAB operon is induced by copper but not by quinones. Two of the proteins encoded by the operon appear to play key roles in alleviating quinone/copper stress: YaiB is a flavoprotein that converts p-benzoquinones to less toxic hydroquinones, using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as reductant; YaiA is a hydroquinone dioxygenase that converts hydroquinone putatively to 4-hydroxymuconic semialdehyde in an oxygen-consuming reaction. Hydroquinone and methylhydroquinone are both substrates of YaiA. Deletion of yaiB causes increased sensitivity of L. lactis to quinones and complete growth arrest under combined quinone and copper stress. Copper induction of the yahCD-yaiAB operon offers protection to copper/quinone toxicity and could provide a growth advantage to L. lactis in some environments.

Reaction of Copper(II) Chloroacetate with Pyrazole. Synthesis of a One-Dimensional Coordination Polymer and Unexpected Dehydrochlorination Reaction

The reaction of copper(II) chloroacetate (1d) with pyrazole (Hpz) mainly yielded the mononuclear compound [Cu(μ-ClCH2COO)2(Hpz)2] (2m), which self-assembled generating a one-dimensional coordination polymer. Moreover, the concomitant isolation of the tetranuclear [{Cu2(μ-pz)(μ-OCH2COO)(Hpz)(MeOH)}2(μ-ClCH2COO)2] (3t) and hexanuclear [{Cu3(μ3-OH)(μ-pz)3(Hpz)2}2(μ-ClCH2COO)2](Cl)2 (4h) species evidenced the occurrence of a peculiar, previously unreported, dehydrochlorination reaction and the formation of the trinuclear triangular moiety [Cu3(μ3-OH)(μ-pz)3]. Theoretical calculations based on density functional theory including solvation effects indicate a possible pathway for the formation of 3t. Interestingly, besides the energy minimum corresponding to 3t, a further relative energy minimum is found for a species which can be considered a possible reaction intermediate.

Chlorophyll Breakdown in Senescent Arabidopsis Leaves. Characterization of Chlorophyll Catabolites and of Chlorophyll Catabolic Enzymes Involved in the Degreening Reaction

During senescence, chlorophyll (chl) is metabolized to colorless nonfluorescent chl catabolites (NCCs). A central reaction of the breakdown pathway is the ring cleavage of pheophorbide (pheide) a to a primary fluorescent chl catabolite. Two enzymes catalyze this reaction, pheide a oxygenase (PAO) and red chl catabolite reductase. Five NCCs and three fluorescent chl catabolites (FCCs) accumulated during dark-induced chl breakdown in Arabidopsis (Arabidopsis thaliana). Three of these NCCs and one FCC (primary fluorescent chl catabolite-1) were identical to known catabolites from canola (Brassica napus). The presence in Arabidopsis of two modified FCCs supports the hypothesis that modifications, as present in NCCs, occur at the level of FCC. Chl degradation in Arabidopsis correlated with the accumulation of FCCs and NCCs, as well as with an increase in PAO activity. This increase was due to an up-regulation of Pao gene expression. In contrast, red chl catabolite reductase is not regulated during leaf development and senescence. A pao1 knockout mutant was identified and analyzed. The mutant showed an age- and light-dependent cell death phenotype on leaves and in flowers caused by the accumulation of photoreactive pheide a. In the dark, pao1 exhibited a stay-green phenotype. The key role of PAO in chl breakdown is discussed.

Reactions of CF3-enones with arenes under superelectrophilic activation: a pathway to trans-1,3-diaryl-1-CF3-indanes, new cannabinoid receptor ligands

4-Aryl-1,1,1-trifluorobut-3-en-2-ones ArCH[double bond, length as m-dash]CHCOCF3 (CF3-enones) react with arenes in excess of Brønsted superacids (TfOH, FSO3H) to give, stereoselectively, trans-1,3-diaryl-1-trifluoromethyl indanes in 35-85% yields. The reaction intermediates, the O-protonated ArCH[double bond, length as m-dash]CHC(OH(+))CF3 and the O,C-diprotonated ArHC(+)CH2C(OH(+))CF3 species, have been studied by means of (1)H, (13)C, (19)F NMR, and DFT calculations. Both types of the cations may participate in the reaction, depending on their electrophilicity and electron-donating properties of the arenes. The formation of CF3-indanes is a result of cascade reaction of protonated CF3-enones to form chemo-, regio- and stereoselectively three new C-C bonds. The obtained trans-1,3-diaryl-1-trifluoromethyl indanes were investigated as potential ligands for cannabinoid receptors CB1 and CB2 types. The most potent compound showed sub-micromolar affinity for both receptor subtypes with a 6-fold selectivity toward the CB2 receptor with no appreciable cytotoxicity toward SHSY5Y cells.

Evaluation of elevator shafts as a pathway for fungal spores and particles to enter a hospital housing immuno-compromised patients

Background. It is estimated that hospitals spend between 28 and 33 billion dollars per year as a result of hospital-acquired infections. (Scott, 2009) The costs continue to rise despite the guidance and controls provided by hospital infection control staff to reduce patient exposures to fungal spores and other infectious agents. With all processes and controls in place, the vented elevator shaft represents an unprotected opening from the top of the building to the lower floors. The hypothesis for this prospective study is that there is a positive correlation between the number of Penicillium/Aspergillus-like spores, Cladosporium, ascospores, basidiospores in spores/m3 as individual spore categories found in the hoistway vent of an elevator shaft and the levels of the same spores, sampled near-simultaneously in the outdoor intake of the elevator shaft. Specific aims of this study include determining if external Penicillium/Aspergillus-like spores are entering the healthcare facility via the elevator shaft and hoistway vents. Additional aims include determining levels of Penicillium/Aspergillus-like spores outdoors, in the elevator shafts, and indoors in areas possibly affected by elevator shaft air; and, finally, to evaluate whether any effect is observed due to the installation of a hoistway vent damper, installed serendipitously during this study. ^ Methods. Between April 2010 and September 2010, a total of 3,521 air samples were collected, including 363 spore trap samples analyzed microscopically for seven spore types, and polymerase chain reaction analyses on 254 air samples. 2178 particle count measurements, 363 temperature readings and 363 relative humidity readings were also obtained from 7 different locations potentially related to the path of air travel inside and near a centrally-located and representative elevator shaft. ^ Results. Mean Penicillium/Aspergillus-like spore values were higher outside the building (530 spores/m3 of air) than inside the hoistway (22.8 spores/m3) during the six month study. Mean values inside the hospital were lower than outside throughout the study, ranging from 15 to 73 spores/m3 of air. Mean Penicillium/Aspergillus-like spore counts inside the hoistway decreased from 40.1 spores/m3 of air to 9 spores/m3 of air following the installation of a back draft damper between the outside air and the elevator shaft. Comparison of samples collected outside the building and inside the hoistway vent prior to installing the damper indicated a strong positive correlation (Spearman's Rho=0.8008, p=0.0001). The similar comparison following the damper installation indicated a moderate non-significant inverse correlation (Spearman's rho = −0.2795, p=0.1347). ^ Conclusion. Elevator shafts are one pathway for mold spores to enter a healthcare facility. A significant correlation was detected between spores and particle counts inside the hoistway and outside prior to changes in the ventilation system. The insertion of the back draft damper appeared to lower the spore counts inside the hoistway and inside the building. The mold spore counts in air outside the study building were higher in the period following the damper installation while the levels inside the hoistway and hospital decreased. Cladosporium and Penicillium/Aspergillus -like spores provided a method for evaluating indoor air quality as a natural tracer from outside the building to inside the building. Ascospores and basidiospores were not a valuable tracer due to low levels of detection during this study. ^ Installation of a back draft damper provides additional protection for the indoor environment of a hospital or healthcare facility, including in particular patients who may be immunocompromised. Current design standards and references do not require the installation of a back draft damper, but evaluation of adding language to relevant building codes should be considered. The data indicate a reduction in levels of Penicillium/Aspergillus -like spores, particle counts and a reduction in relative humidity inside of the elevator shaft after damper installation.^

Molecular switch in signal transduction: Reaction paths of the conformational changes in ras p21

Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change is determined by molecular dynamics simulation with a holonomic constraint directing the system from the known GTP-bound structure (with the γ-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD–Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of α2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of α2 and facilitates the unwinding of a helical turn of α2 (residues 66–69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition. Possible functional implications of the results are discussed.

α-Tocopherol transfer protein stimulates the secretion of α-tocopherol from a cultured liver cell line through a brefeldin A-insensitive pathway

Vitamin E (α-tocopherol) is a fat-soluble antioxidant that is transported by plasma lipoproteins in the body. α-Tocopherol taken up by the liver with lipoprotein is thought to be resecreted into the plasma in very low density lipoprotein (VLDL). α-Tocopherol transfer protein (αTTP), which was recently identified as a product of the causative gene for familial isolated vitamin E deficiency, is a cytosolic liver protein and plays an important role in the efficient recycling of plasma vitamin E. To throw light on the mechanism of αTTP-mediated α-tocopherol transfer in the liver cell, we devised an assay system using the hepatoma cell line McARH7777. Using this system, we found that the secretion of α-tocopherol was more efficient in cells expressing αTTP than in matched cells lacking αTTP. Brefeldin A, which effectively inhibits VLDL secretion by disrupting the Golgi apparatus, had no effect on α-tocopherol secretion, indicating that αTTP-mediated α-tocopherol secretion is not coupled to VLDL secretion. Among other agents tested, only 25-hydroxycholesterol, a modulator of cholesterol metabolism, inhibited α-tocopherol secretion. This inhibition is most likely mediated by oxysterol-binding protein. These results suggest that αTTP present in the liver cytosol functions to stimulate secretion of cellular α-tocopherol into the extracellular medium and that the reaction utilizes a novel non-Golgi-mediated pathway that may be linked to cellular cholesterol metabolism and/or transport.

Nature knows best: An amazing reaction cascade is uncovered by design and discovery

The Daphniphyllum alkaloids are a group of highly complex polycyclic alkaloids. Examination of the structures if several members of this family of natural products led to a hypothesis about their mode of biosynthesis (depicted in Scheme SI). Based on this hypothetical biosynthetic pathway, a laboratory synthesis was designed that incorporated as a key transformation the novel one-pot transformation of dialdehyde 24 to pentacyclic unsaturated amine 25. This process turned out to be an exceptionally efficient way to construct the pentacyclic nucleus of the Daphniphyllum alkaloids. However, a purely fortuitous discovery, resulting from accidental use of methylamine rather than ammonia, led to a great improvement in the synthesis and suggests an even more attractive possible biosynthesis.