On the relationship between energy input to the ionosphere and the ion outflow flux under different solar zenith angles.

The ionosphere is one of the important sources for magnetospheric plasma, particularly for heavy ions with low charge states. We investigate the effect of solar illumination on the number flux of ion outflow using data obtained by the Fast Auroral SnapshoT (FAST) satellite at 3000-4150 km altitude from 7 January 1998 to 5 February 1999. We derive empirical formulas between energy inputs and outflowing ion number fluxes for various solar zenith angle ranges. We found that the outflowing ion number flux under sunlit conditions increases more steeply with increasing electron density in the loss cone or with increasing precipitating electron density (> 50 eV), compared to the ion flux under dark conditions. Under ionospheric dark conditions, weak electron precipitation can drive ion outflow with small averaged fluxes (~ 107 cm-2 s-1). The slopes of relations between the Poynting fluxes and outflowing ion number fluxes show no clear dependence on the solar zenith angle. Intense ion outflow events (> 108 cm-2 s-1) occur mostly under sunlit conditions (solar zenith angle < 90°). Thus, it is presumably difficult to drive intense ion outflows under dark conditions, because of a lack of the solar illumination (low ionospheric density and/or small scale height owing to low plasma temperature).

Performance Evaluation of Proximal Sensors for Soil Assessment in Smallholder Farms in Embu County, Kenya.

Four proximal soil sensors were tested at four smallholder farms in Embu County, Kenya: a portable X-ray fluorescence sensor (PXRF), a mobile phone application for soil color determination by photography, a dual-depth electromagnetic induction (EMI) sensor, and a LED-based soil optical reflectance sensor. Measurements were made at 32-43 locations at each site. Topsoil samples were analyzed for plant-available nutrients (N, P, K, Mg, Ca, S, B, Mn, Zn, Cu, and Fe), pH, total nitrogen (TN) and total carbon (TC), soil texture, cation exchange capacity (CEC), and exchangeable aluminum (Al). Multivariate prediction models of each of the lab-analyzed soil properties were parameterized for 576 sensor-variable combinations. Prediction models for K, N, Ca and S, B, Zn, Mn, Fe, TC, Al, and CEC met the setup criteria for functional, robust, and accurate models. The PXRF sensor was the sensor most often included in successful models. We concluded that the combination of a PXRF and a portable soil reflectance sensor is a promising combination of handheld soil sensors for the development of in situ soil assessments as a field-based alternative or complement to laboratory measurements.

A remarkable short-snouted horned dinosaur from the Late Cretaceous (late Campanian) of southern Laramidia.

The fossil record of centrosaurine ceratopsids is largely restricted to the northern region of western North America (Alberta, Montana and Alaska). Exceptions consist of single taxa from Utah (Diabloceratops) and China (Sinoceratops), plus otherwise fragmentary remains from the southern Western Interior of North America. Here, we describe a remarkable new taxon, Nasutoceratops titusi n. gen. et sp., from the late Campanian Kaiparowits Formation of Utah, represented by multiple specimens, including a nearly complete skull and partial postcranial skeleton. Autapomorphies include an enlarged narial region, pneumatic nasal ornamentation, abbreviated snout and elongate, rostrolaterally directed supraorbital horncores. The subrectangular parietosquamosal frill is relatively unadorned and broadest in the mid-region. A phylogenetic analysis indicates that Nasutoceratops is the sister taxon to Avaceratops, and that a previously unknown subclade of centrosaurines branched off early in the group's history and persisted for several million years during the late Campanian. As the first well-represented southern centrosaurine comparable in age to the bulk of northern forms, Nasutoceratops provides strong support for the provincialism hypothesis, which posits that Laramidia-the western landmass formed by inundation of the central region of North America by the Western Interior Seaway-hosted at least two coeval dinosaur communities for over a million years of late Campanian time.

Zinc trafficking: 1,10-phenanthroline, glutathione, and other metal binding ligands form adducts with proteomic Zn2.

Hypotheses were tested that the proteome of pig kidney LLC-PK1 cells (i) contains Zn-proteins that react with a diversity of native and pharmacologically active metal-binding ligands to form ternary complexes and (ii) includes proteins that bind Zn2+ nonspecifically and together form ternary adducts with a variety of metal-binding agents. The method to observe ternary complex formation with Zn-proteins and proteome•Zn involved preformation of fluorescent TSQ [6-Methoxy-(8-p-toluenesulfonamido)quinoline]-Zn-proteins and/or proteome•Zn-TSQ adducts followed by competitive reaction with selected ligands. The loss of TSQ-dependent fluorescence signaled the replacement of TSQ by the competing ligand in the starting adducts. In vitro, 1,10-phenanthroline competed effectively with TSQ for binding to Zn-proteins in the proteome. The successful competition of 1,10-phenanthroline with TSQ-Zn-proteins was also observed in cells. Similarly, 1,10-phenanthroline was shown to bind to a sizable fraction of Zn2+ associated adventitiously with proteome (proteome•Zn). Other synthetic ligands that bind to Zn-proteins and proteome•Zn include 2,2-bipyridyl, 8-hydroxyquinoline, 2,2'-dicarboxypyridine, and pyrithione. Such results suggest that ligand binding to such sites may play a role in the observed biological effects of these and other metal-binding molecules. Although cysteine does not significantly compete with TSQ, glutathione displaces TSQ from Zn-proteins and proteome•Zn at concentrations well below those found in cells, implying that ternary complex formation involving glutathione may be physiologically significant.

Affinity purification of a chimeric nicotinic acetylcholine receptor in the agonist and antagonist bound states.

Nicotinic acetylcholine receptors (nAChRs) form ligand-gated ion channels that mediate fast signal transmission at synapses. These receptors are members of a large family of pentameric ion channels that are of active medical interest. An expression system utilizing a chimerical construct of the N-terminal extracellular ligand binding domain of alpha7 type nAChR and the C-terminal transmembrane portion of 5HT3 type receptor resulted high level of expressions. Two ligand affinity chromatography purification methods for this receptor have been developed. One method relies on the covalent immobilization of a high affinity small molecule alpha7 nAChR agonist, (R)-5-(4-aminophenyl)-N-(quinuclidin-3-yl) furan-2-carboxamide, and the other uses mono biotinylated alpha-bungarotoxin, an antagonist, that forms a quasi-irreversible complex with alpha7 nAChR. Detergent solubilized alpha7/5HT(3) chimeric receptors were selectively retained on the affinity resins and could be eluted with free ligand or biotin. The proteins purified by both methods were characterized by gel electrophoresis, mass spectra, amino acid composition analysis, and N-terminal sequence determination. These analyses confirmed the isolation of a mature alpha7/5HT(3) receptor with the signal peptide removed. These results suggest a scalable path forward to generate multi-milligram amounts of purified complexes for additional studies including protein crystallization.

Correction: Evolution of high tooth replacement rates in theropod dinosaurs.

[This corrects the article DOI: 10.1371/journal.pone.0224734.].

Evolution of high tooth replacement rates in theropod dinosaurs.

Tooth replacement rate is an important contributor to feeding ecology for polyphyodont animals. Dinosaurs exhibit a wide range of tooth replacement rates, mirroring their diverse craniofacial specializations, but little is known about broad-scale allometric or evolutionary patterns within the group. In the current broad but sparse dinosaurian sample, only three non-avian theropod tooth replacement rates have been estimated. We estimated tooth formation and replacement rates in three additional non-avian theropod dinosaurs, the derived latest Cretaceous abelisaurid Majungasaurus and the more generalized Late Jurassic Allosaurus and Ceratosaurus. We created the largest dental histological and CT dataset for any theropod dinosaur, sectioning and scanning over a dozen toothed elements of Majungasaurus and several additional elements from the other two genera. Using this large sample, we created models of tooth formation time that allow for theropod replacement rates to be estimated non-destructively. In contrast to previous results for theropods, we found high tooth replacement rates in all three genera, with Allosaurus and Ceratosaurus rates of ~100 days and 56 days for Majungasaurus. The latter rate is on par with those of derived herbivorous dinosaurs including some neosauropods, hadrosaurids, and ceratopsians. This elevated rate may be a response to high rates of tooth wear in Majungasaurus. Within Dinosauria, there is no relationship between body mass and tooth replacement rate and no trends in replacement rate over time. Rather, tooth replacement rate is clade-specific, with elevated rates in abelisaurids and diplodocoids and lower rates in coelurosaurs.

Viability, infectivity and fatty acid synthetic activity of Perkinsus marinus meront cells incubated in estuarine and artificial seawater.

We investigated the viability and fatty acid synthetic activity of in vitro cultured Perkinsus marinus (Dermo) in lipid-free medium and estuarine water, and the infectivity of P. marinus maintained in artificial seawater (ASW). Viability and fatty acid synthetic activity in 7 d old P. marinus meronts maintained in lipid-free medium and estuarine water were tested. The infectivity of meronts incubated in ASW was examined by first incubating P. marinus meronts in ASW for 2, 3 or 7 d, and then inoculating viable ASW-incubated meronts into the shell cavity of individual oysters Crassostrea virginica. P. marinus infection prevalence and intensity in oysters were determined 9 wk post-inoculation. Heavy mortality occurred in meronts maintained in estuarine water, a drop from an initial value of 100% viable to 7.8 and 6.1% after 3 and 14 d incubation, respectively. Viability was 85 and 67% in meronts maintained in lipid-free medium for 3 and 24 d, respectively. Meronts kept in lipid-free medium for 14 d retained their ability to synthesize fatty acids. Viable meronts incubated in ASW remained infective for up to 7 d. The infection prevalences were 85, 48 and 100%, in the treatments inoculated with viable meronts that were incubated in ASW for 2, 3 and 7 d, respectively. Infection prevalence in the group inoculated with viable meronts immediately after they were transferred to ASW ranged from 61 to 85%. Our results suggest that in nature meronts can survive for at least 14 d outside the host. Viable meronts are not only infective, but are also able to replicate and retain their fatty acid synthetic ability for 7 d.

A New Centrosaurine Ceratopsid, Machairoceratops cronusi gen et sp. nov., from the Upper Sand Member of the Wahweap Formation (Middle Campanian), Southern Utah.

The Upper Cretaceous (middle-late Campanian) Wahweap Formation of southern Utah contains the oldest diagnostic evidence of ceratopsids (to date, all centrosaurines) in North America, with a number of specimens recovered from throughout a unit that spans between 81 and 77 Ma. Only a single specimen has been formally named, Diabloceratops eatoni, from the lower middle member of the formation. Machairoceratops cronusi gen. et sp. nov., a new centrosaurine ceratopsid from the upper member of the Wahweap Formation, is here described based on cranial material representing a single individual recovered from a calcareous mudstone. The specimen consists of two curved and elongate orbital horncores, a left jugal, a nearly complete, slightly deformed braincase, the left squamosal, and a mostly complete parietal ornamented by posteriorly projected, anterodorsally curved, elongate spikes on either side of a midline embayment. The fan-shaped, stepped-squamosal is diagnostic of Centrosaurinae, however, this element differs from the rectangular squamosal in Diabloceratops. Machairoceratops also differs in the possession of two anterodorsally (rather than laterally) curved epiparietal ornamentations on either side of a midline embayment that are distinguished by a posteromedially-oriented sulcus along the entire length of the epiparietal. Additionally, the parietosquamosal frill is lacking any other epiossifications along its periphery. Machairoceratops shares a triangular (rather than round) frill and spike-like epiparietal loci (p1) ornamentation with the stratigraphically lower Diabloceratops. Both parsimony and Bayesian phylogenetic analyses place Machairoceratops as an early-branching centrosaurine. However, the parsimony-based analysis provides little resolution for the position of the new taxon, placing it in an unresolved polytomy with Diabloceratops. The resultant Bayesian topology yielded better resolution, aligning Machairoceratops as the definitive sister taxon to a clade formed by Diabloceratops and Albertaceratops. Considered together, both phylogenetic methods unequivocally place Machairoceratops as an early-branching centrosaurine, and given the biostratigraphic position of Machairoceratops, these details increase the known ceratopsid diversity from both the Wahweap Formation and the southern portion of Laramidia. Finally, the unique morphology of the parietal ornamentation highlights the evolutionary disparity of frill ornamentation near the base of Centrosaurinae.

Effects of triclosan on growth, viability and fatty acid synthesis of the oyster protozoan parasite Perkinsus marinus.

Perkinsus marinus, a protozoan parasite of the Eastern oyster Crassostrea virginica, has severely impacted oyster populations from the Mid-Atlantic region to the Gulf of Mexico coast of North America for more than 30 yr. Although a chemotherapeutic treatment to reduce or eliminate P. marinus from infected oysters would be useful for research and hatchery operations, an effective and practical drug treatment does not currently exist. In this study, the antimicrobial drug triclosan 5-chloro-2-(2,4 dichlorophenoxy) phenol, a specific inhibitor of Fab1 (enoyl-acyl-carrier-protein reductase), an enzyme in the Type II class of fatty acid synthetases, was tested for its effects on viability, proliferation and fatty acid synthesis of in vitro-cultured P. marinus meronts. Treatment of P. marinus meront cell cultures with concentrations of > or = 2 microM triclosan at 28 degrees C (a temperature favorable for parasite proliferation) for up to 6 d stopped proliferation of the parasite. Treatment at > or = 5 microM at 28 degrees C greatly reduced the viability and fatty acid synthesis of meront cells. Oyster hemocytes treated with > or = 20 microM triclosan exhibited no significant (p < 0.05) reduction in viability relative to controls for up to 24 h at 13 degrees C. P. marinus meronts exposed to > or = 2 microM triclosan for 24 h at 13 degrees C exhibited significantly (p < 0.05) lower viability relative to controls. Exposure of P. marinus meronts to triclosan concentrations of > or = 20 microM resulted in > 50% mortality of P. marinus cells after 24 h. These results suggest that triclosan may be effective in treating P. marinus-infected oysters.

Phospholipid biosynthesis in the oyster protozoan parasite, Perkinsus marinus.

Perkinsus marinus is a protozoan parasite that causes high mortality in its commercially and ecologically important host, the Eastern oyster Crassostrea virginica. In order to understand the host-parasite relationship in lipid metabolism, the ability of P. marinus to synthesize phospholipids from polar headgroup precursors was investigated. Pulse/chase experiments were conducted using radiolabled serine, choline, ethanolamine and inositol. Timecourse incubations revealed that in vitro cultured P. marinus meronts can utilize the cytidine diphosphate-diacylglycerol (CDP-DAG) pathway to synthesize phosphatidylinositol (PI) from inositol and phosphatidylserine (PS) from serine. Serine label was also incorporated into phosphatidylethanolamine (PE), phosphatidylcholine (PC) and lysophosphatidylcholine (LPC). Incubations of P. marinus cells with increasing concentrations of radiolabeled serine resulted in more radioactivity recovered in neutral lipids than in polar lipids at the highest substrate concentration tested (344 microM). This suggests that excess serine label was being utilized for fatty acid synthesis and stored as triacylglycerols. Additional incubations were conducted with radiolabeled choline and ethanolamine at concentrations equimolar to the highest serine concentration tested. Ethanolamine label was also incorporated into PE, PS, PC and LPC. Choline label was incorporated into PC. These results suggest the presence of three pathways for de novo synthesis of phospholipids in P. marinus: CDP-choline, CDP-ethanolamine and CDP-DAG. At equivalent substrate concentrations (344 microM) the highest incorporation of labeled substrate into total phospholipids was with serine followed by ethanolamine and choline, respectively. P. marinus phospholipid biosynthetic capabilities appear to be similar to those of Plasmodium and Trypanosoma species.

Arachidonic acid synthetic pathways of the oyster protozoan parasite, Perkinsus marinus: evidence for usage of a delta-8 pathway.

The meront stage of the oyster protozoan parasite, Perkinsus marinus, is capable of synthesizing saturated and unsaturated fatty acids including the essential fatty acid, arachidonic acid [20:4(n-6)]. Eukaryotes employ either delta-6 (Delta-6) or delta-8 (Delta-8) desaturase pathway or both to synthesize arachidonic acid. To elucidate the arachidonic acid synthetic pathways in P. marinus, meronts were incubated with deuterium-labeled precursors [18:1(n-9)-d6, 18:2(n-6)-d4, 18:3(n-3)-d4, and 20:3(n-3)-d8]. The lipids were extracted, converted to fatty acid methyl esters, and analyzed using gas chromatography/mass spectrometry and gas chromatography/flame ionization detection. Deuterium-labeled 18:2(n-6), 20:2(n-6), 20:3(n-6), and 20:4(n-6) were detected in meront lipids after 1-, 3-, 5-, and 10-day incubation with 18:1(n-9)-d6. Deuterium-labeled 20:2(n-6), 20:3(n-6) and 20:4(n-6) were found in lipids from meronts after incubation with 18:2(n-6)-d4 methyl ester. No labeled 18:3(n-6) was detected in either incubation. Apparently, when incubated with 18:1(n-9)-d6, the parasite first desaturated 18:1(n-9)-d6 to 18:2(n-6)-d6 by Delta-12 desaturase, then to 20:2(n-6)-d6 by elongation, and ultimately desaturated to 20:3(n-6)-d6 and 20:4(n-6)-d6 using the sequential Delta-8 and Delta-5 desaturation. Similarly, when incubated with 18:2(n-6)-d4, P. marinus converted the 18:2(n-6)-d4 to 20:2(n-6)-d4 by elongation and 20:2(n-6)-d4 to 20:3(n-6)-d4 by Delta-8 desaturase then by Delta-5 desaturase to 20:4(n-6)-d4. These results provide evidence that P. marinus employed the Delta-8 rather Delta-6 pathway for arachidonic acid synthesis. Additional support for the presence of a Delta-8 pathway was the demonstrated ability of the parasite to metabolize 18:3(n-3)-d4 to 20:3(n-3)-d4 and 20:4(n-3)-d4, and 20:3(n-3)-d8 to 20:4(n-3)-d6 and 20:5(n-3)-d6 using the sequential position-specific Delta-8 and Delta-5 desaturases.

De novo arachidonic acid synthesis in Perkinsus marinus, a protozoan parasite of the eastern oyster Crassostrea virginica.

The capability of synthesizing fatty acids de novo in the meront stage of the oyster protozoan parasite, Perkinsus marinus, was investigated employing stable-isotope-labeled precursors (1,2 13C-acetate and palmitic-d(31) acid). Fatty acid methyl esters derived from 1,2 13C-acetate and palmitic-d(31) acid were analyzed using gas chromatography/mass spectrometry and gas chromatography/flame ionization detection. Results revealed that in vitro cultured P. marinus meronts utilized 13C-acetate to synthesize a range of saturated and unsaturated fatty acids. The saturated fatty acids 14:0, 16:0, 18:0, 20:0, 22:0, 24:0 and the unsaturated fatty acids, 18:1(n-9), 18:2(n-6), 20:1(n-9), 20:2(n-6), 20:2(n-9), 20:3(n-6), 20:4(n-6) were found to contain 13C, after 7, 14, and 21 days incubation with the precursor. This indicates that meronts can synthesize fatty acid de novo using acetate as a substrate. Meronts efficiently elongated 16:0-d(31) to 18:0, 20:0, 22:0, 24:0, but desaturation activity was limited, after 7 and 14 days cultivation. Only a small quantity of 18:1-d(29) was detected. This suggests that meronts cannot directly convert exogenous palmitic acid or its products of elongation to unsaturated counterparts. The ability to synthesize 20:4(n-6) from acetate is particularly interesting. No parasitic protozoan has been reported to be capable of synthesizing long chain essential fatty acids, such as 20:4(n-6) de novo. Future study will be directed to determine whether the observed in vitro activities indeed reflect the in vivo activities, when meronts are associated with the host.

How are you really feeling? A prospective evaluation of cognitive function following trauma.

BACKGROUND: Mild traumatic brain injury is associated with persistent cognitive difficulties. However, these symptoms may not be specific to the head injury itself. We sought to evaluate the prevalence of these symptoms in patients following trauma. METHODS: A prospective analysis of patients who were seen in the outpatient trauma clinic during a 20-month period and completed self-administered Rivermead Post-Concussion Symptoms Questionnaire was conducted. "Significant" difficulty with cognition was defined by two or more symptoms reported as severe or four or more symptoms reported as moderate. Head injury was defined as head Abbreviated Injury Scale (AIS) score greater than 0, including the diagnosis of concussion. Multivariable logistic regression was used to test associations between head injury, injury severity, sex, and age with significant cognitive difficulties, loss of work/school, and unmet physical, occupational, or psychological therapy needs. RESULTS: A total of 587 completed questionnaires were matched to trauma registry admissions (382 early, 111 mid, 86 late). The incidence of significant cognitive difficulties was 37% at less than 1 month, 40% at 1 month to 3 months, and 45% of patients at more than 3 months following injury. Head injury was not associated with increased odds for significant cognitive difficulties (adjusted odds ratio, 1.21; 95% confidence interval, 0.82-1.77; p = 0.3) There was no significant difference in symptoms in patients who carried a head injury diagnosis and those who did not. CONCLUSION: Cognitive problems occur frequently following injury even in the absence of a head injury diagnosis. Either mild traumatic brain injury is grossly underdiagnosed or these symptoms are not specific to postconcussive states and simply are the cognitive sequelae of traumatic injury. The reporting of moderate-to-severe symptoms suggests a need to better understand the effects of trauma on cognitive function and strongly suggests that services for these patients are badly needed to maximize cognitive function and return to preinjury quality of life. LEVEL OF EVIDENCE: Prognostic/epidemiologic study, level II.

High-throughput bioanalysis of bile acids and their conjugates using UHPLC coupled to HRMS.

BACKGROUND: Quantitative assessment of bile acids in biological matrixes is of growing interest, primarily due to hepatic toxicity resulting from drug interactions with the bile salt export pump. Nevertheless, many bile acids demonstrate poor fragmentation in MS, making conventional MS/MS not a good match for their selective quantitation in biological matrices. RESULTS: The current study was designed to evaluate the feasibility of simultaneous quantitation of 19 bile acids using HRMS coupled to UHPLC separation with minimal instrument optimization. An effective chromatography was developed using an Agilent Zorbax(®) Eclipse XDB-C18 column (1.8 µm, 50 x 2.1 mm internal diameter), achieving separation of 19 compounds in 10 min. Excellent assay reproducibility was demonstrated, with two sets of standard curves, run 42 days apart. CONCLUSIONS: The results show that LC-HRMS is a viable platform for high throughput bioanalysis of bile acids especially in a drug-discovery setting.

Perkinsus marinus, a protozoan parasite of the eastern oyster, has a requirement for dietary sterols.

Perkinsus marinus, a protozoan parasite of the eastern oyster, Crassostrea virginica, causes high mortality in its host along the Atlantic and Gulf coasts of North America. P. marinus meronts cultured in vitro in medium containing complete lipid supplement (cod liver oil, cholesterol and alpha tocopherol acetate in detergent) are able to synthesize a wide variety of lipids, yet cultures cannot be maintained in lipid-free medium. To determine P. marinus lipid requirements meronts were inoculated into media containing different combinations of lipid components in detergent. Treatments included complete lipid supplement (positive control), detergent only (negative control), cholesterol in detergent, alpha tocopherol acetate in detergent and cholesterol+alpha tocopherol acetate in detergent. Meronts proliferated in the positive control medium and media containing cholesterol or cholesterol+alpha tocopherol acetate, but failed to proliferate in the negative control medium and the medium containing just alpha tocopherol acetate. Gas chromatography analysis of P. marinus meronts grown in medium with added (13)C sodium acetate (0.5 mg mL(-1)) revealed the presence of fatty acids containing (13)C, but the only sterol present was cholesterol containing no (13)C. These results suggest that P. marinus cannot synthesize sterols and must sequester them from its host.

Novel mechanistic class of fatty acid amide hydrolase inhibitors with remarkable selectivity.

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic, anti-inflammatory, anxiolytic, and antidepressant phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for treatment of pain, inflammation, and other central nervous system disorders. However, the FAAH inhibitors reported to date lack drug-like pharmacokinetic properties and/or selectivity. Herein we describe piperidine/piperazine ureas represented by N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750) and N-phenyl-4-(quinolin-2-ylmethyl)piperazine-1-carboxamide (PF-622) as a novel mechanistic class of FAAH inhibitors. PF-750 and PF-622 show higher in vitro potencies than previously established classes of FAAH inhibitors. Rather unexpectedly based on the high chemical stability of the urea functional group, PF-750 and PF-622 were found to inhibit FAAH in a time-dependent manner by covalently modifying the enzyme's active site serine nucleophile. Activity-based proteomic profiling revealed that PF-750 and PF-622 were completely selective for FAAH relative to other mammalian serine hydrolases. We hypothesize that this remarkable specificity derives, at least in part, from FAAH's special ability to function as a C(O)-N bond hydrolase, which distinguishes it from the vast majority of metabolic serine hydrolases in mammals that are restricted to hydrolyzing esters and/or thioesters. The piperidine/piperazine urea may thus represent a privileged chemical scaffold for the synthesis of FAAH inhibitors that display an unprecedented combination of potency and selectivity for use as potential analgesic and anxiolytic/antidepressant agents.

Assessment of the cell viability of cultured Perkinsus marinus (Perkinsea), a parasitic protozoan of the Eastern oyster, Crassostrea virginica, using SYBRgreen-propidium iodide double staining and flow cytometry.

A flow cytometry (FCM) assay using SYBRgreen and propidium iodide double staining was tested to assess viability and morphological parameters of Perkinsus marinus under different cold- and heat-shock treatments and at different growth phases. P. marinus meront cells, cultivated at 28 degrees C, were incubated in triplicate for 30 min at -80 degrees C, -20 degrees C, 5 degrees C, and 20 degrees C for cold-shock treatments and at 32 degrees C, 36 degrees C, 40 degrees C, 44 degrees C, 48 degrees C, 52 degrees C, and 60 degrees C for heat-shock treatments. A slight and significant decrease in percentage of viable cells (PVC), from 93.6% to 92.7%, was observed at -20 degrees C and the lowest PVC was obtained at -80 degrees C (54.0%). After 30 min of heat shocks at 40 degrees C and 44 degrees C, PVC decreased slightly but significantly compared to cells maintained at 28 degrees C. When cells were heat shocked at 48 degrees C, 52 degrees C, and 60 degrees C heavy mortality occurred and PVC decreased to 33.8%, 8.0%, and 3.4%, respectively. No change in cell complexity and size was noted until cells were heat shocked at >or=44 degrees C. High cell mortality was detected at stationary phase of P. marinus cell culture. Cell viability dropped below 40% in 28-day-old cultures and ranged 11-25% in 38 to 47-day-old cultures. Results suggest that FCM could be a useful tool for determining viability of cultured P. marinus cells.

Identification of a novel mitochondrial protein ("mitoNEET") cross-linked specifically by a thiazolidinedione photoprobe.

Thiazolidinediones address underlying causes of type 2 diabetes, although their mechanism of action is not clearly understood. The compounds are thought to function as direct activators of the nuclear receptor PPARgamma (peroxisome proliferator-activated receptor-gamma), although pioglitazone, the weaker agonist of the two thiazolidinediones now in clinical use, seems to have more useful effects on circulating lipids. We have used tritiated pioglitazone and a photoaffinity cross-linker to identify a novel binding site in mitochondria. A saturable binding site for [3H]pioglitazone was solubilized from the membranes with CHAPS and migrated as a large complex by size exclusion chromatography. The binding correlated with a <17-kDa protein (m17), marked by a photoaffinity cross-linker, in both subcellular location and selectivity of competition by analogs. The protein was isolated and identified by mass spectrometry analysis and NH2-terminal sequencing. Three synthetic peptides with potential antigenic properties were synthesized from the predicted nontransmembrane sequence to generate antibodies in rabbits. Western blots show that this protein, which we have termed "mitoNEET," is located in the mitochondrial fraction of rodent brain, liver, and skeletal muscle, showing the identical subcellular location and migration on SDS-PAGE as the protein cross-linked specifically by the thiazolidinedione photoprobe. The protein exists in low levels in preadipocytes, and expression increases exponentially in differentiated adipocytes. The synthetic protein bound to solid phase associated with a complex of solubilized mitochondrial proteins, including the trifunctional beta-oxidation protein. It is possible that thiazolidinedione modification of the function of the mitochondrial target may contribute to lipid lowering and/or antidiabetic actions.

Cross-linking in the living cell locates the site of action of oxazolidinone antibiotics.

Oxazolidinone antibiotics, an important new class of synthetic antibacterials, inhibit protein synthesis by interfering with ribosomal function. The exact site and mechanism of oxazolidinone action has not been elucidated. Although genetic data pointed to the ribosomal peptidyltransferase as the primary site of drug action, some biochemical studies conducted in vitro suggested interaction with different regions of the ribosome. These inconsistent observations obtained in vivo and in vitro have complicated the understanding of oxazolidinone action. To localize the site of oxazolidinone action in the living cell, we have cross-linked a photoactive drug analog to its target in intact, actively growing Staphylococcus aureus. The oxazolidinone cross-linked specifically to 23 S rRNA, tRNA, and two polypeptides. The site of cross-linking to 23 S rRNA was mapped to the universally conserved A-2602. Polypeptides cross-linked were the ribosomal protein L27, whose N terminus may reach the peptidyltransferase center, and LepA, a protein homologous to translation factors. Only ribosome-associated LepA, but not free protein, was cross-linked, indicating that LepA was cross-linked by the ribosome-bound antibiotic. The evidence suggests that a specific oxazolidinone binding site is formed in the translating ribosome in the immediate vicinity of the peptidyltransferase center.