CYP1B1-derived epoxides modulate the TRPA1 channel in chronic pain.

Pain is often debilitating, and current treatments are neither universally efficacious nor without risks. Transient receptor potential (TRP) ion channels offer alternative targets for pain relief, but little is known about the regulation or identities of endogenous TRP ligands that affect inflammation and pain. Here, transcriptomic and targeted lipidomic analysis of damaged tissue from the mouse spinal nerve ligation (SNL)-induced chronic pain model revealed a time-dependent increase in Cyp1b1 mRNA and a concurrent accumulation of 8,9-epoxyeicosatrienoic acid (EET) and 19,20-EpDPA post injury. Production of 8,9-EET and 19,20-EpDPA by human/mouse CYP1B1 was confirmed in vitro, and 8,9-EET and 19,20-EpDPA selectively and dose-dependently sensitized and activated TRPA1 in overexpressing HEK-293 cells and Trpa1-expressing/AITC-responsive cultured mouse peptidergic dorsal root ganglia (DRG) neurons. TRPA1 activation by 8,9-EET and 19,20-EpDPA was attenuated by the antagonist A967079, and mouse TRPA1 was more responsive to 8,9-EET and 19,20-EpDPA than human TRPA1. This latter effect mapped to residues Y933, G939, and S921 of TRPA1. Intra-plantar injection of 19,20-EpDPA induced acute mechanical, but not thermal hypersensitivity in mice, which was also blocked by A967079. Similarly, Cyp1b1-knockout mice displayed a reduced chronic pain phenotype following SNL injury. These data suggest that manipulation of the CYP1B1-oxylipin-TRPA1 axis might have therapeutic benefit.

Gene and protein expression of dorsal root ganglion sensory receptors in normotensive and hypertensive male rats.

The exercise pressor reflex (EPR), a neurocirculatory control mechanism, is exaggerated in hypertensive humans and rats. Disease-related abnormalities within the afferent arm of the reflex loop, including mechano- and metabosensitive receptors located at the terminal end of group III/IV muscle afferents, may contribute to the dysfunctional EPR in hypertension. Using control (WKY) and spontaneous hypertensive (SHR) rats, we examined dorsal root ganglion (DRG) gene and protein expression of molecular receptors recognized as significant determinants of the EPR. Twelve lumbar DRGs (6 left, 6 right) were harvested from each of 10 WKY [arterial blood pressure (MAP): 96 ± 9 mmHg] and 10 SHR (MAP: 144 ± 9 mmHg). DRGs from the left side were used for protein expression (Western blotting; normalized to GAPDH), whereas right-side DRGs (i.e., parallel structure) were used to determine mRNA levels (RNA-sequencing, normalized to TPM). Analyses focused on metabosensitive (ASIC3, Bradykinin receptor B2, EP4, P2X3, TRPv1) and mechanosensitive (Piezo1/2) receptors. Although Piezo1 was similar in both groups (P = 0.75), protein expression for all other receptors was significantly higher in SHR compared with WKY. With the exception of a greater Bradykinin-receptor B2 in SHR (P < 0.05), mRNA expression of all other receptors was not different between groups (P > 0.18). The higher protein content of these sensory receptors in SHR indirectly supports the previously proposed hypothesis that the exaggerated EPR in hypertension is, in part, due to disease-related abnormalities within the afferent arm of the reflex loop. The upregulated receptor content, combined with normal mRNA levels, insinuates that posttranscriptional regulation of sensory receptor protein expression might be impaired in hypertension.

Nicotinic Acetylcholine Receptor Partial Antagonist Polyamides from Tunicates and Their Predatory Sea Slugs.

In our efforts to discover new drugs to treat pain, we identified molleamines A-E (1-5) as major neuroactive components of the sea slug, Pleurobranchus forskalii, and their prey, Didemnum molle, tunicates. The chemical structures of molleamines were elucidated by spectroscopy and confirmed by the total synthesis of molleamines A (1) and C (3). Synthetic 3 completely blocked acetylcholine-induced calcium flux in peptidergic nociceptors (PNs) in the somatosensory nervous system. Compound 3 affected neither the α7 nAChR nor the muscarinic acetylcholine receptors in calcium flux assays. In addition to nociceptors, 3 partially blocked the acetylcholine-induced calcium flux in the sympathetic nervous system, including neurons from the superior cervical ganglion. Electrophysiology revealed a block of α3ß4 (mouse) and α6/α3ß4 (rat) nicotinic acetylcholine receptors (nAChRs), with IC50 values of 1.4 and 3.1 µM, respectively. Molleamine C (3) is a partial antagonist, reaching a maximum block of 76-82% of the acetylcholine signal and showing no partial agonist response. Molleamine C (3) may thus provide a lead compound for the development of neuroactive compounds with unique biological properties.

Neuroactive Type-A γ-Aminobutyric Acid Receptor Allosteric Modulator Steroids from the Hypobranchial Gland of Marine Mollusk, Conus geographus.

In a program to identify pain treatments with low addiction potential, we isolated five steroids, conosteroids A-E (1-5), from the hypobranchial gland of the mollusk Conus geographus. Compounds 1-5 were active in a mouse dorsal root ganglion (DRG) assay that suggested that they might be analgesic. A synthetic analogue 6 was used for a detailed pharmacological study. Compound 6 significantly increased the pain threshold in mice in the hot-plate test at 2 and 50 mg/kg. Compound 6 at 500 nM antagonizes type-A γ-aminobutyric acid receptors (GABAARs). In a patch-clamp experiment, out of the six subunit combinations tested, 6 exhibited subtype selectivity, most strongly antagonizing α1ß1γ2 and α4ß3γ2 receptors (IC50 1.5 and 1.0 µM, respectively). Although the structures of 1-6 differ from those of known neuroactive steroids, they are cell-type-selective modulators of GABAARs, expanding the known chemical space of neuroactive steroids.

Acylcarnitines as markers of exercise-associated fuel partitioning, xenometabolism, and potential signals to muscle afferent neurons.

NEW FINDINGS: What is the central question of this study? Does improved metabolic health and insulin sensitivity following a weight-loss and fitness intervention in sedentary, obese women alter exercise-associated fuel metabolism and incomplete mitochondrial fatty acid oxidation (FAO), as tracked by blood acylcarnitine patterns? What is the main finding and its importance? Despite improved fitness and blood sugar control, indices of incomplete mitochondrial FAO increased in a similar manner in response to a fixed load acute exercise bout; this indicates that intramitochondrial muscle FAO is inherently inefficient and is tethered directly to ATP turnover. With insulin resistance or type 2 diabetes mellitus, mismatches between mitochondrial fatty acid fuel delivery and oxidative phosphorylation/tricarboxylic acid cycle activity may contribute to inordinate accumulation of short- or medium-chain acylcarnitine fatty acid derivatives [markers of incomplete long-chain fatty acid oxidation (FAO)]. We reasoned that incomplete FAO in muscle would be ameliorated concurrent with improved insulin sensitivity and fitness following a ∼14 week training and weight-loss intervention in obese, sedentary, insulin-resistant women. Contrary to this hypothesis, overnight-fasted and exercise-induced plasma C4-C14 acylcarnitines did not differ between pre- and postintervention phases. These metabolites all increased robustly with exercise (∼45% of pre-intervention peak oxygen consumption) and decreased during a 20 min cool-down. This supports the idea that, regardless of insulin sensitivity and fitness, intramitochondrial muscle ß-oxidation and attendant incomplete FAO are closely tethered to absolute ATP turnover rate. Acute exercise also led to branched-chain amino acid acylcarnitine derivative patterns suggestive of rapid and transient diminution of branched-chain amino acid flux through the mitochondrial branched-chain ketoacid dehydrogenase complex. We confirmed our prior novel observation that a weight-loss/fitness intervention alters plasma xenometabolites [i.e. cis-3,4-methylene-heptanoylcarnitine and γ-butyrobetaine (a co-metabolite possibly derived in part from gut bacteria)], suggesting that host metabolic health regulated gut microbe metabolism. Finally, we considered whether acylcarnitine metabolites signal to muscle-innervating afferents; palmitoylcarnitine at concentrations as low as 1-10 µm activated a subset (∼2.5-5%) of these neurons ex vivo. This supports the hypothesis that in addition to tracking exercise-associated shifts in fuel metabolism, muscle acylcarnitines act as signals of exertion to short-loop somatosensory-motor circuits or to the brain.

Gene Expression Factor Analysis to Differentiate Pathways Linked to Fibromyalgia, Chronic Fatigue Syndrome, and Depression in a Diverse Patient Sample.

OBJECTIVE: To determine if independent candidate genes can be grouped into meaningful biologic factors, and whether these factors are associated with the diagnosis of chronic fatigue syndrome (CFS) and fibromyalgia syndrome (FMS), while controlling for comorbid depression, sex, and age. METHODS: We included leukocyte messenger RNA gene expression from a total of 261 individuals, including healthy controls (n = 61), patients with FMS only (n = 15), with CFS only (n = 33), with comorbid CFS and FMS (n = 79), and with medication-resistant (n = 42) or medication-responsive (n = 31) depression. We used exploratory factor analysis (EFA) on 34 candidate genes to determine factor scores and regression analysis to examine whether these factors were associated with specific diagnoses. RESULTS: EFA resulted in 4 independent factors with minimal overlap of genes between factors, explaining 51% of the variance. We labeled these factors by function as 1) purinergic and cellular modulators, 2) neuronal growth and immune function, 3) nociception and stress mediators, and 4) energy and mitochondrial function. Regression analysis predicting these biologic factors using FMS, CFS, depression severity, age, and sex revealed that greater expression in factors 1 and 3 was positively associated with CFS and negatively associated with depression severity (Quick Inventory for Depression Symptomatology score), but not associated with FMS. CONCLUSION: Expression of candidate genes can be grouped into meaningful clusters, and CFS and depression are associated with the same 2 clusters, but in opposite directions, when controlling for comorbid FMS. Given high comorbid disease and interrelationships between biomarkers, EFA may help determine patient subgroups in this population based on gene expression.

Fatigue sensation and gene expression in trained cyclists following a 40 km time trial in the heat.

PURPOSE: We examined the effect of race-effort cycling exercise with and without heat stress on post-exercise perceptions of fatigue and pain, as well as mRNA expression in genes related to exercise responses. METHODS: Trained cyclists (n = 20) completed 40 km time trials during temperate (TC, 21 °C) and hot (HC, 35 °C) conditions. Blood lactates were measured 1 and 5 min post-exercise. Venous blood samples and ratings of fatigue and pain perceptions were obtained at baseline and at 0.5, 8, 24, and 48 h post-exercise. Leukocyte mRNA expression was performed for metabolite detecting, adrenergic, monoamine, and immune receptors using qPCR. RESULTS: Significantly lower mean power (157 ± 32.3 vs 187 ± 40 W) and lactates (6.4 ± 1.7 vs 8.8 ± 3.2 and 4.2 ± 1.5 vs 6.6 ± 2.7 mmol L(-1) at 1- and 5-min post-exercise) were observed for HC versus TC, respectively (p < 0.05). Increases (p < 0.05) in physical fatigue and pain perception during TTs did not differ between TC and HC (p > 0.30). Both trials resulted in significant post-exercise decreases in metabolite detecting receptors ASIC3, P2X4, TRPV1, and TRPV4; increases in adrenergic receptors α2a, α2c, and ß1; decreases in adrenergic ß2, the immune receptor TLR4, and dopamine (DRD4); and increases in serotonin (HTR1D) and IL-10 (p < 0.05). Post-exercise IL-6 differed between TC and HC, with significantly greater increases observed following HC (p < 0.05). CONCLUSIONS: Both TT performances appeared to be regulated around a specific sensory perception of fatigue and pain. Heat stress may have compensated for lower lactate during HC, thereby matching changes in metabolite detecting and other mRNAs across conditions.

Effect of Pregabalin on Cardiovascular Responses to Exercise and Postexercise Pain and Fatigue in Fibromyalgia: A Randomized, Double-Blind, Crossover Pilot Study.

Pregabalin, an approved treatment for fibromyalgia (FM), has been shown to decrease sympathetic nervous system (SNS) activity and inhibit sympathetically maintained pain, but its effects on exercise responses have not been reported. Methods. Using a randomized double-blind crossover design, we assessed the effect of 5 weeks of pregabalin (versus placebo) on acute cardiovascular and subjective responses to moderate exercise in 19 FM patients. Blood pressure (BP), heart rate (HR), and ratings of perceived exertion (RPE) during exercise and ratings of pain, physical fatigue, and mental fatigue before, during, and for 48 hours after exercise were compared in patients on pregabalin versus placebo and also versus 18 healthy controls. Results. On placebo, exercise RPE and BP were significantly higher in FM patients than controls (p < 0.04). Pregabalin responders (n = 12, defined by patient satisfaction and symptom changes) had significantly lower exercise BP, HR, and RPE on pregabalin versus placebo (p < 0.03) and no longer differed from controls (p > 0.26). Cardiovascular responses of nonresponders (n = 7) were not altered by pregabalin. In responders, pregabalin improved ratings of fatigue and pain (p < 0.04), but negative effects on pain and fatigue were seen in nonresponders. Conclusions. These preliminary findings suggest that pregabalin may normalize cardiovascular and subjective responses to exercise in many FM patients.

Griseorhodins D-F, neuroactive intermediates and end products of post-PKS tailoring modification in Griseorhodin biosynthesis.

The griseorhodins belong to a family of extensively modified aromatic polyketides that exhibit activities such as inhibition of HIV reverse transcriptase and human telomerase. The vast structural diversity of this group of polyketides is largely introduced by enzymatic oxidations, which can significantly influence the bioactivity profile. Four new compounds, griseorhodins D-F, were isolated from a griseorhodin producer, Streptomyces sp. CN48+, based upon their enhancement of calcium uptake in a mouse dorsal root ganglion primary cell culture assay. Two of these compounds, griseorhodins D1 and D2, were shown to be identical to the major, previously uncharacterized products of a grhM mutant in an earlier griseorhodin biosynthesis study. Their structures enabled the establishment of a more complete hypothesis for the biosynthesis of griseorhodins and related compounds. The other two compounds, griseorhodins E and F, represent new products of post-polyketide synthase tailoring in griseorhodin biosynthesis and showed significant binding activity in a human dopamine active transporter assay.

Exogenously applied muscle metabolites synergistically evoke sensations of muscle fatigue and pain in human subjects.

NEW FINDINGS: What is the central question of this study? Can physiological concentrations of metabolite combinations evoke sensations of fatigue and pain when injected into skeletal muscle? If so, what sensations are evoked? What is the main finding and its importance? Low concentrations of protons, lactate and ATP evoked sensations related to fatigue. Higher concentrations of these metabolites evoked pain. Single metabolites evoked no sensations. This suggests that the combination of an ASIC receptor and a purinergic P2X receptor is required for signalling fatigue and pain. The results also suggest that two types of sensory neurons encode metabolites; one detects low concentrations of metabolites and signals sensations of fatigue, whereas the other detects higher levels of metabolites and signals ache and hot. The perception of fatigue is common in many disease states; however, the mechanisms of sensory muscle fatigue are not understood. In mice, rats and cats, muscle afferents signal metabolite production in skeletal muscle using a complex of ASIC, P2X and TRPV1 receptors. Endogenous muscle agonists for these receptors are combinations of protons, lactate and ATP. Here we applied physiological concentrations of these agonists to muscle interstitium in human subjects to determine whether this combination could activate sensations and, if so, to determine how the subjects described these sensations. Ten volunteers received infusions (0.2 ml over 30 s) containing protons, lactate and ATP under the fascia of a thumb muscle, abductor pollicis brevis. Infusion of individual metabolites at maximal amounts evoked no fatigue or pain. Metabolite combinations found in resting muscles (pH 7.4 + 300 nm ATP + 1 mm lactate) also evoked no sensation. The infusion of a metabolite combination found in muscle during moderate endurance exercise (pH 7.3 + 400 nm ATP + 5 mm lactate) produced significant fatigue sensations. Infusion of a metabolite combination associated with vigorous exercise (pH 7.2 + 500 nm ATP + 10 mm lactate) produced stronger sensations of fatigue and some ache. Higher levels of metabolites (as found with ischaemic exercise) caused more ache but no additional fatigue sensation. Thus, in a dose-dependent manner, intramuscular infusion of combinations of protons, lactate and ATP leads to fatigue sensation and eventually pain, probably through activation of ASIC, P2X and TRPV1 receptors. This is the first demonstration in humans that metabolites normally produced by exercise act in combination to activate sensory neurons that signal sensations of fatigue and muscle pain.

Dysregulation of leukocyte gene expression in women with medication-refractory depression versus healthy non-depressed controls.

BACKGROUND: Depressive Disorders (DD) are a great financial and social burden. Females display 70% higher rate of depression than males and more than 30% of these patients do not respond to conventional medications. Thus medication-refractory female patients are a large, under-served, group where new biological targets for intervention are greatly needed. METHODS: We used real-time quantitative polymerase chain reaction (qPCR) to evaluate mRNA gene expression from peripheral blood leukocytes for 27 genes, including immune, HPA-axis, ion channels, and growth and transcription factors. Our sample included 23 females with medication refractory DD: 13 with major depressive disorder (MDD), 10 with bipolar disorder (BPD). Our comparison group was 19 healthy, non-depressed female controls. We examined differences in mRNA expression in DD vs. controls, in MDD vs. BPD, and in patients with greater vs. lesser depression severity. RESULTS: DD patients showed increased expression for IL-10, IL-6, OXTR, P2RX7, P2RY1, and TRPV1. BPD patients showed increased APP, CREB1, NFKB1, NR3C1, and SPARC and decreased TNF expression. Depression severity was related to increased IL-10, P2RY1, P2RX1, and TRPV4 expression. CONCLUSIONS: These results support prior findings of dysregulation in immune genes, and provide preliminary evidence of dysregulation in purinergic and other ion channels in females with medication-refractory depression, and in transcription and growth factors in those with BPD. If replicated in future research examining protein levels as well as mRNA, these pathways could potentially be used to explore biological mechanisms of depression and to develop new drug targets.

Differing leukocyte gene expression profiles associated with fatigue in patients with prostate cancer versus chronic fatigue syndrome.

BACKGROUND: Androgen deprivation therapy (ADT) often worsens fatigue in patients with prostate cancer, producing symptoms similar to chronic fatigue syndrome (CFS). Comparing expression (mRNA) of many fatigue-related genes in patients with ADT-treated prostate cancer versus with CFS versus healthy controls, and correlating mRNA with fatigue severity may clarify the differing pathways underlying fatigue in these conditions. METHODS: Quantitative real-time PCR was performed on leukocytes from 30 fatigued, ADT-treated prostate cancer patients (PCF), 39 patients with CFS and 22 controls aged 40-79, together with ratings of fatigue and pain severity. 46 genes from these pathways were included: (1) adrenergic/monoamine/neuropeptides, (2) immune, (3) metabolite-detecting, (4) mitochondrial/energy, (5) transcription factors. RESULTS: PCF patients showed higher expression than controls or CFS of 2 immune transcription genes (NR3C1 and TLR4), chemokine CXCR4, and mitochondrial gene SOD2. They showed lower expression of 2 vasodilation-related genes (ADRB2 and VIPR2), 2 cytokines (TNF and LTA), and 2 metabolite-detecting receptors (ASIC3 and P2RX7). CFS patients showed higher P2RX7 and lower HSPA2 versus controls and PCF. Correlations with fatigue severity were similar in PCF and CFS for only DBI, the GABA-A receptor modulator (r=-0.50, p<0.005 and r=-0.34, p<0.05). Purinergic P2RY1 was correlated only with PCF fatigue and pain severity (r=+0.43 and +0.59, p=0.025 and p=0.001). CONCLUSIONS: PCF patients differed from controls and CFS in mean expression of 10 genes from all 5 pathways. Correlations with fatigue severity implicated DBI for both patient groups and P2RY1 for PCF only. These pathways may provide new targets for interventions to reduce fatigue.

Neuroactive diol and acyloin metabolites from cone snail-associated bacteria.

The bacterium Gordonia sp. 647W.R.1a.05 was cultivated from the venom duct of the cone snail, Conus circumcisus. The Gordonia sp. organic extract modulated the action potential of mouse dorsal root ganglion neurons. Assay-guided fractionation led to the identification of the new compound circumcin A (1) and 11 known analogs (2-12). Two of these compounds, kurasoin B (7) and soraphinol A (8), were active in a human norepinephrine transporter assay with Ki values of 2575 and 867 nM, respectively. No neuroactivity had previously been reported for compounds in this structural class. Gordonia species have been reproducibly isolated from four different cone snail species, indicating a consistent association between these organisms.

Activation of transient receptor potential ankyrin-1 (TRPA1) in lung cells by wood smoke particulate material.

Cigarette smoke, diesel exhaust, and other combustion-derived particles activate the calcium channel transient receptor potential ankyrin-1 (TRPA1), causing irritation and inflammation in the respiratory tract. It was hypothesized that wood smoke particulate and select chemical constituents thereof would also activate TRPA1 in lung cells, potentially explaining the adverse effects of wood and other forms of biomass smoke on the respiratory system. TRPA1 activation was assessed using calcium imaging assays in TRPA1-overexpressing HEK-293 cells, mouse primary trigeminal neurons, and human adenocarcinoma (A549) lung cells. Particles from pine and mesquite smoke were less potent agonists of TRPA1 than an equivalent mass concentration of an ethanol extract of diesel exhaust particles; pine particles were comparable in potency to cigarette smoke condensate, and mesquite particles were the least potent. The fine particulate (PM < 2.5 µm) of wood smoke were the most potent TRPA1 agonists and several chemical constituents of wood smoke particulate, 3,5-ditert-butylphenol, coniferaldehyde, formaldehyde, perinaphthenone, agathic acid, and isocupressic acid, were TRPA1 agonists. Pine particulate activated TRPA1 in mouse trigeminal neurons and A549 cells in a concentration-dependent manner, which was inhibited by the TRPA1 antagonist HC-030031. TRPA1 activation by wood smoke particles occurred through the electrophile/oxidant-sensing domain (i.e., C621/C641/C665/K710), based on the inhibition of cellular responses when the particles were pretreated with glutathione; a role for the menthol-binding site of TRPA1 (S873/T874) was demonstrated for 3,5-ditert-butylphenol. This study demonstrated that TRPA1 is a molecular sensor for wood smoke particulate and several chemical constituents thereof, in sensory neurons and A549 cells, suggesting that TRPA1 may mediate some of the adverse effects of wood smoke in humans.

A bacterial source for mollusk pyrone polyketides.

In the oceans, secondary metabolites often protect otherwise poorly defended invertebrates, such as shell-less mollusks, from predation. The origins of these metabolites are largely unknown, but many of them are thought to be made by symbiotic bacteria. In contrast, mollusks with thick shells and toxic venoms are thought to lack these secondary metabolites because of reduced defensive needs. Here, we show that heavily defended cone snails also occasionally contain abundant secondary metabolites, γ-pyrones known as nocapyrones, which are synthesized by symbiotic bacteria. The bacteria, Nocardiopsis alba CR167, are related to widespread actinomycetes that we propose to be casual symbionts of invertebrates on land and in the sea. The natural roles of nocapyrones are unknown, but they are active in neurological assays, revealing that mollusks with external shells are an overlooked source of secondary metabolite diversity.

Differences in metabolite-detecting, adrenergic, and immune gene expression after moderate exercise in patients with chronic fatigue syndrome, patients with multiple sclerosis, and healthy controls.

OBJECTIVE: Chronic fatigue syndrome (CFS) and multiple sclerosis (MS) are characterized by debilitating fatigue, yet evaluation of this symptom is subjective. We examined metabolite-detecting, adrenergic, and immune gene expression (messenger ribonucleic acid [mRNA]) in patients with CFS (n = 22) versus patients with MS (n = 20) versus healthy controls (n = 23) and determined their relationship to fatigue and pain before and after exercise. METHODS: Blood samples and fatigue and pain ratings were obtained at baseline and 0.5, 8, 24, and 48 hours after sustained moderate exercise. Leukocyte mRNA of four metabolite-detecting receptors (acid-sensing ion channel 3, purinergic type 2X4 and 2X5 receptors, and transient receptor potential vanilloid type 1) and four adrenergic (α-2a, ß-1, and ß-2 receptors and catechol-O-methyltransferase) and five immune markers (CD14, toll-like receptor 4 [TLR4], interleukin [IL] 6, IL-10, and lymphotoxin α) was examined using quantitative polymerase chain reaction. RESULTS: Patients with CFS had greater postexercise increases in fatigue and pain (10-29 points above baseline, p < .001) and greater mRNA increases in purinergic type 2X4 receptor, transient receptor potential vanilloid type 1, CD14, and all adrenergic receptors than controls (mean ± standard error = 1.3 ± 0.14- to 3.4 ± 0.90-fold increase above baseline, p = .04-.005). Patients with CFS with comorbid fibromyalgia (n = 18) also showed greater increases in acid-sensing ion channel 3 and purinergic type 2X5 receptors (p < .05). Patients with MS had greater postexercise increases than controls in ß-1 and ß-2 adrenergic receptor expressions (1.4 ± 0.27- and 1.3 ± 0.06-fold increases, respectively, p = .02 and p < .001) and greater decreases in TLR4 (p = .02). In MS, IL-10 and TLR4 decreases correlated with higher fatigue scores. CONCLUSIONS: Postexercise mRNA increases in metabolite-detecting receptors were unique to patients with CFS, whereas both patients with MS and patients with CFS showed abnormal increases in adrenergic receptors. Among patients with MS, greater fatigue was correlated with blunted immune marker expression.

Electrophilic components of diesel exhaust particles (DEP) activate transient receptor potential ankyrin-1 (TRPA1): a probable mechanism of acute pulmonary toxicity for DEP.

Inhalation of environmental particulate matter (PM) is correlated with adverse health effects in humans, but gene products that couple detection with cellular responses, and the specific properties of PM that target different pathways, have not been fully elucidated. TRPA1 and V1 are two cation channels expressed by sensory neurons and non-neuronal cells of the respiratory tract that have been implicated as possible mediators of PM toxicity. The goals of this research were to determine if environmental PM preferentially activated TRPA1 and to elucidate the criteria responsible for selectivity. Quantification of TRPA1 activation by 4 model PM revealed that diesel exhaust PM (DEP) and coal fly ash PM (CFA1) were TRPA1 agonists at concentrations >0.077 mg/mL. DEP was more potent, and approximately 97% of the activity of DEP was recovered by serial extraction of the solid DEP with ethanol and hexane/n-butyl chloride. Modification of the electrophile/agonist binding sites on TRPA1 (C621, C641, C665, and K710) to non-nucleophilic residues reduced TRPA1 activation by DEP and abolished activation by DEP extracts as well as multiple individual electrophilic chemical components of DEP. However, responses to CFA1 and DEP solids were not affected by these mutations. Activity-guided fractionation of DEP and high resolution mass spectroscopy identified several new DEP-derived TRPA1 agonists, and activation of mouse dorsal root ganglion neurons demonstrated that TRPA1 is a primary target for DEP in a heterogeneous population of primary sensory nerves. It is concluded that TRPA1 is a specific target for electrophilic chemical components of DEP and proposed that activation of TRPA1 in the respiratory tract is likely to be an important mechanism for DEP pneumotoxicity.

Absence of XMRV retrovirus and other murine leukemia virus-related viruses in patients with chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a multisystem disorder characterized by prolonged and severe fatigue that is not relieved by rest. Attempts to treat CFS have been largely ineffective primarily because the etiology of the disorder is unknown. Recently, CFS has been associated with xenotropic murine leukemia virus-related virus (XMRV) as well as other murine leukemia virus (MLV)-related viruses, though not all studies have found these associations. We collected blood samples from 100 CFS patients and 200 self-reported healthy volunteers from the same geographical area. We analyzed these in a blind manner using molecular, serological, and viral replication assays. We also analyzed samples from patients in the original study that reported XMRV in CFS patients. We did not find XMRV or related MLVs either as viral sequences or infectious viruses, nor did we find antibodies to these viruses in any of the patient samples, including those from the original study. We show that at least some of the discrepancy with previous studies is due to the presence of trace amounts of mouse DNA in the Taq polymerase enzymes used in these previous studies. Our findings do not support an association between CFS and MLV-related viruses, including XMRV, and the off-label use of antiretrovirals for the treatment of CFS does not seem justified at present.

Nobilamides A-H, long-acting transient receptor potential vanilloid-1 (TRPV1) antagonists from mollusk-associated bacteria.

New compounds nobilamides A-H and related known compounds A-3302-A and A-3302-B were isolated based upon their suppression of capsaicin-induced calcium uptake in a mouse dorsal root ganglion primary cell culture assay. Two of these compounds, nobilamide B and A-3302-A, were shown to be long-acting antagonists of mouse and human TRPV1 channels, abolishing activity for >1 h after removal of drug presumably via a covalent attachment. Other derivatives also inhibited the TRPV1 channel, albeit with low potency, affording a structure-activity profile to support the proposed mechanism of action. While the activities were modest, we propose a new mechanism of action and a new site of binding for these inhibitors that may spur development of related analogues for treatment of pain.

Pulicatins A-E, neuroactive thiazoline metabolites from cone snail-associated bacteria.

The cone snail Conus pulicarius from the Philippines provides a specific habitat for actinomycetes and other bacteria. A phenotypic screen using primary cultures of mouse dorsal root ganglion neurons revealed that one C. pulicarius associate, Streptomyces sp. CP32, produces a series of natural products that enhance or diminish whole-cell Ca(2+) flux. These compounds include known thiazoline compounds and a series of new derivatives, pulicatins A-E (6-10). Individual compounds were shown to bind to a series of human receptors, with selective binding to the human serotonin 5-HT(2B) receptor. Here, we report the structure elucidation of the new compounds and results of the neurological assays.