Spoxazomicin D and Oxachelin C, Potent Neuroprotective Carboxamides from the Appalachian Coal Fire-Associated Isolate Streptomyces sp. RM-14-6.

The isolation and structure elucidation of six new bacterial metabolites [spoxazomicin D (2), oxachelins B and C (4, 5), and carboxamides 6-8] and 11 previously reported bacterial metabolites (1, 3, 9-12a, and 14-18) from Streptomyces sp. RM-14-6 is reported. Structures were elucidated on the basis of comprehensive 1D and 2D NMR and mass spectrometry data analysis, along with direct comparison to synthetic standards for 2, 11, and 12a,b. Complete 2D NMR assignments for the known metabolites lenoremycin (9) and lenoremycin sodium salt (10) were also provided for the first time. Comparative analysis also provided the basis for structural revision of several previously reported putative aziridine-containing compounds [exemplified by madurastatins A1, B1, C1 (also known as MBJ-0034), and MBJ-0035] as phenol-dihydrooxazoles. Bioactivity analysis [including antibacterial, antifungal, cancer cell line cytotoxicity, unfolded protein response (UPR) modulation, and EtOH damage neuroprotection] revealed 2 and 5 as potent neuroprotectives and lenoremycin (9) and its sodium salt (10) as potent UPR modulators, highlighting new functions for phenol-oxazolines/salicylates and polyether pharmacophores.

Identification of Neuroprotective Spoxazomicin and Oxachelin Glycosides via Chemoenzymatic Glycosyl-Scanning.

The assessment of glycosyl-scanning to expand the molecular and functional diversity of metabolites from the underground coal mine fire-associated Streptomyces sp. RM-14-6 is reported. Using the engineered glycosyltransferase OleD Loki and a 2-chloro-4-nitrophenylglycoside-based screen, six metabolites were identified as substrates of OleD Loki, from which 12 corresponding metabolite glycosides were produced and characterized. This study highlights the first application of the 2-chloro-4-nitrophenylglycoside-based screen toward an unbiased set of unique microbial natural products and the first reported application of the 2-chloro-4-nitrophenylglycoside-based transglycosylation reaction for the corresponding preparative synthesis of target glycosides. Bioactivity analysis (including antibacterial, antifungal, anticancer, and EtOH damage neuroprotection assays) revealed glycosylation to attenuate the neuroprotective potency of 4, while glycosylation of the structurally related inactive spoxazomicin C (3) remarkably invoked neuroprotective activity.

Functional AdoMet Isosteres Resistant to Classical AdoMet Degradation Pathways.

S-adenosyl-l-methionine (AdoMet) is an essential enzyme cosubstrate in fundamental biology with an expanding range of biocatalytic and therapeutic applications. We report the design, synthesis, and evaluation of stable, functional AdoMet isosteres that are resistant to the primary contributors to AdoMet degradation (depurination, intramolecular cyclization, and sulfonium epimerization). Corresponding biochemical and structural studies demonstrate the AdoMet surrogates to serve as competent enzyme cosubstrates and to bind a prototypical class I model methyltransferase (DnrK) in a manner nearly identical to AdoMet. Given this conservation in function and molecular recognition, the isosteres presented are anticipated to serve as useful surrogates in other AdoMet-dependent processes and may also be resistant to, and/or potentially even inhibit, other therapeutically relevant AdoMet-dependent metabolic transformations (such as the validated drug target AdoMet decarboxylase). This work also highlights the ability of the prototypical class I model methyltransferase DnrK to accept non-native surrogate acceptors as an enabling feature of a new high-throughput methyltransferase assay.

Steroid binding to Autotaxin links bile salts and lysophosphatidic acid signalling.

Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors of ATX, thereby attenuating LPA receptor activation. This unexpected interplay between ATX-LPA signalling and select steroids, notably natural bile salts, provides a molecular basis for the emerging association of ATX with disorders associated with increased circulating levels of bile salts. Furthermore, our findings suggest potential clinical implications in the use of steroid drugs.

Pharmacological Elevation of Circulating Bioactive Phosphosphingolipids Enhances Myocardial Recovery After Acute Infarction.

UNLABELLED: Acute myocardial infarction (AMI) triggers mobilization of bone marrow (BM)-derived stem/progenitor cells (BMSPCs) through poorly understood processes. Recently, we postulated a major role for bioactive lipids such as sphingosine-1 phosphate (S1P) in mobilization of BMSPCs into the peripheral blood (PB). We hypothesized that elevating S1P levels after AMI could augment BMSPC mobilization and enhance cardiac recovery after AMI. After AMI, elevating bioactive lipid levels was achieved by treating mice with the S1P lyase inhibitor tetrahydroxybutylimidazole (THI) for 3 days (starting at day 4 after AMI) to differentiate between stem cell mobilization and the known effects of S1P on myocardial ischemic pre- and postconditioning. Cardiac function was assessed using echocardiography, and myocardial scar size evolution was examined using cardiac magnetic resonance imaging. PB S1P and BMSPCs peaked at 5 days after AMI and returned to baseline levels within 10 days (p < .05 for 5 days vs. baseline). Elevated S1P paralleled a significant increase in circulating BMSPCs (p < .05 vs. controls). We observed a greater than twofold increase in plasma S1P and circulating BMSPCs after THI treatment. Mechanistically, enhanced BMSPC mobilization was associated with significant increases in angiogenesis, BM cell homing, cardiomyocytes, and c-Kit cell proliferation in THI-treated mice. Mice treated with THI demonstrated better recovery of cardiac functional parameters and a reduction in scar size. Pharmacological elevation of plasma bioactive lipids after AMI could contribute to BMSPC mobilization and could represent an attractive strategy for enhancing myocardial recovery and improving BMSC targeting. SIGNIFICANCE: Acute myocardial infarction (AMI) initiates innate immune and reparatory mechanisms through which bone marrow-derived stem/progenitor cells (BMSPCs) are mobilized toward the ischemic myocardium and contribute to myocardial regeneration. Although it is clear that the magnitude of BMSPC mobilization after AMI correlates with cardiac recovery, the molecular events driving BMSPC mobilization and homing are poorly understood. The present study confirms the role of bioactive lipids in BMSPC mobilization after AMI and proposes a new strategy that improves cardiac recovery. Inhibiting sphingosine-1 phosphate (S1P) lyase (SPL) allows for the augmentation of the plasma levels of S1P and stem cell mobilization. These findings demonstrate that early transient SPL inhibition after MI correlates with increased stem cell mobilization and their homing to the infarct border zones. Augmenting BMSPC mobilization correlated with the formation of new blood vessels and cardiomyocytes and c-Kit cell proliferation. These novel findings on the cellular level were associated with functional cardiac recovery, reduced adverse remodeling, and a decrease in scar size. Taken together, these data indicate that pharmacological elevation of bioactive lipid levels can be beneficial in the early phase after cardiac ischemic injury. These findings provide the first evidence that a carefully timed transient pharmacological upregulation of bioactive lipids after AMI could be therapeutic, because it results in significant cardiac structural and functional improvements.

Loss of multidrug resistance-associated protein 1 potentiates chronic doxorubicin-induced cardiac dysfunction in mice.

Doxorubicin (DOX), an effective cancer chemotherapeutic agent, induces dose-dependent cardiotoxicity, in part due to its ability to cause oxidative stress. We investigated the role of multidrug resistance-associated protein 1 (Mrp1/Abcc1) in DOX-induced cardiotoxicity in C57BL wild-type (WT) mice and their Mrp1 null (Mrp1(-/-)) littermates. Male mice were administered intraperitoneal DOX (3 or 2 mg/kg body weight) or saline twice a week for 3 weeks and examined 2 weeks after the last dose (protocol A total dose: 18 mg/kg) or for 5 weeks, and mice were examined 48 hours and 2 weeks after the last dose (protocol B total dose: 20 mg/kg). Chronic DOX induced body weight loss and hemotoxicity, adverse effects significantly exacerbated in Mrp1(-/-) versus WT mice. In the heart, significantly higher basal levels of glutathione (1.41-fold ± 0.27-fold) and glutathione disulfide (1.35-fold ± 0.16-fold) were detected in Mrp1(-/-) versus WT mice, and there were comparable decreases in the glutathione/glutathione disulfide ratio in WT and Mrp1(-/-) mice after DOX administration. Surprisingly, DOX induced comparable increases in 4-hydroxynonenal glutathione conjugate concentration in hearts from WT and Mrp1(-/-) mice. However, more DOX-induced apoptosis was detected in Mrp1(-/-) versus WT hearts (P < 0.05) (protocol A), and cardiac function, assessed by measurement of fractional shortening and ejection fraction with echocardiography, was significantly decreased by DOX in Mrp1(-/-) versus WT mice (P < 0.05; 95% confidence intervals of 20.0%-24.3% versus 23.7%-29.5% for fractional shortening, and 41.5%-48.4% versus 47.7%-56.7% for ejection fraction; protocol B). Together, these data indicate that Mrp1 protects the mouse heart against chronic DOX-induced cardiotoxicity.

Elevated glutathione is not sufficient to protect against doxorubicin-induced nuclear damage in heart in multidrug resistance-associated protein 1 (Mrp1/Abcc1) null mice.

Cardiotoxicity is a major dose-limiting adverse effect of doxorubicin (DOX), mediated in part by overproduction of reactive oxygen species and oxidative stress. Abcc1 (Mrp1) mediates the efflux of reduced and oxidized glutathione (GSH, GSSG) and is also a major transporter that effluxes the GSH conjugate of 4-hydroxy-2-nonenal (HNE; GS-HNE), a toxic product of lipid peroxidation formed during oxidative stress. To assess the role of Mrp1 in protecting the heart from DOX-induced cardiac injury, wild-type (WT) and Mrp1 null (Mrp1(-/-)) C57BL/6 littermate mice were administered DOX (15 mg/kg) or saline (7.5 ml/kg) i.v., and heart ventricles were examined at 72 hours. Morphometric analysis by electron microscopy revealed extensive injuries in cytosol, mitochondria, and nuclei of DOX-treated mice in both genotypes. Significantly more severely injured nuclei were observed in Mrp1(-/-) versus WT mice (P = 0.031). GSH and the GSH/GSSG ratio were significantly increased in treatment-naïve Mrp1(-/-) versus WT mice; GSH remained significantly higher in Mrp1(-/-) versus WT mice after saline and DOX treatment, with no changes in GSSG or GSH/GSSG. GS-HNE, measured by mass spectrometry, was lower in the hearts of treatment-naïve Mrp1(-/-) versus WT mice (P < 0.05). DOX treatment decreased GS-HNE in WT but not Mrp1(-/-) mice, so that GS-HNE was modestly but significantly higher in Mrp1(-/-) versus WT hearts after DOX. Expression of enzymes mediating GSH synthesis and antioxidant proteins did not differ between genotypes. Thus, despite elevated GSH levels in Mrp1(-/-) hearts, DOX induced significantly more injury in the nuclei of Mrp1(-/-) versus WT hearts.

Effects of Adipocyte Aryl Hydrocarbon Receptor Deficiency on PCB-Induced Disruption of Glucose Homeostasis in Lean and Obese Mice.

BACKGROUND: Coplanar polychlorinated biphenyls (PCBs) promote adipocyte inflammation and impair glucose homeostasis in lean mice. The diabetes-promoting effects of lipophilic PCBs have been observed only during weight loss in obese mice. The molecular mechanisms linking PCB exposures to impaired glucose metabolism are unclear. OBJECTIVES: In this study we tested the hypothesis that coplanar PCBs act at adipocyte aryl hydrocarbon receptors (AhRs) to promote adipose inflammation and impair glucose homeostasis in lean mice and in obese mice during weight loss. METHODS AND RESULTS: PCB-77 administration impaired glucose and insulin tolerance in LF (low fat diet)-fed control (AhR(fl/fl)) mice but not in adipocyte AhR-deficient mice (AhR(AdQ)). Unexpectedly, AhR(AdQ) mice exhibited increased fat mass when fed a standard LF or high fat (HF) diet. In mice fed a HF diet, both genotypes became obese, but AhR(AdQ) mice administered vehicle (VEH) exhibited increased body weight, adipose mass, adipose inflammation, and impaired glucose tolerance compared with AhR(fl/fl) controls. Impairment of glucose homeostasis in response to PCB-77 was not observed in obese mice of either genotype. However, upon weight loss, AhR(fl/fl) mice administered PCB-77 exhibited increased abundance of adipose tumor necrosis factor-α (TNF-α) mRNA and impaired glucose homeostasis compared with those administered VEH. In contrast, PCB-77 had no effect on TNF-α or glucose homeostasis in AhR(AdQ) mice exhibiting weight loss. CONCLUSIONS: Our results demonstrate that adipocyte AhR mediates PCB-induced adipose inflammation and impairment of glucose homeostasis in mice. Moreover, deficiency of AhR in adipocytes augmented the development of obesity, indicating that endogenous ligand(s) for AhR regulate adipose homeostasis.

ORMDL/serine palmitoyltransferase stoichiometry determines effects of ORMDL3 expression on sphingolipid biosynthesis.

The ORM1 (Saccharomyces cerevisiae)-like proteins (ORMDLs) and their yeast orthologs, the Orms, are negative homeostatic regulators of the initiating enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT). Genome-wide association studies have established a strong correlation between elevated expression of the endoplasmic reticulum protein ORMDL3 and risk for childhood asthma. Here we test the notion that elevated levels of ORMDL3 decrease sphingolipid biosynthesis. This was tested in cultured human bronchial epithelial cells (HBECs) (an immortalized, but untransformed, airway epithelial cell line) and in HeLa cells (a cervical adenocarcinoma cell line). Surprisingly, elevated ORMDL3 expression did not suppress de novo biosynthesis of sphingolipids. We determined that ORMDL is expressed in functional excess relative to SPT at normal levels of expression. ORMDLs and SPT form stable complexes that are not increased by elevated ORMDL3 expression. Although sphingolipid biosynthesis was not decreased by elevated ORMDL3 expression, the steady state mass levels of all major sphingolipids were marginally decreased by low level ORMDL3 over-expression in HBECs. These data indicate that the contribution of ORMDL3 to asthma risk may involve changes in sphingolipid metabolism, but that the connection is complex.

Regulation of de novo sphingolipid biosynthesis by the ORMDL proteins and sphingosine kinase-1.

Sphingolipids are a diverse set of structurally and metabolically related lipids that have numerous functions in cell structure and signaling. The regulation of these lipids is critical for normal cell function and disregulation has been implicated in pathophysiological conditions such as cancer and inflammation. Here we examine control of the initiating, and rate limiting, enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT). We find that de novo synthesis of sphingolipid is stimulated by a number of cancer chemotherapeutics, suggesting that this may be an important aspect of their cytotoxic effects. The three ORMDL proteins are membrane proteins of the endoplasmic reticulum related to the yeast Orm proteins, which have been shown to be homeostatic regulators of SPT. We find that the ORMDL proteins are also negative regulators of SPT that transmit cellular levels of sphingolipids to SPT. The three isoforms have redundant functions in this system. The sphingosine kinases (sphingosine kinase-1 and -2) phosphorylate both sphingosine, which is released from ceramide, but also dihydrosphingosine, which is in the de novo biosynthetic pathway. We therefore examined the role of the sphingosine kinases in controlling de novo ceramide biosynthesis and find that sphingosine kinase-1 does indeed act as a negative regulator of this pathway. This establishes that sphingosine kinase, in addition to producing sphingosine-1-phosphate as a signaling molecule, also consumes dihydrosphingosine to regulate ceramide synthesis. Our studies demonstrate that there are multiple mechanisms of regulation of SPT and suggest that these regulators are important mediators of cell stress responses.

Granule cargo release from bone marrow-derived cells sustains cardiac hypertrophy.

Bone marrow-derived inflammatory cells, including platelets, may contribute to the progression of pressure overload-induced left ventricular hypertrophy (LVH). However, the underlying mechanisms for this are still unclear. One potential mechanism is through release of granule cargo. Unc13-d(Jinx) (Jinx) mice, which lack Munc13-4, a limiting factor in vesicular priming and fusion, have granule secretion defects in a variety of hematopoietic cells, including platelets. In the current study, we investigated the role of granule secretion in the development of LVH and cardiac remodeling using chimeric mice specifically lacking Munc13-4 in marrow-derived cells. Pressure overload was elicited by transverse aortic constriction (TAC). Chimeric mice were created by bone marrow transplantation. Echocardiography, histology staining, immunohistochemistry, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and mass spectrometry were used to study LVH progression and inflammatory responses. Wild-type (WT) mice that were transplanted with WT bone marrow (WT→WT) and WT mice that received Jinx bone marrow (Jinx→WT) developed LVH and a classic fetal reprogramming response early (7 days) after TAC. However, at late times (5 wk), mice lacking Munc13-4 in bone marrow-derived cells (Jinx→WT) failed to sustain the cardiac hypertrophy observed in WT chimeric mice. No difference in cardiac fibrosis was observed at early or late time points. Reinjection of WT platelets or platelet releasate partially restored cardiac hypertrophy in Jinx chimeric mice. These results suggest that sustained LVH in the setting of pressure overload depends on one or more factors secreted from bone marrow-derived cells, possibly from platelets. Inhibiting granule cargo release may represent a novel target for preventing sustained LVH.

Effect of procysteine on aging-associated changes in hepatic GSH and SMase: evidence for transcriptional regulation of smpd3.

In hepatocytes, aging-associated decline in GSH has been linked to activation of neutral SMase (nSMase), accumulation of bioactive ceramide, and inflammation. In this study, we seek to test whether dietary supplementation with the cysteine precursor, L-2-oxothiazolidine-4-carboxylic acid (OTC), would correct the aging-associated differences in hepatic GSH, nSMase, and ceramide. Young and aged mice were placed on a diet that either lacked sulfur-containing amino acids (SAAs) or had 0.5% OTC for 4 weeks. Mice fed standard chow were used as an additional control. SAA-deficient mice exhibited significant aging-associated differences in hepatic GSH, GSH/GSSG, ceramide, and nSMase. C24:1 ceramide, the major ceramide species in liver, was affected the most by aging, followed by the less abundant C16:0 ceramide. OTC supplementation eliminated the aging-associated differences in hepatic GSH and GSH/GSSG ratio. Surprisingly, however, instead of decreasing, the nSMase activity and ceramide increased in the OTC-fed mice irrespective of their age. These effects were due to elevated nSMase-2 mRNA and protein and appeared to be direct. Similar increases were seen in HepG2 cells following treatment with OTC. The OTC-fed aged mice also exhibited hepatic steatosis and triacylglyceride accumulation. These results suggest that OTC is a potent stimulant of nSMase-2 expression and that there may be unanticipated complications of OTC supplementation.

Bisphenol A increases atherosclerosis in pregnane X receptor-humanized ApoE deficient mice.

BACKGROUND: Bisphenol A (BPA) is a base chemical used extensively in many consumer products. BPA has recently been associated with increased risk of cardiovascular disease (CVD) in multiple large-scale human population studies, but the underlying mechanisms remain elusive. We previously reported that BPA activates the pregnane X receptor (PXR), which acts as a xenobiotic sensor to regulate xenobiotic metabolism and has pro-atherogenic effects in animal models upon activation. Interestingly, BPA is a potent agonist of human PXR but does not activate mouse or rat PXR signaling, which confounds the use of rodent models to evaluate mechanisms of BPA-mediated CVD risk. This study aimed to investigate the atherogenic mechanism of BPA using a PXR-humanized mouse model. METHODS AND RESULTS: A PXR-humanized ApoE deficient (huPXR•ApoE(-/-)) mouse line was generated that respond to human PXR ligands and feeding studies were performed to determine the effects of BPA exposure on atherosclerosis development. Exposure to BPA significantly increased atherosclerotic lesion area in the aortic root and brachiocephalic artery of huPXR•ApoE(-/-) mice by 104% (P<0.001) and 120% (P<0.05), respectively. By contrast, BPA did not affect atherosclerosis development in the control littermates without human PXR. BPA exposure did not affect plasma lipid levels but increased CD36 expression and lipid accumulation in macrophages of huPXR•ApoE(-/-) mice. CONCLUSION: These findings identify a molecular mechanism that could link BPA exposure to increased risk of CVD in exposed individuals. PXR is therefore a relevant target for future risk assessment of BPA and related environmental chemicals in humans.

Facile chemoenzymatic strategies for the synthesis and utilization of S-adenosyl-(L)-methionine analogues.

A chemoenzymatic platform for the synthesis of S-adenosyl-L-methionine (SAM) analogues compatible with downstream SAM-utilizing enzymes is reported. Forty-four non-native S/Se-alkylated Met analogues were synthesized and applied to probing the substrate specificity of five diverse methionine adenosyltransferases (MATs). Human MAT II was among the most permissive of the MATs analyzed and enabled the chemoenzymatic synthesis of 29 non-native SAM analogues. As a proof of concept for the feasibility of natural product "alkylrandomization", a small set of differentially-alkylated indolocarbazole analogues was generated by using a coupled hMAT2-RebM system (RebM is the sugar C4'-O-methyltransferase that is involved in rebeccamycin biosynthesis). The ability to couple SAM synthesis and utilization in a single vessel circumvents issues associated with the rapid decomposition of SAM analogues and thereby opens the door for the further interrogation of a wide range of SAM utilizing enzymes.

Understanding molecular recognition of promiscuity of thermophilic methionine adenosyltransferase sMAT from Sulfolobus solfataricus.

UNLABELLED: Methionine adenosyltransferase (MAT) is a family of enzymes that utilizes ATP and methionine to produce S-adenosylmethionine (AdoMet), the most crucial methyl donor in the biological methylation of biomolecules and bioactive natural products. Here, we report that the MAT from Sulfolobus solfataricus (sMAT), an enzyme from a poorly explored class of the MAT family, has the ability to produce a range of differentially alkylated AdoMet analogs in the presence of non-native methionine analogs and ATP. To investigate the molecular basis for AdoMet analog production, we have crystallized the sMAT in the AdoMet bound, S-adenosylethionine (AdoEth) bound and unbound forms. Notably, among these structures, the AdoEth bound form offers the first MAT structure containing a non-native product, and cumulatively these structures add new structural insight into the MAT family and allow for detailed active site comparison with its homologs in Escherichia coli and human. As a thermostable MAT structure from archaea, the structures herein also provide a basis for future engineering to potentially broaden AdoMet analog production as reagents for methyltransferase-catalyzed 'alkylrandomization' and/or the study of methylation in the context of biological processes. DATABASES: PDB IDs: 4HPV, 4L7I, 4K0B and 4L2Z. EC 2.5.1.6 STRUCTURED DIGITAL ABSTRACT: • sMAT and sMAT bind by x-ray crystallography (View interaction).

Mice with targeted inactivation of ppap2b in endothelial and hematopoietic cells display enhanced vascular inflammation and permeability.

OBJECTIVE: Lipid phosphate phosphatase 3 (LPP3), encoded by the PPAP2B gene, is an integral membrane enzyme that dephosphorylates, and thereby terminates, the G-protein-coupled receptor-mediated signaling actions of lysophosphatidic acid (LPA) and sphingosine-1-phosphate. LPP3 is essential for normal vascular development in mice, and a common PPAP2B polymorphism is associated with increased risk of coronary artery disease in humans. Herein, we investigate the function of endothelial LPP3 to understand its role in the development and human disease. APPROACH AND RESULTS: We developed mouse models with selective LPP3 deficiency in endothelial and hematopoietic cells. Tyrosine kinase Tek promoter-mediated inactivation of Ppap2b resulted in embryonic lethality because of vascular defects. LPP3 deficiency in adult mice, achieved using a tamoxifen-inducible Cre transgene under the control of the Tyrosine kinase Tek promoter, enhanced local and systemic inflammatory responses. Endothelial, but not hematopoietic, cell LPP3 deficiency led to significant increases in vascular permeability at baseline and enhanced sensitivity to inflammation-induced vascular leak. Endothelial barrier function was restored by pharmacological or genetic inhibition of either LPA production by the circulating lysophospholipase D autotaxin or of G-protein-coupled receptor-dependent LPA signaling. CONCLUSIONS: Our results identify a role for the autotaxin/LPA-signaling nexus as a mediator of endothelial permeability in inflammation and demonstrate that LPP3 limits these effects. These findings have implications for therapeutic targets to maintain vascular barrier function in inflammatory states.

Green tea diet decreases PCB 126-induced oxidative stress in mice by up-regulating antioxidant enzymes.

Superfund chemicals such as polychlorinated biphenyls pose a serious human health risk due to their environmental persistence and link to multiple diseases. Selective bioactive food components such as flavonoids have been shown to ameliorate PCB toxicity, but primarily in an in vitro setting. Here, we show that mice fed a green tea-enriched diet and subsequently exposed to environmentally relevant doses of coplanar PCB exhibit decreased overall oxidative stress primarily due to the up-regulation of a battery of antioxidant enzymes. C57BL/6 mice were fed a low-fat diet supplemented with green tea extract (GTE) for 12 weeks and exposed to 5 µmol PCB 126/kg mouse weight (1.63 mg/kg-day) on weeks 10, 11 and 12 (total body burden: 4.9 mg/kg). F2-isoprostane and its metabolites, established markers of in vivo oxidative stress, measured in plasma via HPLC-MS/MS exhibited fivefold decreased levels in mice supplemented with GTE and subsequently exposed to PCB compared to animals on a control diet exposed to PCB. Livers were collected and harvested for both messenger RNA and protein analyses, and it was determined that many genes transcriptionally controlled by aryl hydrocarbon receptor and nuclear factor (erythroid-derived 2)-like 2 proteins were up-regulated in PCB-exposed mice fed the green tea-supplemented diet. An increased induction of genes such as SOD1, GSR, NQO1 and GST, key antioxidant enzymes, in these mice (green tea plus PCB) may explain the observed decrease in overall oxidative stress. A diet supplemented with green tea allows for an efficient antioxidant response in the presence of PCB 126, which supports the emerging paradigm that healthful nutrition may be able to bolster and buffer a physiological system against the toxicities of environmental pollutants.

Venturicidin C, a new 20-membered macrolide produced by Streptomyces sp. TS-2-2.

Venturicidin C (1), a new 20-membered macrolide along with the known venturicidins A (2) and B (3) were isolated from the crude extract of the Appalachian bacterial strain Streptomyces sp. TS-2-2. Additionally, nine other known compounds namely nocardamine, dehydroxynocardamine, desmethylenylnocardamine, ferrioxamine E, adenosine, riboflavin, cyclo(D)-trans-4-OH-Pro-(D)-Phe, cyclo(D)-Pro-(D)-Phe and N-(2-phenylethyl)-acetamide were also isolated and identified. The structure of the new macrolide 1 was elucidated by the cumulative analyses of NMR spectroscopy and HR-MS data. Complete NMR assignments for the known venturicidins A (2) and B (3) are also provided, for the first time, in this report. Venturicidins A-C did not inhibit the proliferation of A549 lung cancer cell line but all displayed potent antifungal activity.

Autophagy and oxidative stress in gliomas with IDH1 mutations.

IDH1 mutations in gliomas associate with longer survival. Prooxidant and antiproliferative effects of IDH1 mutations and its D-2-hydroxyglutarate (2-HG) product have been described in vitro, but inconsistently observed. It is also unclear whether overexpression of mutant IDH1 in wild-type cells accurately phenocopies the effects of endogenous IDH1-mutations on tumor apoptosis and autophagy. Herein we investigated the effects of 2-HG and mutant IDH1 overexpression on proliferation, apoptosis, oxidative stress, and autophagy in IDH1 wild-type glioma cells, and compared those results with patient-derived tumors. 2-HG reduced viability and proliferation of U87MG and LN18 cells, triggered apoptosis in LN18 cells, and autophagy in U87MG cells. In vitro studies and flank xenografts of U87MG cells overexpressing R132H IDH1 exhibited increased oxidative stress, including increases of both manganese superoxide dismutase (MnSOD) and p62. Patient-derived IDH1-mutant tumors showed no significant differences in apoptosis or autophagy, but showed p62 accumulation and actually trended toward reduced MnSOD expression. These data indicate that mutant IDH1 and 2-HG can induce oxidative stress, autophagy, and apoptosis, but these effects vary greatly according to cell type.

Resveratrol protects against polychlorinated biphenyl-mediated impairment of glucose homeostasis in adipocytes.

Resveratrol (RSV) is a plant polyphenol that exhibits several favorable effects on glucose homeostasis in adipocytes. Recent studies from our laboratory demonstrated that coplanar polychlorinated biphenyls (PCBs) that are ligands of the aryl hydrocarbon receptor impair glucose homeostasis in mice. PCB-induced impairment of glucose homeostasis was associated with augmented expression of inflammatory cytokines in adipose tissue, a site for accumulation of lipophilic PCBs. This study determined if RSV protects against PCB-77 induced impairment of glucose disposal in vitro and in vivo and if these beneficial effects are associated with enhanced nuclear factor erythoid 2-related factor 2 (Nrf2) signaling in adipose tissue. PCB-77 increased oxidative stress and abolished insulin stimulated 2-deoxy-d-glucose uptake in 3 T3-L1 adipocytes. These effects were restored by RSV, which resulted in a concentration-dependent increase in NAD(P)H:quinone oxidoreductase 1 (NQO1), the downstream target of Nrf2 signaling. We quantified glucose and insulin tolerance and components of Nrf2 and insulin signaling cascades in adipose tissue of male C57BL/6 mice administered vehicle or PCB-77 (50 mg/kg) and fed a diet with or without resVida (0.1%, or 160 mg/kg per day). PCB-77 impaired glucose and insulin tolerance, and these effects were reversed by RSV. PCB-77 induced reductions in insulin signaling in adipose tissue were also abolished by RSV, which increased NQO1 expression. These results demonstrate that coplanar PCB-induced impairment of glucose homeostasis in mice can be prevented by RSV, potentially through stimulation of Nrf2 signaling and enhanced insulin stimulated glucose disposal in adipose tissue.