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Aims We developed a novel mouse model with increased skeletal muscle estrogen content via inducible, skeletal-muscle–specific aromatase overexpression (SkM-Arom↑). We proposed to examine the effect that increased skeletal muscle estrogen both in gonadally intact and ovariectomized (OVX) female mice has on preventing or rescuing high-fat diet (HFD)-induced obesity. Methods In the prevention experiment, gonadally intact and OVX SkM-Arom↑ mice and littermate controls were fed a low-fat diet (LFD) or HFD for 13 weeks. SkM-Arom↑ was induced at the initiation of dietary treatment. In the intervention experiment, gonadally intact and OVX SkM-Arom↑ mice and littermate controls were fed an HFD for 14 weeks before induction of SkM-Arom↑ for 6 weeks. Glucose tolerance, insulin action, adipose tissue inflammation, and body composition were assessed. Liquid chromatography–mass spectrometry was used to determine circulating and skeletal muscle steroid content. Results SkM-Arom↑ significantly increased skeletal muscle 17β-estradiol (E2) and estrone (E1) in both experiments. Interestingly, this resulted in leakage of estrogens into circulation, producing a physiologically relevant E2 concentration. Consequently, bone mineral density (BMD) was enhanced and adipose tissue inflammation was reduced in the prevention experiment only. However, no benefits were seen with respect to changes in adiposity or metabolic outcomes. Conclusion We show that increasing skeletal muscle estrogen content does not provide a metabolic benefit in gonadally intact and OVX female mice in the setting of obesity. However, a chronic physiological concentration of circulating E2 can improve BMD and reduce adipose tissue inflammation independently of a metabolic benefit or changes in adiposity.

The sphingomyelin pathway is important in cell regulation and determining cellular fate. Inhibition of sphingosine kinase isoform 1 (SK1) within this pathway, leads to a buildup of sphingosine and ceramide, two molecules directly linked to cell apoptosis, while decreasing the intracellular concentration of sphingosine-1-phosphate (S1P), a molecule linked to cellular proliferation. Recently, an inhibitor capable of inhibiting SK1 in vitro was identified, but also shown to be ineffective in vivo. A set of compounds designed to assess the impact of synthetic modifications to the hydroxynaphthalene ring region of the template inhibitor with SK1 to obtain a compound with increased efficacy in vivo. Of these fifteen compounds, 4A was shown to have an IC50 = 6.55 μM with improved solubility and in vivo potential.

Mauritius Island possesses unique plant biodiversity with a potential reservoir of biologically active compounds of pharmacological interest. In the current study, we investigated Mauritius endemic plant families Asteraceae, Ebenaceae, Sapotaceae, and Erythroxylaceae, for anti-cancer properties on T cell lymphoma and B16F10 Melanoma cells and immunomodulatory properties on primary T and B cells. The cytotoxicity of methanolic plant extracts at 1, 10, 25 µg/ml was determined. The most active plant species were evaluated for their apoptosis-inducing effects. The immunomodulatory properties of the plants were also studied, and preliminary phytochemical screening of selected plants was done by LC–MS analysis. Psiadia lithospermifolia (Lam.) Cordem (Asteraceae) at 25 µg/ml was the most cytotoxic on both EL4 and B16 cells and triggered apoptosis by the death receptor pathway, and at least in part, by the mitochondrial pathway. Most plant species from Asteraceae, Ebenaceae, Erythroxylaceae, and Sapotaceae inhibited the proliferation of activated T and B cells, although some promoted T cell proliferation. LC–MS profile of Asteraceae plants showed the presence of terpenes, terpenoids, fatty acids, and phenolic. Flavonoids and phenolic acid were also detected from Ebenaceae and Sapotaceae plants. Together, our study demonstrated that Mauritius endemic flora exhibit potential anti-cancer and anti-inflammatory properties worthy of further in-depth studies.

Background Emodin, a natural anthraquinone, has shown potential as an effective therapeutic agent in the treatment of many diseases including cancer. However, its clinical development is hindered by uncertainties surrounding its potential toxicity. The primary purpose of this study was to uncover any potential toxic properties of emodin in mice at doses that have been shown to have efficacy in our cancer studies. In addition, we sought to assess the time course of emodin clearance when administered both intraperitoneally (I.P.) and orally (P.O.) in order to begin to establish effective dosing intervals. Methods We performed a subchronic (12 week) toxicity study using 3 different doses of emodin (~ 20 mg/kg, 40 mg/kg, and 80 mg/kg) infused into the AIN-76A diet of male and female C57BL/6 mice (n = 5/group/sex). Body weight and composition were assessed following the 12-week feeding regime. Tissues were harvested and assessed for gross pathological changes and blood was collected for a complete blood count and evaluation of alanine transaminase (ALT), aspartate transaminase (AST) and creatinine. For the pharmacokinetic study, emodin was delivered intraperitoneally I.P. or P.O. at 20 mg/kg or 40 mg/kg doses to male and female mice (n = 4/group/sex/time-point) and circulating levels of emodin were determined at 1, 4 and 12 h following administration via liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. Results We found that 12 weeks of low (20 mg/kg), medium (40 mg/kg), or high (80 mg/kg) emodin feeding did not cause pathophysiological perturbations in major organs. We also found that glucuronidated emodin peaks at 1 h for both I.P. and P.O. administered emodin and is eliminated by 12 h. Interestingly, female mice appear to metabolize emodin at a faster rate than male mice as evidenced by greater levels of glucuronidated emodin at the 1 h time-point (40 mg/kg for both I.P. and P.O. and 20 mg/kg I.P.) and the 4-h time-point (20 mg/kg I.P.). Conclusions In summary, our studies establish that 1) emodin is safe for use in both male and female mice when given at 20, 40, and 80 mg/kg doses for 12 weeks and 2) sex differences should be considered when establishing dosing intervals for emodin treatment.

SummaryThe NDUFS4 knockout (KO) mouse phenotype resembles the human Complex I deficiency Leigh Syndrome. The irreversible succination of protein thiols by fumarate is increased in select regions of the NDUFS4 KO brain affected by neurodegeneration, suggesting a mechanistic role in neurodegenerative decline. We report that dihydrolipoyllysine-residue succinyltransferase (DLST), a component of the α-ketoglutarate dehydrogenase complex (KGDHC) of the tricarboxylic acid (TCA) cycle, is succinated in the NDUFS4 KO brain. Succination of DLST reduced KGDHC activity in the brainstem (BS) and olfactory bulb (OB) of KO mice. The defective production of KGDHC derived succinyl-CoA resulted in decreased mitochondrial substrate level phosphorylation, further aggravating the OXPHOS ATP deficit. Protein succinylation, an acylation modification that requires succinyl-CoA, was reduced in the KO mice. Our data demonstrate that the biochemical deficit extends beyond impaired Complex I assembly and OXPHOS deficiency, functionally impairing select components of the TCA cycle to drive metabolic perturbations in affected neurons.HighlightsCysteine succination by fumarate is increased in model of Complex I deficiencySuccination of DLST impairs KGDHC activity, limits SLP, and reduces lysine succinylationIrreversible succination drives neuropathology independent of the bioenergetic defect

Glycogen synthase kinase 3 (GSK-3) has been linked to regulation of kinesin-dependent axonal transport in squid and flies, and to indirect regulation of cytoplasmic dynein. We have now found evidence for direct regulation of dynein by mammalian GSK-3β in both neurons and non-neuronal cells. GSK-3β coprecipitates with and phosphorylates mammalian dynein. Phosphorylation of dynein intermediate chain (IC) reduces its interaction with Ndel1, a protein that contributes to dynein force generation. Two conserved residues, S87/T88 in IC-1B and S88/T89 in IC-2C, have been identified as GSK-3 targets by both mass spectrometry and site-directed mutagenesis. These sites are within an Ndel1-binding domain, and mutation of both sites alters the interaction of IC's with Ndel1. Dynein motility is stimulated by (i) pharmacological and genetic inhibition of GSK-3β, (ii) an insulin-sensitizing agent (rosiglitazone) and (iii) manipulating an insulin response pathway that leads to GSK-3β inactivation. Thus, our study connects a well-characterized insulin-signaling pathway directly to dynein stimulation via GSK-3 inhibition.

Analysis of the broad range of trace chemical modifications of proteins in biological samples is a significant challenge for modern mass spectrometry. Modification at lysine and arginine residues, in particular, causes resistance to digestion by trypsin, producing large tryptic peptides that are not readily sequenced by mass spectrometry. In this work, we describe the analysis of ribonuclease (RNase) modified by methylglyoxal (MGO) under physiological conditions. For detection of modifications, we use comparative analysis of the single combined spectra extracted from the full-scan MS data of the tryptic digests from native and modified proteins. This approach revealed 11 ions unique to MGO-modified RNase, including a 32-amino acid peptide containing a modified Arg-85 residue. Sequential digestion of MGO-modified RNase by endoproteinase Glu-C and trypsin was required to obtain peptides that were amenable to sequencing analysis. Arg-39 was identified as the main site of modification (35% modification) on MGO-modified Rnase, and the dihydroxyimidazolidine and hydroimidazolone derivatives were the main adducts formed, with minor amounts of the tetrahydropyrimidine and argpyrimidine derivatives. For identification of these products, we used variations in source voltage and collision energy to obtain the dehydration and decarboxylation products of the tetrahydropyrimidine-containing peptides and dehydration of the dihydroxyimidazoline-containing peptides. The resultant spectra were dependent on the cone voltage and collision energy, and analysis of spectra at various settings permitted structural assignments. These studies illustrate the usefulness of single combined mass spectra extracted from full-scan data and variations in source and collision cell voltages for detection and structural characterization of chemical adducts on proteins.

Accumulation of advanced glycation end-products (AGEs) on proteins is associated with the development of diabetic complications. Although the overall extent of modification of protein by AGEs is limited, localization of these modifications at a few critical sites might have a significant effect on protein structure and function. In the present study, we describe the sites of modification of RNase by glyoxal under physiological conditions. Arg39 and Arg85, which are closest to the active site of the enzyme, were identified as the primary sites of formation of the glyoxal-derived dihydroxyimidazolidine and hydroimidazolone adducts. Lower amounts of modification were detected at Arg10, while Arg33 appeared to be unmodified. We conclude that dihydroxyimidazolidine adducts are the primary products of modification of protein by glyoxal, that Arg39 and Arg85 are the primary sites of modification of RNase by glyoxal, and that modification of arginine residues during Maillard reactions of proteins is a highly selective process.

Proteomic analysis using electrospray liquid chromatography-mass spectrometry (ESI-LC-MS) has been used to compare the sites of glycation (Amadori adduct formation) and carboxymethylation of RNase and to assess the role of the Amadori adduct in the formation of the advanced glycation end-product (AGE), N(epsilon)-(carboxymethyl)lysine (CML). RNase (13.7 mg/mL, 1 mM) was incubated with glucose (0.4 M) at 37 degrees C for 14 days in phosphate buffer (0.2 M, pH 7.4) under air. On the basis of ESI-LC-MS of tryptic peptides, the major sites of glycation of RNase were, in order, K41, K7, K1, and K37. Three of these, in order, K41, K7, and K37 were also the major sites of CML formation. In other experiments, RNase was incubated under anaerobic conditions (1 mM DTPA, N2 purged) to form Amadori-modified protein, which was then incubated under aerobic conditions to allow AGE formation. Again, the major sites of glycation were, in order, K41, K7, K1, and K37 and the major sites of carboxymethylation were K41, K7, and K37. RNase was also incubated with 1-5 mM glyoxal, substantially more than is formed by autoxidation of glucose under experimental conditions, but there was only trace modification of lysine residues, primarily at K41. We conclude the following: (1) that the primary route to formation of CML is by autoxidation of Amadori adducts on protein, rather than by glyoxal generated on autoxidation of glucose; and (2) that carboxymethylation, like glycation, is a site-specific modification of protein affected by neighboring amino acids and bound ligands, such as phosphate or phosphorylated compounds. Even when the overall extent of protein modification is low, localization of a high proportion of the modifications at a few reactive sites might have important implications for understanding losses in protein functionality in aging and diabetes and also for the design of AGE inhibitors.

Our investigations on the discovery of novel natural metabolites using type II polyketide synthase gene probes (actI/III) yielded an unusual angucyclinone, oviedomycin (2), when applied to the oleandomycin (1) producer Streptomyces antibioticus ATCC11891. The novel natural product was produced using S. albus R(-)M(-) as a host strain, into which a cosmid containing the oviedomycin gene cluster was transformed. Its structure was elucidated by NMR spectroscopy and mass spectrometry.

Single nucleotide polymorphisms (SNPs) and mutations were genotyped for both homozygous and heterozygous PCR products of p53, a tumor suppressor gene, and K-ras, an oncogene, using electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) and ESI-quadrupole MS analysis. Mass accuracy was adequate for both instruments to detect genetic changes in homozygous PCR products, including the most difficult to distinguish (adenine [A] --> thymine [T] transversion). However, for the detection of A --> T shifts (9.0 Da difference) in heterozygous PCR products, the increased resolution of ESI-Q-TOFMS proved essential. Although, greater mass differences in heterozygotes (e.g. cytosine [C] T or guanine [G] A) can be discriminated using ESI-quadrupole MS analysis.

Protein succination is a stable post-translational modification that occurs when fumarate reacts with cysteine residues to generate 2SC [S-(2-succino)cysteine]. We demonstrate that both α- and β-tubulin are increasingly modified by succination in 3T3-L1 adipocytes and in the adipose tissue of db / db mice. Incubation of purified tubulin from porcine brain with fumarate (50 mM) or the pharmacological compound DMF (dimethylfumarate, 500 μM) inhibited polymerization up to 35% and 59% respectively. Using MS we identified Cys347α, Cys376α, Cys12β and Cys303β as sites of succination in porcine brain tubulin and the relative abundance of succination at these cysteine residues increased in association with fumarate concentration. The increase in succination after incubation with fumarate altered tubulin recognition by an anti-α-tubulin antibody. Succinated tubulin in adipocytes cultured in high glucose compared with normal glucose also had reduced reactivity with the anti-α-tubulin antibody; suggesting that succination may interfere with tubulin–protein interactions. DMF reacted rapidly with 11 of the 20 cysteine residues in the αβ-tubulin dimer, decreased the number of free thiols and inhibited the proliferation of 3T3-L1 fibroblasts. Our data suggest that inhibition of tubulin polymerization is an important undocumented mechanism of action of DMF. Taken together, our results demonstrate that succination is a novel post-translational modification of tubulin and suggest that extensive modification by fumarate, either physiologically or pharmacologically, may alter microtubule dynamics. Abbreviations: C2SC, cysteine succination by fumarate; CID, collision-induced dissociation; CTT, C-terminal tail of tubulin; DMF, dimethylfumarate; DMEM, Dulbecco’s modified Eagle’s medium; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); DTPA, diethylenetriaminepenta-acetic acid; GI, gastrointestinal; 4-HNE, 4-hydroxynonenal; MAP, microtubule-associated protein; NEM, N-ethylmaleimide; PE, pyridylethylation; PTM, post-translational modification; RIPA, radioimmunoprecipitation assay; 2SC, S-2-(succino)cysteine; TFA, trifluoroacetic acid; TOF, time-of-flight

Organochlorine pesticides and polychlorinated biphenyls were determined in high volume air samples collected in Green Bay from the University of Wisconsin (1989), and from shipboard over the four lower Great Lakes (1990). Average concentrations of Σchlordane, DDT, DDE, toxaphene and ΣPCBs in Green Bay were 35, 8.7, 15, 59 and 330 pg m−3, respectively. Air concentrations of the pesticides were positively correlated to each other (R2=0.896–0.997), and analysis of back trajectory data from Green Bay showed that higher atmospheric concentrations of organochlorine insecticides were associated with air masses originating in southern USA. Average concentrations from the Great Lakes were: Σchlordane, 187 pg m−3; DDT, 38 pg m−3; pg m−3; DDE, 59 pg m−3; toxaphene, 33 pg m−3; and ΣPCBs, 385 pg m−3. Higher air concentrations of PCBs were found in areas of industrial and urban development. There was no strong positive correlation of concentration values between any pesticides, with the exception of the chlordane isomers, or between any pesticide and ΣPCB from the Great Lakes cruise. Results from 5-day air mass back trajectory data suggest that, at the time of the cruise, atmospheric sources of organochlorines were not long-range transport from southern USA, but local or regional volatilization.

A rapid extraction method has been developed for the quantitation of (Z,E)-propanethial S-oxide, the lachrymatory factor (LF), and other flavor chemicals in onion (Allium cepa) using gas chromatogr...

Air samples were collected at an urban (Chicago, IL) and a rural (University of Wisconsin at Green Bay, UWGB) site during February 1988 to determine concentrations and particle/gas distributions of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). Geometric mean ΣPAHs of molecular weights ≥166 were 195 ng/m 3 in Chicago and 14 ng/m 3 at UWGB. Ratios among PAHs suggested that some labile compounds were depleted during atmospheric transport. Geometric mean ΣPCBs (45 congeners) were 1.3 ng/m 3 in Chicago and 0.13 ng/m 3 at UWGB. PCB profiles of most samples in Chicago and all at UWGB were skewed toward the lower molecular weight congeners, as in mixtures of Aroclors 1242 and 1254. Two Chicago samples showed a heavy PCB profile similar to Aroclor 1260. Apparent particle/gas partition coefficients (K p ) were well correlated with liquid-phase vapor pressures (po L ) within a class of compounds (PAHs or PCBs). PAHs were sorbed to a greater degree than PCBs for compounds of the same volatility. Slopes of log K p vs log p o L plots deviated from the expected value of -1 in Chicago, suggesting nonequilibrium effects. Aerosols at UWGB appeared to be closer to equilibrium with the gas phase, as indicated by slopes close to -1. Measured particulate percentages in the two locations were within a factor of 2 of those predicted by the Junge-Pankow adsorption model.

Two experimental techniques were used to test the hypothesis that mono- and non-ortho-substituted polychlorinated biphenyls (PCBs) are sorbed to urban aerosols to a greater extent than multi-ortho congeners of the same homolog. Particulate and gaseous PCBs in Chicago air were operationally defined by sampling with a glass fiber filter followed by a polyurethane foam trap. After separation from organochlorine pesticides, PCBs were fractionated into non-ortho, mono-ortho, and multi-ortho groups on an activated carbon-silicic acid column and determined by capillary gas chromatography (GC) with electron capture detection or GC/mass spectrometry. Within a homolog group (e.g., pentachlorobiphenyls), the percentage of particulate PCBs increased in the order : multi-ortho < mono-ortho < non-ortho. This order was explained for the most part by the lower liquid-phase vapor pressures (p° L ) of mono- and non-ortho-PCBs, although the non-ortho congeners 77 and 126 showed slightly enhanced sorption relative to expectations based on vapor pressure. To gauge the relative extent of sorption for PCBs having different numbers of ortho-chlorines, filters loaded with Chicago aerosols were exposed to gaseous PCBs at a constant temperature. Particle/gas partition coefficients (K p ) were inversely related to p° L , but distinct differences were seen among the ortho-chlorine classes. For a given value of log p° L , values of log K p increased in the order : multi-ortho < mono-ortho < non-ortho. A multiple linear regression model using log p° L and the dihedral angle between the biphenyl rings explained 98% of the variance in sorption.

Electrospray-ionization mass spectrometry is used for the effective analysis of post-synthetically modified oligodeoxynucleotides, wherein thiol groups present on 4-thio-2′-deoxyuridine residues within the oligomers are used as site-specific points of attachment for functionalized tethers.

Concentrations of hexachlorocyclohexanes (HCHs) in air and water were measured in order to estimate the direction and magnitude of gas flux across the air-water interface in the four lower Great Lakes. Samples were collected in Green Bay, Lake Michigan during June, 1989, and in lakes Michigan, Huron, Erie, and Ontario during Aug 1990. Green Bay water was undersaturated with respect to the air due to low surface water temperatures (6-11 o C) and a well-mixed water column, and the flux direction was from air to water for both α- and γ-HCH

Air samples were collected at Sable Island in January 1988 and 1989 for organochlorine (OC) compounds, and July-August 1989 for OCs and polycyclic aromatic hydrocarbons (PAHs). Mean concentrations of OCs (pg/m3) were: hexachlorocyclohexanes (α-HCH + γ-HCH) = 250, polychlorinated biphenyls (PCBs, sum of 38 congeners) = 52, polychlorocamphenes (PCCs) = 35, chlordanes + nonachlors = 15, heptachlor epoxide = 5.2, p,p′-DDT + p,p′-DDE = 4.5. Endosulfan (sum of two isomers) and dieldrin concentrations were 24–159 and 9–12 pg/m3 in summer samples. The sum of 3–5 ring PAHs ranged from 553 – 842 pg/m3, and was dominated by 3-ring (92%) followed by 4-ring (6%) and 5-ring (2%) compounds. HCHs in rain (3.6–7.3 ng/L) and seawater (1.2-2.1 ng/L) were similar to other values reported for the North Atlantic and eastern Canada. Results show the importance of atmospheric transport in delivering contaminants to the Scotian Shelf and the advantages of Sable Island as a monitoring site.

An experimental system was designed to equilibrate urban air particulate matter on a glass fiber filter (GFF) with gas-phase organochlorine (OC) pesticides and polychlorinated biphenyls (PCBs) at controlled temperature, humidity, and vapor concentrations. Particle-loaded filters (PLFs) were backed up by clean filters (CFs) to correct for gaseous semivolatile organic compound (SOC) adsorption to the filter matrix. Analysis of the CF indicated the presence of a complex mixture of alkanes and other organic compounds in addition to the OC pesticides introduced in the experiment

This work predicts the flux of several OC compounds: α- and γ-HCH, hexachlorobenzene (HCB), chlordane (cis- + trans- chlordane), diedlrin, p,p′-DDT and toxaphene, to the Arctic in the summer and winter. These estimates are based on known deposition mechanisms using atmospheric OC concentrations, gas-aerosol partition data, and OC physical properties typical of the polar environment.

We recently detected the formation of estradiol-17β (estradiol) dimers, linked together through a diaryl ether bond between the C-3 phenolic oxygen of one estradiol molecule and the 2- or 4-position aromatic carbon of another estradiol, following incubations of [ 3 H ]estradiol with human liver microsomes or cytochrome P450 enzymes in the presence of NADPH. Using estradiol as the starting material, we designed a four-step method for the chemical synthesis of these two estrogen dimers with the Ullmann condensation reaction as a key step. Step 1: Synthesis of 2- or 4-bromoestradiol from estradiol. Step 2 : Protection of the C-3 phenolic hydroxyl group of the 2- or 4-bromoestradiol. Step 3 : The Ullmann condensation reaction between the phenol-protected bromoestradiol and the estradiol potassium salt under our modified reaction conditions (with a 41% product yield). Step 4 : Removal of the C-3 benzyl group by catalytic hydrogenation. The chromatographic and various spectrometric properties of the two synthesized compounds were identical to those metabolically formed by human cytochrome P450 3A4.

Cu(I) catalyzed alkyne-azide cycloaddition reaction was employed to synthesize a series of anthracene-based human thymidylate synthase (hTS) inhibitor analogues. The triazolo-anthracene derivatives were characterized by ESI-MS/MS and a novel rearrangement reaction in ESI-MS/MS was observed. The mechanism is proposed whereby the protonated triazolo-anthracene derivative forms a carbocation, and then the carbocation electrophilically attacks an anthracene moiety resulting in formation of a rearrangement ion. Moreover, the carbocation prefers to attack the gamma position rather than the alpha or beta position of the anthracene moiety by an electrophilic substitution mechanism.