Fumigaclavine C ameliorates liver steatosis by attenuating hepatic de novo lipogenesis via modulation of the RhoA/ROCK signaling pathway.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) has been well defined as a common chronic liver metabolism disorder. Statins as a first-line therapeutic treatment had some side effects. Here, we found that Fumigaclavine C (FC) was collected from endophytic Aspergillus terreus via the root of Rhizophora stylosa (Rhizophoraceae), had potential anti-adipogenic and hepatoprotective effects both in vitro and in vivo without obvious adverse side effects. However, the mechanisms of the prevention and management of FC for hepatic steatosis are incompletely delineated. METHODS: The pharmacodynamic effects of FC were measured in high-fat diet (HFD)-induced obese mice. Liver index and blood biochemical were examined. Histopathological examination in the liver was performed by hematoxylin & eosin or oil red O. The levels of serum TG, TC, LDL-c, HDL-c, FFA, T-bili, ALT, AST, creatinine, and creatine kinase were estimated via diagnostic assay kits. The levels of hepatic lipid metabolism-related genes were detected via qRT-PCR. The expression levels of hepatic de novo lipogenesis were quantitated with Western blot analysis.  RESULTS: FC-treatment markedly reduced hepatic lipid accumulation in HFD-induced obese mice. FC significantly attenuated the hepatic lipid metabolism and ameliorated liver injury without obvious adverse side effects. Moreover, FC also could dose-dependently modulate the expressions of lipid metabolism-related transcription genes. Mechanically, FC notably suppressed sterol response element binding protein-1c mediated de novo lipogenesis via interfering with the RhoA/ROCK signaling pathway by decreasing the levels of geranylgeranyl diphosphate and farnesyl diphosphate. CONCLUSIONS: These findings suggested that FC could improve hepatic steatosis through inhibiting de novo lipogenesis via modulating the RhoA/ROCK signaling pathway.

Effect of Field-applied Fungicides on Claviceps purpurea Sclerotia and Associated Toxins in Wheat.

Claviceps purpurea (Fr.) Tul is the causal organism for ergot impacting grass hosts, including wheat. The pathogen produces ergot alkaloids (EAs) during the development of mature sclerotia leading to potential wheat quality discounts or rejection at the point of sale. Cultural practices are recommended for the management of ergot in wheat, but there is limited information pertaining to the use of in-season fungicides to help reduce ergot. The objective of this research was to evaluate the efficacy of four fungicides (prothioconazole + metconazole, pydiflumetofen + propiconazole, azoxystrobin + propiconazole, and fluxapyroxad + pyraclostrobin) on sclerotia characteristics, and EAs associated with C. purpurea. A field experiment was established using a male-sterile hard red spring line with fungicide applications occurring at complete full head emergence (Feekes Growth Stage 10.5). Individual plots were harvested and cleaned, and ergot sclerotia were collected. Physical characteristics and toxin production were examined. Fungicides had a significant (p < .05) impact on total ergot body weight (EBW), with all fungicides having lower EBW than the nontreated control. The fungicide premixture of pydiflumetofen + propiconazole had the lowest EBW among all treatments. Fluxapyroxad + pyraclostrobin had the lowest levels of EAs among fungicides. Results suggest that fungicide premixtures can potentially reduce EBW and influence EA production in wheat.

Chronic ergot exposure in adult bulls suppresses prolactin but minimally impacts results of typical breeding soundness exams.

Canadian standards allow ≤3000 µg ergot alkaloids/kg cattle feed. A concentration-response relationship was hypothesized between ergot in feed and reductions in plasma prolactin, sperm motility, sperm function, and increase in sperm abnormalities. The study consisted of pre-treatment (12 weeks), treatment (9 weeks), and post-treatment periods (10 weeks). Adult bulls were fed 1113 (n = 8; low ergot group) or 2227 (n = 6; high) µg/kg of dry matter intake. Endpoints were measured every two weeks. Ejaculates were analyzed for sperm concentration, total and progressive motility, plasma membrane and acrosome integrity, mitochondrial membrane potential and sperm abnormalities. Data were analyzed by repeated measures MIXED PROC in SAS. Average outside ambient temperature during the pre-treatment, treatment, and post-treatment periods was -13 (-31 to 1), 0.5 (-18 to 19), and 21 (13-28) °C. Plasma prolactin decreased markedly during treatment (-52.4%; Experimental period p < 0.01). Rectal temperature increased during the treatment and post-treatment periods (EP p < 0.01) but was within the normal physiological range. Bull weight increased during the study (EP p < 0.01). Scrotal circumference in low ergot group increased during treatment (+0.8 cm; Tx∗EP p = 0.05). Progressive motility in high ergot group decreased during treatment (-7%; Tx∗EP p = 0.05), however, semen volume and sperm concentrations were unaffected (p ≥ 0.11). Live sperm with high and medium MMP decreased during treatment (-1.4 and -3.7%; EP p < 0.01). Results suggest that feeding ≤2227 µg ergot alkaloids/kg has only minor effects on adult bull semen quality.

Effect of ergot alkaloids and a mycotoxin deactivating product on in vitro ruminal fermentation using the Rumen simulation technique (RUSITEC).

The rumen simulation technique (RUSITEC) was used to investigate the effect of ergot alkaloids (EA) and a mycotoxin deactivating product (Biomin AA; MDP) on nutrient digestion, ruminal fermentation parameters, total gas, methane, and microbial nitrogen production. Ruminal fermentation vessels received a feedlot finishing diet of 90:10 concentrate:barley silage (DM basis). Using a randomized complete block design, treatments were assigned (n = 4 vessels/treatment) within two RUSITEC apparatuses in a 2 × 2 factorial arrangement. Treatments included: (1) control (CON) diet (no EA and no MDP); (2) CON diet + 1 g/d MDP; (3) CON diet + 20 mg/kg EA; and (4) CON diet + 20 mg/kg EA + 1 g/d MDP. The study was conducted over 14 d with 7 d of adaptation and 7 d of sample collection. Data were analyzed in SAS using PROC MIXED including fixed effects of EA, MDP, and the EA×MDP interaction. Random effects included RUSITEC apparatus and cow rumen inoculum (n = 4). Ergot alkaloids decreased dry matter (DMD) (P = 0.01; 87.9 vs. 87.2%) and organic matter disappearance (OMD) (P = 0.02; 88.8 vs. 88.4%). Inclusion of MDP increased OMD (P = 0.01; 88.3 vs. 88.9%). Neutral detergent fiber disappearance (NDFD) was improved with MDP; however, an EA×MDP interaction was observed with MDP increasing (P < 0.001) NDFD more with EA diet compared to CON. Acetate proportion decreased (P = 0.01) and isovalerate increased (P = 0.03) with EA. Consequently, acetate:propionate was reduced (P = 0.03) with EA. Inclusion of MDP increased total volatile fatty acid (VFA) production (P < 0.001), and proportions of acetate (P = 0.03) and propionate (P = 0.03), and decreased valerate (P < 0.001), isovalerate (P = 0.04), and caproate (P = 0.002). Treatments did not affect (P ≥ 0.17) ammonia, total gas, or methane production (mg/d or mg/g of organic matter fermented). The inclusion of MDP reduced (P < 0.001) microbial nitrogen (MN) production in the effluent and increased (P = 0.01) feed particle-bound MN. Consequently, total MN decreased (P = 0.001) with MDP. In all treatments, the dominant microbial phyla were Firmicutes, Bacteroidota, and Proteobacteria, and the major microbial genus was Prevotella. Inclusion of MDP further increased the abundance of Bacteroidota (P = 0.04) as it increased both Prevotella (P = 0.04) and Prevotellaceae_UCG-003 (P = 0.001). In conclusion, EA reduced OMD and acetate production due to impaired rumen function, these responses were successfully reversed by the addition of MDP.

Ergot formed from a parasitic fungus (Claviceps purpurea) affects various types of grains (rye, wheat, or oats) and may contain several toxic ergot alkaloids (EA). Individual EA may impact the rumen microorganisms, and cattle feed intake, digestibility, health, and overall performance. A common method to alleviate toxicity in mycotoxin-contaminated feed is through the addition of mycotoxin binders (MDP); however, their efficacy against EA is unknown. To better understand the effect of EA in cattle, we performed an in vitro experiment to examine the impact of EA on the ruminal microbial populations and fermentation of a finishing feedlot diet using an artificial rumen (RUSITEC). Additionally, an MDP was added to test if it could reduce the detrimental effects of EA on rumen fermentation. MDP increased total volatile fatty acids (VFA) and reduced total microbial protein synthesis. Furthermore, EA reduced microbial diversity and the acetate:propionate ratio. Although EA reduced organic matter digestibility and acetate production, these negative effects were reversed by the addition of the MDP.

Identification, semisynthesis, and anti-inflammatory evaluation of 2,3-seco-clavine-type ergot alkaloids from human intestinal fungus Aspergillus fumigatus CY018.

Intestinal commensal fungi are vital to human health, and their secondary metabolites play a key role in the reciprocal relationship. In the present study, the first example of 2,3-seco ergot alkaloids belonging to clavine-type were isolated from the fermentation of human intestinal fungus Aspergillus fumigatus CY018, including two pairs of diastereoisomers, secofumigaclavines A (3) and B (4) and secofumigaclavines C (5) and D (6), one analogue features a highly unsaturated skeleton, secofumigaclavine E (7), along with two known ones, fumigaclavines C (1) and D (2). Their structures were identified based on extensive spectroscopic data in a combination of quantum chemical calculations. Moreover, a single-step operation of semi-synthetic reaction based on riboflavin (RF)-dependent photocatalysis was performed to obtain the novel 2,3-seco ergot alkaloids 3 and 5 from their biosynthetic precursors 1 and 2. All the isolated compounds were evaluated for their anti-inflammatory activity. Among them, secofumigaclavine B (4) could bind to MD2 with a low micromole level of the equilibrium dissociation constant measured by surface plasmon resonance (SPR), and suppress TLR4-mediated NF-κB signaling pathway in RAW264.7 cells, resulting in its anti-inflammatory effect. Molecular dynamics revealed that amino acid residue Tyr131 played a key role in the interaction of secofumigaclavine B (4) with MD2. These findings suggested that secofumigaclavine B (4) could be considered as a potential candidate for the development of MD2 inhibitors.

The effect of ergot alkaloid exposure during gestation on the microscopic morphology and vasculature of the ovine placenta.

Ergot alkaloids, a class of mycotoxins associated with ergotism, act as agonists on serotonin (5HT) receptors, specifically 5HT2a, which mediate smooth muscle contraction and vasoconstriction. The objective of this study was to examine the impact of ergot alkaloid exposure during mid and late gestation on microscopic placental structure and vascular development. Ewes were fed endophyte-infected tall fescue seed containing ergot alkaloids (E+/E+, 1.77 mg ewe-1 d-1) or endophyte-free tall fescue seed (E-/E-, 0 mg ergot alkaloids) during both mid (d 35 to d 85) and late gestation (d 86 to d 133). On d 133 of gestation, a terminal surgery was performed and two placentomes of the type B morphology were collected for microscopic analyses. Amorphous connective tissue regions were larger (p < 0.0001) and more numerous (p = 0.025) in the placentome of ergot alkaloid exposed ewes. Staining showed no difference (p = 0.83) in the number of vessels present, but luminal area of maternal vasculature was 117% greater (p < 0.0001) in ergot alkaloid exposed ewes. Results showed that exposure to ergot alkaloids during gestation slowed maturation of the fetal villi as indicated by greater amorphous connective tissue regions, and altered size and shape of blood vessels to counteract reductions in blood flow caused by vasoconstriction.

Assessment of the vasoactive effects of the (S)-epimers of ergot alkaloids in vitro.

Ergot alkaloids are produced by the fungus Claviceps purpurea and their levels are carefully monitored in animal and human diets due to their harmful effects and widespread contamination of cereal crops. Ergot alkaloids exist in two forms known as the (R)- and (S)-epimers with only the former being monitored in diets in North America. The (S)-epimers of ergot alkaloids are thought to be biologically inactive and, therefore, harmless. A major mechanism by which the (R)-epimers of ergot alkaloids produce their toxic effect is through vasoconstriction. Therefore, the objective of this study was to examine the vasoactivity potential (contractile response) of four (S)-epimers, namely ergocryptinine, ergocristinine, ergocorninine, and ergotaminine utilizing an in vitro arterial tissue bath system. Bovine metatarsal arteries (n = 6, ergocryptinine and ergocorninine; n = 6, ergocristinine and ergotaminine; n = 6 arteries/(S)-epimer, total n = 12) were collected from healthy mixed-breed beef steers immediately after slaughter, cut into 3-mm arterial cross sections, and suspended in a tissue bath with continuously oxygenated Krebs-Henseleit buffer. To assess the contractile response of each (S)-epimer, a cumulative contractile dose-response curve was constructed by incubating arteries with increasing concentrations (1 × 10-11 to 1 × 10-6 M) of that (S)-epimer. Contractile responses were recorded as grams of tension and were normalized to an initial contraction of phenylephrine. Contrary to the widespread belief, all tested (S)-epimers were found vasoactive and produced a concentration-dependent arterial contractile response similar to what has been reported for the (R)-epimers. The arterial contractile response to ergotaminine was strongest and was significantly greater than that of ergocryptinine and ergocristinine at the highest concentration used (P ≤ 0.01). Our results indicate that the (S)-epimers are biologically active and are likely harmful similar to the (R)-epimers. The levels of (S)-epimers should be carefully monitored in human and animal diets worldwide.

Ergot alkaloids induce vasoconstriction of bovine uterine and ovarian blood vessels.

Fescue toxicosis is a syndrome that impairs growth and reproduction in cattle grazing endophyte-infected tall fescue [Lolium arundinaceum [(Schreb.].) Darbysh)] in the United States, resulting in approximately $1 billion in annual economic loss in species that utilize this forage resource. Approximately 90% of tall fescue contains an endophytic fungus (Epichloë coenophiala) that produces ergot alkaloids. Ergot alkaloids cause vasoconstriction and reduced blood flow to the extremities; however, it remains unknown how blood flow to the reproductive organs is affected in cattle. Therefore, the objective of this study was to determine if ergot alkaloids from endophyte-infected tall fescue reduce blood flow to the reproductive organs, thus hindering reproductive function. Angus heifers (n = 36) naïve to ergot alkaloids were placed in Calan gates and randomly assigned to receive either endophyte-infected fescue seed (E+) or noninfected fescue seed (E-; control) in a total mixed ration for 63 d. Weekly measurements were taken to monitor heifer growth and response to ergot alkaloid exposure. Reproductive measurements, including ovarian structures, uterine and ovarian vessel diameter, and hormone concentrations were determined after heifers were synchronized using the standard CO-Synch + 7 d CIDR protocol to ensure all measurements were collected at the same stages of the estrous cycle (0, 4, 10, and 17 d). Data were analyzed using repeated measures in PROC MIXED of SAS. Average daily gain was decreased for the E+ group (0.8 kg/d) compared to control heifers (1.0 kg/d). Body condition scores tended to be greater in control heifers compared to the E+ group (P = 0.053). Additionally, hair coat and hair shedding scores were greater in E+ heifers compared to controls (P < 0.05). Heart rate, rectal temperature, respiration rate, and blood pressure did not differ between treatments (P > 0.05). Vasoconstriction was observed in the caudal artery, but not the caudal vein, in heifers consuming the E+ fescue seed (P < 0.05). No differences were observed in antral follicle counts, corpus luteum area or circulating progesterone concentrations in E+ heifers compared to controls (P > 0.05). There was a significant decrease in the diameter of arteries and veins servicing the ovary and uterus on day 10 and 17 of the estrous cycle. Reduction in blood flow to the reproductive organs during critical times in the estrous cycle may contribute to the reduced ovarian function and pregnancy rates associated with fescue toxicosis.

Pharmacologic assessment of bovine ruminal and mesenteric vascular serotonin receptor populations.

Prior work using a contractility bioassay determined that the serotonin (5-HT) receptor subtype 5-HT2A is present in bovine lateral saphenous veins and plays a role in ergot alkaloid (EA)-induced vascular contraction in steers grazing endophyte-infected (Epichloë coenophiala) tall fescue (Lolium arundinaceum). Ergot alkaloids have also been shown to be vasoactive in bovine gut vasculature. To determine what 5-HT receptors are involved in vasoconstriction of gut vasculature, contractility of ruminal and mesenteric arteries and veins collected from cattle was evaluated in the presence of agonists selective for 5-HT1B (CP 93129), 5-HT1D (L-694, 247), 5-HT2A (TCB-2), 5-HT2B (BW 723C86), 5-HT4 (BIMU-8), and 5-HT7 (LP 44) receptors. Segments of ruminal and mesenteric veins and arteries were collected and suspended in a multimyograph containing continuously oxygenated Krebs-Henseleit buffer. Blood vessels were exposed to increasing concentrations of 5-HT agonists every 15 min and contractile response data were normalized as a percentage of the maximum contractile response induced by 120 mM KCl. Analysis of variance was evaluated using mixed models procedure of SAS for effects of agonist concentration for each vessel type. Receptor agonists for 5-HT2B, 5-HT1D, and 5-HT7 did not induce a contractile response for ruminal or mesenteric vasculature (P > 0.05). However, when exposed to agonists for 5-HT2B or 5-HT1D, mesenteric veins relaxed below zero (P < 0.05). Exposure of all 4 blood vessel types to 5-HT2A agonist induced contractile responses (P < 0.05). The findings of this study indicate that 5-HT1D and 5-HT2B are present in mesenteric veins and may play a role in vasorelaxation. Further, 5-HT2A is present in ruminal and mesenteric vasculature, plays a role in vasoconstriction of these vessels, and could be influenced by EA exposure as has been demonstrated in peripheral blood vessels.

Fumigaclavine C exhibits anti-inflammatory effects by suppressing high mobility group box protein 1 relocation and release.

A previous study demonstrated that Fumigaclavine C (FC) had a potential immunosuppressive activity against concanavalin A-induced hepatitis in mice. However, the precise mechanisms of the anti-inflammatory and hepatoprotective effects of FC are incompletely delineated. This study further investigated the protective effects of FC on lipopolysaccharide (LPS)-induced murine RAW264.7 cells and the underlying molecular mechanism in liver kupffer cells. FC differentially attenuated the production of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), interferon gamma (IFN-γ), and high mobility group box protein 1 (HMGB-1). Intriguingly, FC significantly suppressed LPS-induced HMGB-1 nucleo-cytoplasmic shuttling relocation and release. FC notably decreased the phosphorylation levels of phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent kinase 1 (PDK1), protein kinase C beta II (PKCßII), and protein kinase C gamma (PKCγ). PKCßII/γ played an important part in this signaling pathway cascade. Furthermore, the docking simulation revealed that FC could directly bind to the HMGB-1 B box interfering with Lys90 and Leu145. All of these results indicated that FC exhibited anti-inflammatory and hepatoprotective effects through suppressing HMGB-1 relocation and release by interfering with Lys90 and Leu145.

Tall fescue ergot alkaloids are vasoactive in equine vasculature.

Mares grazing endophyte-infected () tall fescue () typically exhibit reproductive dysfunction rather than problems associated with peripheral vasoconstriction as a primary sign of the fescue toxicosis syndrome. Research using Doppler ultrasonography demonstrated that consumption of endophyte-infected tall fescue seed causes measurable vasoconstriction in the medial palmar artery. The objective of this study was to evaluate contractile responses of medial palmar artery and vein to increasing concentrations of various tall fescue alkaloids. Medial palmar arteries and veins were collected immediately following euthanasia from 23 horses of mixed breed, age, and gender from both forelimbs, and uterine arteries were collected from females ( = 12). Vessels were separated, cleaned of excess connective and adipose tissue, divided into 2- to 3-mm cross-sections, and suspended in a multimyograph chamber with continuously oxygenated Krebs-Henseleit buffer (95% O/5% CO; pH 7.4; 37°C). Following a 90-min equilibration and recovery from reference compound exposure, increasing concentrations of norepinephrine, 5-hydroxytryptamine, ergotamine, and ergonovine for the palmar artery and vein and uterine artery and ergovaline, ergocryptine, ergocristine, ergocornine, and lysergic acid for the palmar artery and vein were added to assess vasoactivity. Data were normalized as a percentage of contractile response induced by the reference compound addition and analyzed as a completely randomized design. Both norepinephrine and serotonin were vasoactive in all 3 types of blood vessels. Neither ergotamine nor ergonovine were vasoactive in the uterine artery. All alkaloids tested with the palmar artery and vein produced a contractile response, except that neither the palmar artery nor the palmar vein responded to lysergic acid ( > 0.05). Ergovaline was the most vasoactive ergot alkaloid in both the palmar artery and the palmar vein ( < 0.05) followed by ergonovine, whereas out of the 4 remaining ergopeptine alkaloids tested, ergocristine induced the lowest contractile response. Although horses do not outwardly appear to be affected by peripheral vasoconstriction as observed in cattle, these data indicate that tall fescue alkaloids are vasoactive and suggest that potential exists for peripheral vascular effects of tall fescue alkaloids in horses. This does not appear to be the case for the uterine artery, and future research should be directed at understanding how ergot alkaloids cause equine reproductive dysfunction.

A fumigaclavine C isostere alleviates Th1-mediated experimental colitis via competing with IFN-γ for binding to IFN-γ receptor 1.

Interferon gamma (IFN-γ) signaling in T cells plays an important role in developing T helper 1 (Th1)-mediated inflammation. Selective regulation of IFN-γ signaling is an attractive strategy for treating Th1-mediated immune diseases. In this study, we aimed to explore possible means of targeting IFN-γ signaling by using small molecule compound. A synthetic small molecule FC9 was identified as it selectively inhibited IFN-γ signaling in T cells without suppressing interleukin 4 (IL-4) signaling. Furthermore, FC9 inhibited IFN-γ-induced Janus kinase 2 (JAK2) activation via competing with IFN-γ for binding to IFN-γ receptor 1 (IFN-γ R1). Interestingly, we found that FC9 bound to IFN-γ R1 and selectively suppressed Th1 but not Th2 immune response in T cells, resulting in an improvement in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. In conclusion, FC9-induced competitive blockade of IFN-γ R1 for selective inhibition of IFN-γ signaling, demonstrated a novel mean of targeting IFN-γ signaling. These findings could lead to increased options for the treatment of Crohn's disease and other Th1-mediated inflammatory diseases.

Three New Ergot Alkaloids from the Fruiting Bodies of Xylaria nigripes (Kl.) Sacc.

Three new ergot alkaloids, xylanigripones A - C (1 - 3) together with three known compounds, agroclavine (4), 8,9-didehydro-10-hydroxy-6,8-dimethylergolin (5), and (6S)-agroclavine N-oxide (6) were isolated from the fungus Xylaria nigripes (Kl.) Sacc. Their structures were elucidated by comprehensive spectroscopic analyses and high-resolution mass spectrometry as well as by comparison with the literature. The absolute configuration was determined by Density Functional Theory (DFT) calculation methods. In addition, all of the compounds were evaluated for bioactivity via a cytotoxicity assay, an acetylcholinesterase inhibition assay and a cholesterol ester transfer protein inhibition assay.

Vasoactivity and Vasoconstriction Changes in Cattle Related to Time off Toxic Endophyte-Infected Tall Fescue.

Previous research has indicated that serotonergic and α-adrenergic receptors in peripheral vasculature are affected by exposure of cattle grazing toxic endophyte-infected (E+; Epichlöe coenophialia) tall fescue (Lolium arundinaceum). The objective of this experiment was to determine the period of time necessary for the vascular effects of ergot alkaloids to subside. Two experiments were conducted to investigate changes in vascular contractile response and vasoconstriction over time relative to removal from an ergot alkaloid-containing E+ tall fescue pasture. In Experiment 1, lateral saphenous vein biopsies were conducted on 21 predominantly Angus steers (357 ± 3 kg body weight) at 0 (n = 6), 7 (n = 6), 14 (n = 5), or 28 days (n = 4) after removal from grazing pasture (3.0 ha; endpoint ergovaline + ergovalinine = 1.35 mg/kg DM) for 126 days. In Experiment 2, lateral saphenous veins were biopsied from 24 Angus-cross steers (361 ± 4 kg body weight) at 0, 21, 42, and 63 days (n = 6 per time point) following removal from grazing tall fescue pastures (3.0 ha; first 88 days endpoint ergovaline + ergovalinine = 0.15 mg/kg DM; last 18 days endpoint ergovaline + ergovalinine = 0.57 mg/kg DM) for 106 total days. Six steers (370 ± 18 kg body weight) off of bermudagrass pasture for the same time interval were also biopsied on Day 0 and Day 63 (n = 3 per time point). Additionally, in Experiment 2, cross-sectional ultrasound scans of caudal artery at the fourth coccygeal vertebra were taken on Days 0, 8, 15, 21, 29, 36, 42, and 45 to determine mean artery luminal area to evaluate vasoconstriction. In both experiments, steers were removed from pasture and housed in a dry lot and fed a corn silage diet for the duration of biopsies and ultrasound scans. Biopsied vessels used to evaluate vasoactivity were cleaned, incubated in a multimyograph, and exposed to increasing concentrations of 4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl) methylamine hydrobromide (TCB2; 5HT2A agonist), guanfacine (GF; α2A-adrenergic agonist), and (R)-(+)-m-nitrobiphenyline oxalate (NBP; α2C-adrenergic agonist) in both experiments and ergovaline (ERV) and ergotamine (ERT) in Experiments 1 and 2, respectively. In Experiment 1, days off pasture × agonist concentration was not significant (p > 0.1) for all four compounds tested. In Experiment 2, GF, NBP, TCB2 and ERT were significant for days off pasture × agonist concentration interaction (p < 0.02) and vasoactivity increased over time. Vasoactivity to agonists was reduced (p < 0.05) when steers were initially removed from E+ tall fescue pasture compared to bermudagrass, but did not differ by Day 63 for any variable. Luminal areas of caudal arteries in steers grazed on E+ tall fescue relaxed and were similar to steers that had grazed bermudagrass for 36 days on non-toxic diet (p = 0.15). These data demonstrate changes in peripheral vasoactivity and recovery from vasoconstriction occur beyond five weeks off toxic pasture and 5HT2A receptors appear to be more dramatically affected in the lateral saphenous vein by grazing E+ tall fescue pasture than adrenergic receptors.

[Conservative treatment of pelvic varicose veins: indications for and possibilities of therapy]. / Konservativnoe lechenie varikoznoi bolezni taza: pokazaniia i vozmozhnosti terapii.

The article is a literature review containing the data on various conservative methods of treatment for pelvic varicose veins. The authors present herein analysis of efficacy of using non-steroidal anti-inflammatory drugs, derivatives of ergot alkaloids, hormonal drugs, phleboprotectors, compression therapy in treatment of pelvic varicose veins, as well as indications for carrying out pharmacotherapy. Attention is drawn to the critically scarce number of studies dedicated to this issue, underlying the necessity of carrying out large multidisciplinary studies aimed at investigating the possibilities of non-surgical treatment of pelvic varicose veins.

Total synthesis, biosynthesis and biological profiles of clavine alkaloids.

This review highlights noteworthy synthetic and biological aspects of the clavine subfamily of ergot alkaloids. Recent biosynthetic insights have laid the groundwork for a better understanding of the diverse biological pathways leading to these indole derivatives. Ergot alkaloids were among the first fungal-derived natural products identified, inspiring pharmaceutical applications in CNS disorders, migraine, infective diseases, and cancer. Pergolide, for example, is a semi-synthetic clavine alkaloid that has been used to treat Parkinson's disease. Synthetic activities have been particularly valuable to facilitate access to rare members of the Clavine family and empower medicinal chemistry research. Improved molecular target identification tools and a better understanding of signaling pathways can now be deployed to further extend the biological and medical utility of Clavine alkaloids.

Ergot Alkaloids (Re)generate New Leads as Antiparasitics.

Praziquantel (PZQ) is a key therapy for treatment of parasitic flatworm infections of humans and livestock, but the mechanism of action of this drug is unresolved. Resolving PZQ-engaged targets and effectors is important for identifying new druggable pathways that may yield novel antiparasitic agents. Here we use functional, genetic and pharmacological approaches to reveal that serotonergic signals antagonize PZQ action in vivo. Exogenous 5-hydroxytryptamine (5-HT) rescued PZQ-evoked polarity and mobility defects in free-living planarian flatworms. In contrast, knockdown of a prevalently expressed planarian 5-HT receptor potentiated or phenocopied PZQ action in different functional assays. Subsequent screening of serotonergic ligands revealed that several ergot alkaloids possessed broad efficacy at modulating regenerative outcomes and the mobility of both free living and parasitic flatworms. Ergot alkaloids that phenocopied PZQ in regenerative assays to cause bipolar regeneration exhibited structural modifications consistent with serotonergic blockade. These data suggest that serotonergic activation blocks PZQ action in vivo, while serotonergic antagonists phenocopy PZQ action. Importantly these studies identify the ergot alkaloid scaffold as a promising structural framework for designing potent agents targeting parasitic bioaminergic G protein coupled receptors.

Fumigaclavine C ameliorates dextran sulfate sodium-induced murine experimental colitis via NLRP3 inflammasome inhibition.

In the present study, the effect of Fumigaclavine C, a fungal metabolite, on murine experimental colitis induced by dextran sulfate sodium (DSS) and its possible mechanism were examined in vivo and vitro. Oral administration of Fumigaclavine C dose-dependently attenuated the loss of body weight and shortening of colon length induced by DSS. The disease activity index, histopathologic scores of musco was also significantly reduced by Fumigaclavine C treatment. Protein and mRNA levels of DSS-induced pro-inflammatory cytokines in colon, including TNF-α, IL-1ß and IL-17A, were markedly suppressed by Fumigaclavine C. At the same time, decreased activation of caspase-1 in peritoneal macrophages was detected in Fumigaclavine C -treated mice which suggested that the NLRP3 inflammasome activation was suppressed. Furthermore, in the LPS plus ATP cell model, we found that Fumigaclavine C dose-dependent inhibited IL-1ß release and caspase-1 activation. Taken together, our results demonstrate the ability of Fumigaclavine C to inhibit NLRP3 inflammasome activation and give some evidence for its potential use in the treatment of inflammatory bowel diseases.

Identification of cellular and molecular factors determining the response of cancer cells to six ergot alkaloids.

Ergot alkaloids are psychoactive and vasoconstricting agents of the fungus Claviceps purpurea causing poisoning such as ergotism in medieval times (St. Anthony's Fire). This class of substances also inhibits tumor growth in vitro and in vivo, though the underlying mechanisms are unclear as yet. We investigated six ergot alkaloids (agroclavine, ergosterol, ergocornin E, ergotamine, dihydroergocristine, and 1-propylagroclavine tartrate) for their cytotoxicity towards tumor cell lines of the National Cancer Institute, USA. 1-Propylagroclavine tartrate (1-PAT) revealed the strongest cytotoxicity. Out of 76 clinically established anticancer drugs, cross-resistance was found between the ergot alkaloids and 6/7 anti-hormonal drugs (=85.7 %) and 5/15 DNA-alkylating drugs (=33.3 %). The IC50 values for the six alkaloids were not correlated to well-known determinants of drug resistance, such as proliferative activity (as measured by cell doubling times, PCNA expression, and cell cycle distribution), the multidrug resistance-mediating P-glycoprotein/MDR1 and expression or mutations of oncogenes and tumor suppressor genes (EGFR, RAS, TP53). While resistance of control drugs (daunorubicin, cisplatin, erlotinib) correlated with these classical resistance mechanisms, ergot alkaloids did not. Furthermore, COMPARE and hierarchical cluster analyses were performed of mRNA microarray data to identify genes correlating with sensitivity or resistance to 1-PAT. Twenty-three genes were found with different biological functions (signal transducers, RNA metabolism, ribosome constituents, cell cycle and apoptosis regulators etc.). The expression of only 3/66 neuroreceptor genes correlated with the IC50 values for 1-PAT, suggesting that the psychoactive effects of ergot alkaloids may not play a major role for the cytotoxic activity against cancer cells. In conclusion, the cytotoxicity of ergot alkaloids is not involved in classical mechanisms of drug resistance opening the possibility to bypass resistance and to treat otherwise drug-resistant and refractory tumors. The modes of action are multifactorial, which is a typical feature of many natural compounds.

Isolating and using sections of bovine mesenteric artery and vein as a bioassay to test for vasoactivity in the small intestine.

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.