Liver protection and hemostatic effects of medicinal plant Arnebia euchroma (Royle) I.M.Johnst extract in a rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: Arnebia euchroma (Royle) I.M.Johnst. (AE) is a Chinese medicinal herb that is traditionally used to treat various circulatory diseases. It exhibits certain effects, such as the promotion of blood circulation and cooling, rash clearance, and detoxification. AIM OF THE STUDY: This study was designed to explore the hepatoprotective and hemostatic effects of the ethyl acetate extract of AE in rats with carbon tetrachloride (CCl4)-induced liver injury. MATERIALS AND METHODS: Wistar rats were treated via oral gavage with different doses of the ethyl acetate extract of AE (3.5, 7, or 14 g kg-1·day-1) for 14 consecutive days, following which hemostatic and liver function tests were conducted. For the hemostatic tests, the platelet count, blood platelet aggregation, blood platelet adhesion to fibrinogen, platelet factor 4 (PF-4) secretion from blood platelets, prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen levels were measured at the end of the treatment period. For the liver function tests, 0.25 mL/200 g (1.25 mL kg-1·day-1) of olive oil was injected into the abdominal cavity of the control rats, whereas 15% CCl4 plus olive oil (prescription: 7.5 mL CCl4 + 42.5 olive oil) was injected into that of the treated rats at 1 h after extract administration on day 6, 13, and 20. Additionally, food and water were withheld from all the animals. On the following day, the rats were anesthetized and their albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), gamma-glutamyl transpeptidase (GGT), lactate dehydrogenase (LDH), reactive oxygen species (ROS), methane dicarboxylic aldehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) levels were measured. Glutathione S-transferase (GST), glutathione reductase (GR), and glutathione peroxidase (GPx) levels among the groups were determined using a one-way analysis of variance. RESULTS: The platelet count and blood platelet aggregation, blood platelet adhesion to fibrinogen and PF-4 secretion levels were significantly increased in the (3.5 g kg-1 day-1) AE group as compared to those in the control group (all p < 0.001; for the 7 and 14 g kg-1 day-1 AE groups, all p > 0.05, respectively). Although the PT and aPTT were not affected by the AE extract (all p > 0.05), the TT was reduced and the FIB levels were significantly increased in all AE groups (p < 0.05). Liver function tests showed that CCl4 caused significant liver damage, thereby decreasing the albumin, SOD, CAT, GSH, GST, GR, and GPx levels, while increasing the AST, ALT, ALP, SGOT, SGPT, GGT, LDH, ROS, and MDA levels (all p < 0.001). By contrast, treatment with the different doses of AE extract reversed the CCl4 effects on all these parameters. Compared with the levels in the CCl4 group, the GSH and GR levels in the three AE groups (3.5, 7, and 14 g kg-1·day-1) were significantly higher (p < 0.05, p < 0.01, and p < 0.001, respectively), whereas the differences in the other parameters for these three groups were all at the significance levels of p < 0.05, p < 0.05, and p < 0.01, respectively. CONCLUSIONS: AE extracts administered orally exhibited hepatoprotective activity by affecting platelet production and blood coagulation and ameliorating liver function-damaging modifications. Specifically, a dosage of 3.5 g kg-1·day-1 resulted in the most optimal effects.

Green synthesis of selenium based N-heterocyclic carbene compounds; structural, in-vitro anticancer and molecular docking studies.

Benzimidazolium salts (3-6) were synthesized as stable N-Heterocyclic Carbene (NHC) precursors and their selenium-NHC compounds/Selenones (7-10) were prepared using water as a solvent. Characterization of each of the synthesized compounds was carried out by various analytical and spectroscopic (FT-IR, 1H-, 13C NMR) methods. X-ray crystallographic analyses of single crystals obtained for salts 3 and 5 were carried out. Synthesized salts and their Se-NHCs were tested in-vitro for their anticancer potential against Cervical Cancer Cell line from Henrietta Lacks (HeLa), Breast cancer cell line (MDA-MB-231), Adenocarcinoma cell line (A549) and human normal endothelial cell line (EA.hy926). MTT assay was used for analysis and compared with standard drug 5-flourouracil. Benzimidazolium salts (3-6) and their selenium counter parts (7-10) were found potent anticancer agents. Salt 3-5 were found to be potent anticancer against HeLa with IC50 values 0.072, 0.017 and 0.241 µM, respectively, which are less than standard drug (4.9 µM). The Se-NHCs (7-10) had also shown significant anticancer potential against HeLa with IC50 values less than standard drug. Salts 3, 4 against EA.hy926, compounds 3,5,6, and 10 against MDA-MB-321, and compounds 4, 10 against A-549 cell line were found more potent anticancer agents with IC50 values less than standard drug. Molecular docking for (7-10) showed their good anti-angiogenic potential having low binding energy and significant inhibition constant values with VEGFA (vascular endothelial growth factor), EGF (human epidermal growth factor), COX1 (cyclooxygenase-1) and HIF (hypoxia inducible factor).

Paternal low protein diet and the supplementation of methyl-donors impact fetal growth and placental development in mice.

INTRODUCTION: Paternal low-protein diet can alter sperm methylation status, fetal growth and program offspring ill-health, however its impact on the placenta remains poorly defined. Here we examine the influence paternal low-protein diet has on fetal and placental development and the additional impact of supplementary methyl-donors on fetoplacental physiology. METHODS: Male C57BL/6J mice were fed a control normal protein diet (NPD; 18% protein), a low-protein diet (LPD; 9% protein) or LPD with methyl-donor supplementation (MD-LPD; choline chloride, betaine, methionine, folic acid, vitamin B12) for a minimum of 8 weeks. Males were mated with 8-11 week old female C57BL/6J mice and fetal and placental tissue collected on embryonic day 17.5. RESULTS: Paternal LPD was associated with increased fetal weights compared to NPD and MD-LPD with 22% fetuses being above the 90th centile for fetal weight. However, LPD and MD-LPD placental weights were reduced when compared to NPD. Placentas from LPD fathers demonstrated a reduced junctional zone area and reduced free-fatty acid content. MD-LPD placentas did not mirror these finding, demonstrating an increased chorion area, a reduction in junctional-specific glycogen staining and reduced placental Dnmt3bexpression, none of which were apparent in either NPD or LPD placentas. DISCUSSION: A sub-optimal paternal diet can influence fetal growth and placental development, and dietary methyl-donor supplementation alters placental morphology and gene expression differentially to that observed with LPD alone. Understanding how paternal diet and micro-nutrient supplementation influence placental development is crucial for determining connections between paternal well-being and future offspring health.

Antibacterial effect and DNA delivery using a combination of an arsonium-containing lipophosphoramide with an N-heterocyclic carbene-silver complex - Potential benefits for cystic fibrosis lung gene therapy.

Cystic Fibrosis (CF), the most common chronic genetic disorder among the Caucasian population, is a life-threatening disease mainly due to respiratory failures resulting from chronic infections and inflammation. Although research in the pharmacological field has recently made significant progress, gene therapy still remains a promising strategy to cure CF, especially because it should be applicable to any patient whatever the mutation profile. Until now, little attention has been paid to bacterial lung infections with regard to gene delivery to the airways; yet, this could greatly impact on the success of gene therapy. Previously, we have reported arsonium-containing lipophosphoramides as poly-functional nanocarriers capable of simultaneous antibacterial action against Gram-positive bacteria and gene transfer into eukaryotic cells. In the present work, we show that such nanoparticles can also be combined with an N-heterocyclic carbene-silver complex in order to extend the spectrum of antibacterial activity, including towards the Gram-negative Pseudomonas aeruginosa. Importantly, this is demonstrated not only using standard in vitro protocols but also a clinically-relevant aerosol delivery method. Furthermore, antibacterial effects are compatible with efficient and safe gene delivery into human bronchial epithelial cells. The poly-functionality of combinations of such chemical compounds may thus show benefits for CF lung gene therapy.

Methane ameliorates spinal cord ischemia-reperfusion injury in rats: Antioxidant, anti-inflammatory and anti-apoptotic activity mediated by Nrf2 activation.

Methane is reported to have antioxidant, anti-inflammatory and anti-apoptotic properties. We investigated the potential neuroprotective effects of methane-rich saline (MS) on spinal cord ischemia-reperfusion injury and determined that its therapeutic benefits are associated with the activation of nuclear factor erythroid 2-related factor 2 (Nrf2). Rats received 9min of spinal cord ischemia induced by occlusion of the descending thoracic aorta plus systemic hypotension followed by a single MS treatment (10ml/kg, ip) and 72h reperfusion. MS treatment attenuated motor sensory deficits and produced high concentrations of methane in spinal cords during reperfusion, which increased Nrf2 expression and transcriptional activity in neurons, microglia and astrocytes in the ventral, intermediate and dorsal gray matter of lumbar segments. Heme oxygenase-1, superoxide dismutase, catalase and glutathione were upregulated; and glutathione disulfide, superoxide, hydrogen peroxide, malondialdehyde, 8-hydroxy-2-deoxyguanosine and 3-nitrotyrosine were downregulated in MS-treated spinal cords. MS treatment reduced neuronal apoptosis in gray matter zones, which was consistent with the suppression of cytochrome c release to the cytosol from the mitochondria and the activation of caspase-9 and -3. Throughout the gray matter, the activation of microglia and astrocytes was inhibited; the nuclear accumulation of phosphorylated nuclear factor-kappa B p65 was reduced; and tumor necrosis factor α, interleukin 1ß, chemokine (C-X-C motif) ligand 1, intercellular adhesion molecule 1 and myeloperoxidase were decreased. MS treatment attenuated blood-spinal cord barrier dysfunction by preventing the expression and activity of matrix metallopeptidase-9 and disrupting tight junction proteins. Consecutive intrathecal injection of specific siRNAs targeting Nrf2 at 24-h intervals 3 days before ischemia reduced the beneficial effects of MS. Our data indicate that MS treatment prevents IR-induced spinal cord damage via antioxidant, anti-inflammatory and anti-apoptotic activities that involve the activation of Nrf2 signaling. Thus, methane may serve as a novel promising therapeutic agent for treating ischemic spinal cord injury.

Methane digester effluent from swine excreta as a nutrient and water source for growing and finishing swine.

The experimental objective was to evaluate swine methane digester effluent (SMDE) as a water and nutrient source for swine. The mesophilic methane digester was loaded daily with manure from finishing swine fed a corn-soybean meal diet. Dry diet was mixed with SMDE (3.7% DM) and fed twice daily in troughs. Tap water was provided and consumption measured. Barrows were group fed (3 pigs/pen) and adapted to SMDE by increasing SMDE for 7 d, with the full amount fed from d 8 to the end of the feeding phase (d 21, 14, 23, or 37 for Exp. 1 to 4, respectively). Blood samples were collected on d 0, 10, 21, and 31 to determine plasma concentrations of glucose and plasma urea N (PUN). Barrows were placed in individual metabolism cages for a 5-d acclimation and a 5-d fecal and urine collection to determine apparent N and energy utilization. For Exp. 1, 18 pigs averaging 75 kg BW were allotted to diets with 0, 48.6, or 63.7% SMDE, as-fed basis. For Exp. 2 and 3, 12 pigs/experiment averaged 117 and 70 kg, respectively, and were allotted to diets with 0 or 63.7% SMDE, as-fed basis. At the end of Exp. 2 and 3, pigs were sacrificed and liver samples were collected to determine urea cycle enzyme activity, and loin was saved for taste panel evaluation. For Exp. 4, pigs averaged 40 kg and were allotted to diets with 0 or 57.5% SMDE, as-fed basis. The ADFI, ADG, and G:F of finishing swine (Exp. 1 to 3) were not reduced by feeding diets containing 63.7% SMDE (as-fed basis), whereas ADG and G:F of growing swine (Exp. 4) were reduced (P < 0.01) by feeding a diet containing 57.5% SMDE. Pigs fed diets containing SMDE consumed 31 to 56% less (P < 0.05) water and had greater (P < 0.01) PUN concentrations than pigs fed control diets. Pigs fed diets containing SMDE excreted more (g, P < 0.05) fecal N and absorbed and retained less N (%; P < 0.01) and energy (DE and ME) than pigs fed control diets. Treatment had no effect on urea cycle enzyme activity. In conclusion, finishing swine adapted to diets containing 63.7% SMDE (as-fed basis) based on growth performance, whereas growing swine did not adapt to a diet containing 57.5% SMDE because of the large content of nonprotein N in SMDE. Recycling SMDE to swine greatly reduced fresh water consumption, whereas the protein and energy values of SMDE were approximately 0 for swine. Therefore, SMDE is more appropriately recycled as a source of water and N for ruminant nutrition or crop production.

Solid-phase synthesis of azidomethylene inhibitors targeting cysteine proteases.

An efficient strategy for the solid-phase synthesis of azidomethylene inhibitors targeting cysteine proteases is described. The method is highlighted by its compatibility with readily available building blocks, as well as its ability to accommodate different functional groups. A 249-member library has thus far been successfully synthesized, characterized, and screened against Caspase-1, -3 and -7.

The hepatic vagus nerve attenuates Fas-induced apoptosis in the mouse liver via alpha7 nicotinic acetylcholine receptor.

BACKGROUND & AIMS: Although accumulating evidence has recently shown that the efferent vagus nerve attenuates systemic inflammation, it remains unclear whether or not the vagus nerve can affect Fas-induced liver apoptosis. We investigated the effect of the vagus nerve by using a selective hepatic vagotomy. METHODS: We assessed the mortality and apoptosis in Fas-induced fulminant hepatitis in sham-operated and vagotomized male C57BL/6 mice. To determine how the nerve influences hepatocyte apoptosis, hepatitis was preceded by pretreatment with nicotine; PNU-282987, an alpha7 nicotinic acetylcholine receptor (AChR) agonist; liposome-encapsulated dichloromethylene diphosphonate (lipo-Cl(2)MDP), a macrophage eliminator; and Mn (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP), an oxidative inhibitor. RESULTS: Mortality in the vagotomized mice was significantly higher than that in the sham-operated mice following intravenous administration with the anti-Fas antibody Jo-2. This result was also supported by the data from both terminal deoxynucleotidyl-transferase mediated dUTP nick-end labeling and caspase-3 assay, in which vagotomized livers showed a significant elevation in the number of apoptotic hepatocytes and increased caspase-3 activity following Jo-2 treatment compared with the sham-operated livers. Supplementation with nicotine and PNU-282987 dose dependently inhibited this detrimental effect of the vagotomy. Moreover, the vagotomy-triggered exacerbation of Fas-induced hepatitis was completely blocked by lipo-Cl(2)MDP. Similarly, pretreatment with MnTBAP also completely suppressed the vagotomy-triggered exacerbation. CONCLUSIONS: The hepatic vagus nerve appears to play an important role in attenuating Fas-induced hepatocyte apoptosis through alpha7 nicotinic AChR, perhaps by causing the Kupffer cells to reduce their generation of an excessive amount of reactive oxygen species.

Sediment treatment with a nitrate-storing compound to reduce phosphorus release.

The application of Fe(III), in combination with sediment oxidation by NO(3)(-), is an accepted procedure to manage stratified eutrophic lakes by controlling the phosphorus release from sediments into overlying water. Depox(R), a newly developed compound, consisting of Fe(III) and NO(3)(-), has a storage effect for NO(3)(-). NO(3)(-) is released slowly, hence the disadvantageous high solubility of NO(3)(-) in water can be retarded. The compound was added to water as a suspension which quickly flocculated and precipitated. Within 3 weeks, NO(3)(-) was desorbed from the Depox(R) compound in deionized water. After application in lakes, the NO(3)(-) availability on the sediment surface was prolonged for 2 months. After treatment, P release from the sediment and microbial metabolism were investigated under laboratory conditions as well as in the mesocosm. P release was almost stopped in both cases during the experiment. SO(4)(2-) consumption was significantly lower after Depox(R) addition, and CH(4) production was completely suppressed by Depox(R) treatment in the laboratory, whereas in the enclosures SO(4)(2-) and also CH(4) concentrations at the sediment water interface did not change significantly between treated enclosures and controls.

Human cytochrome p450 inhibition and metabolic-intermediate complex formation by goldenseal extract and its methylenedioxyphenyl components.

The concurrent use of herbal medicinals with prescription and over-the-counter drugs carries a risk for unanticipated adverse drug-botanical pharmacokinetic interactions, particularly as a result of cytochrome P450 (P450) inhibition. Extracts of goldenseal (Hydrastis canadensis) containing approximately equal concentrations ( approximately 17 mM) of two methylenedioxyphenyl alkaloids, berberine and hydrastine, inhibited with increasing potency (CYP2C9) diclofenac 4'-hydroxylation, (CYP2D6) bufuralol 1'-hydroxylation, and (CYP3A4) testosterone 6beta-hydroxylation activities in human hepatic microsomes. The inhibition of testosterone 6beta-hydroxylation activity was noncompetitive with an apparent Ki of 0.11% extract. Of the methylenedioxyphenyl alkaloids, berberine (IC50 = 45 microM) was the more inhibitory toward bufuralol 1'-hydroxylation and hydrastine (IC50 approximately 350 microM for both isomers), toward diclofenac 4'-hydroxylation. For testosterone 6beta-hydroxylation, berberine was the least inhibitory component (IC50 approximately 400 microM). Hydrastine inhibited testosterone 6beta-hydroxylation with IC50 values for the (+)- and (-)-isomers of 25 and 30 microM, respectively. For (-)-hydrastine, an apparent Ki value of 18 microM without preincubation and an NADPH-dependent mechanism-based inhibition with a kinactivation of 0.23 min(-1) and a KI of approximately 110 microM were determined. Cytochrome P450 metabolic-intermediate (MI) complex formation could be demonstrated for both hydrastine isomers. With expressed P450 isoforms, hydrastine formed a P450 MI complex with CYP2C9, CYP2D6, and CYP3A4. Coexpression of cytochrome b5 with the P450 isoforms enhanced the rate but not the extent of P450 MI complex formation.

Stimulation of hybrid poplar growth in petroleum-contaminated soils through oxygen addition and soil nutrient amendments.

Hybrid poplar trees (Populus deltoides x nigra DN34) were grown in a green-house using hydrocarbon-contaminated soil from a phytoremediation demonstration site in Health, Ohio. Two independent experiments investigated the effect of nutrient addition on poplar growth and the importance of oxygen addition to root development and plant growth. Biomass measurements, poplar height, and leaf color were used as indicators of plant health in the selection of a 10/5/5 NPK fertilizer applied at 1121 kg/ha (112 kg-N, 24.4 kg-P, 46.5 kg-K per ha) to enhance hybrid poplar growth at the Health site. Five passive methods of oxygen delivery were examined, including aeration tubes, gravel addition, and an Oxygen Release Compound (ORC). When ORC was placed in coffee filters above hydrocarbon-contaminated soil, a statistically significant increase of 145% was observed in poplar biomass growth, relative to unamended controls. The ORC in filters also stimulated significant increases in root density. A 15.2-cm interval of soil directly below ORC addition exhibited an increase from 2.6 +/- 1.0 mg/cm3 to 4.8 +/- 1.0 mg/cm3, showing stimulation of root growth in hydrocarbon-stained soil. The positive response of hybrid poplars to oxygen amendments suggests that overcoming oxygen limitation to plants should be considered in phytoremediation projects when soil contamination exerts a high biochemical oxygen demand, such as in former refinery sites.

Effects of nutrient dosing on subsurface methanotrophic populations and trichloroethylene degradation.

In in situ bioremediation demonstration at the Savannah River Site in Aiken, South Carolina, trichloroethyle degrading microorganisms were stimulated by delivering nutrients to the TCE-contaminated subsurface via horizontal injection wells. Microbial and chemical monitoring of groundwater from 12 vertical wells was used to examine the effects of methane and nutrient (nitrogen and phosphorus) dosing on the methanotrophic populations and on the potential of the subsurface microbial communities to degrade TCE. Densities of methanotrophs increased 3-5 orders of magnitude during the methane- and nutrient-injection phases; this increase coinclded with the higher methane levels observed in the monitoring wells. TCE degradation capacity, although not directly tied to methane concentration, responded to the methane injection, and responded more dramatically to the multiple-nutrient injection. tion. These results support the crucial role of methane, nitrogen, and phosphorus as amended nutrients in TCE bioremediation. The enhancing effects of nutrient dosing on microbial abundance and degradative potentials, coupled with increased chloride concentrations, provided multiple lines of evidence substantiating the effectiveness of this integrated in situ bioremediation process.

Optimization and maintenance of soluble methane monooxygenase activity in Methylosinus trichosporium OB3b.

Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater. A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity in Methylosinus trichosporium OB3b. Methylosinus trichosporium OB3b attained peak sMMO activity (275-300 nmol of naphthol formed h-1 mg of protein-1 at 25 degrees C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium. With the onset of methane limitation however, sMMO activity rapidly declined. It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (mu max 0.08 h-1) and growth yields (0.4-0.5 g cells/g CH4) and near maximal activities of sMMO. In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45-50% of the initial peak level and this was maintained over several weeks. The addition of d-biotin, pyridoxine, and vitamin B12 (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25-75%. The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.

Activation of lordotic responding in female rats by suppression of serotonergic activity.

After systemic administration of several serotonergic antagonists, female rats that had been ovariectomized, adrenalectomized, and estrogen-primed showed lordotic responding. Lordosis could also be elicited after direct placement of serotonergic receptor blockers into hypothalamic sites known to contain serotonergic terminals. None of the treatments activated the soliciting component of the estrous behavior pattern of the female rat. It is postulated that the hypothalamus contains serotonergic terminals which suppress lordotic responding.