Activation of AhR-NQO1 Signaling Pathway Protects Against Alcohol-Induced Liver Injury by Improving Redox Balance.

BACKGROUND & AIMS: Aryl hydrocarbon receptor (AhR) is a liver-enriched xenobiotic receptor that plays important role in detoxification response in liver. This study aimed to investigate how AhR signaling may impact the pathogenesis of alcohol-related liver disease (ALD). METHODS: Chronic alcohol feeding animal studies were conducted with mouse models of hepatocyte-specific AhR knockout (AhRΔhep) and NAD(P)H quinone dehydrogenase 1 (NQO1) overexpression, and dietary supplementation of the AhR ligand indole-3-carbinol. Cell studies were conducted to define the causal role of AhR and NQO1 in regulation of redox balance and apoptosis. RESULTS: Chronic alcohol consumption induced AhR activation and nuclear enrichment of NQO1 in hepatocytes of both alcoholic hepatitis patients and ALD mice. AhR deficiency exacerbated alcohol-induced liver injury, along with reduction of NQO1. Consistently, in vitro studies demonstrated that NQO1 expression was dependent on AhR. However, alcohol-induced NQO1 nuclear translocation was triggered by decreased cellular oxidized nicotinamide adenine dinucleotide (NAD+)-to-NADH ratio, rather than by AhR activation. Furthermore, both in vitro and in vivo overexpression NQO1 prevented alcohol-induced hepatic NAD+ depletion, thereby enhancing activities of NAD+-dependent enzymes and reversing alcohol-induced liver injury. In addition, therapeutic targeting of AhR in the liver with dietary indole-3-carbinol supplementation efficiently reversed alcoholic liver injury by AhR-NQO1 signaling activation. CONCLUSIONS: This study demonstrated that AhR activation is a protective response to counteract alcohol-induced hepatic NAD+ depletion through induction of NQO1, and targeting the hepatic AhR-NQO1 pathway may serve as a novel therapeutic approach for ALD.

Discovery of pyrazolo-thieno[3,2-d]pyrimidinylamino-phenyl acetamides as type-II pan-tropomyosin receptor kinase (TRK) inhibitors: Design, synthesis, and biological evaluation.

Tropomyosin receptor kinase (TRK) represents an attractive oncology target for cancer therapy related to its critical role in cancer formation and progression. NTRK fusions are found to occur in 3.3% of lung cancers, 2.2% of colorectal cancers, 16.7% of thyroid cancers, 2.5% of glioblastomas, and 7.1% of pediatric gliomas. In this paper, we described the discovery of the type-II pan-TRK inhibitor 4c through the structure-based drug design strategy from the original hits 1b and 2b. Compound 4c exhibited excellent in vitro TRKA, TRKB, and TRKC kinase inhibitory activity and anti-proliferative activity against human colorectal carcinoma derived cell line KM12. In the NCI-60 human cancer cell lines screen, compound 4g demonstrated nearly 80% of growth inhibition for KM12, while only minimal inhibitory activity was observed for the remaining 59 cancer cell lines. Western blot analysis demonstrated that 4c and its urea cousin 4k suppressed the TPM3-TRKA autophosphorylation at the concentrations of 100 nM and 10 nM, respectively. The work presented that 2-(4-(thieno[3,2-d]pyrimidin-4-ylamino)phenyl)acetamides could serve as a novel scaffold for the discovery and development of type-II pan-TRK inhibitors for the treatment of TRK driven cancers.

Discovery of indane propanamides as potent and selective TRPV1 antagonists.

A series of indane-type acetamide and propanamide analogues were investigated as TRPV1 antagonists. The analysis of structure-activity relationship indicated that indane A-region analogues exhibited better antagonism than did the corresponding 2,3-dihydrobenzofuran and 1,3-benzodioxole surrogates. Among them, antagonist 36 exhibited potent and selective antagonism toward capsaicin for hTRPV1 and mTRPV1. Further, in vivo studies indicated that antagonist 36 showed excellent analgesic activity in both phases of the formalin mouse pain model and inhibited the pain behavior completely at a dose of 1 mg/kg in the 2nd phase.

Structural basis of ligand recognition at the human MT1 melatonin receptor.

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.

Metabolite profiling and isolation of biologically active compounds from Scadoxus puniceus, a highly traded South African medicinal plant.

Scadoxus puniceus (Amaryllidaceae), a medicinal plant of high value in South Africa, is used as a component of a traditional herbal tonic prescribed to treat several ailments. Ultra-high performance liquid chromatography-tandem mass spectrometry quantified the phenolic compounds in different organs of S. puniceus. Gravity column chromatography was used to separate fractions and active compounds. The structure of these compounds was determined using 1D and 2D nuclear magnetic resonance and mass spectroscopic techniques. A microplate technique was used to determine the acetylcholinesterase inhibitory activity of the pure compounds. Metabolite profiling revealed a greater profusion of hydroxycinnamic acids (69.5%), as opposed to hydroxybenzoic acids (30.5%). Chlorogenic acid was the most abundant (49.6% of hydroxycinnamic acids) compound. In addition to chlorogenic acid, the study is the first to report the presence of sinapic, gallic, and m-hydroxybenzoic acids in the Amaryllidaceae. Chromatographic separation of S. puniceus led to the isolation of haemanthamine (1), haemanthidine (2), and a rare chlorinated amide, metolachlor (3), the natural occurrence of which is described for the first time. Haemanthamine, haemanthidine, and metolachlor displayed strong acetylcholinesterase inhibitory activity (IC50 ; 23.1, 23.7, and 11.5 µM, respectively). These results substantiate the frequent use of S. puniceus as a medicinal plant and hold much promise for further pharmaceutical development.

Multivalent Siderophore-DOTAM Conjugates as Theranostics for Imaging and Treatment of Bacterial Infections.

There is a strong need to better diagnose infections at deep body sites through noninvasive molecular imaging methods. Herein, we describe the synthesis and characterization of probes based on siderophore conjugates with catechol moieties and a central DOTAM scaffold. The probes can accommodate a metal ion as well as an antibiotic moiety and are therefore suited for theranostic purposes. The translocation of the conjugates across the outer and inner cell membranes of E. coli was confirmed by growth recovery experiments with enterobactin-deficient strains, by the antibacterial activity of ampicillin conjugates, and by confocal imaging using a fluorogen-activating protein-malachite green system adapted to E. coli. The suitability of the probes for in vivo imaging was demonstrated with a Cy5.5 conjugate in mice infected with P. aeruginosa.

Synthesis, Computational and Pharmacological Evaluation of N-(2-benzoyl- 4-chlorophenyl)-2-(4-(Substituted Phenyl) Piperazin-1-yl) Acetamides as CNS Agents.

BACKGROUND: A series of new N-(2-benzoyl-4-chlorophenyl)-2-(4-(substituted phenyl) piperazin-1-yl) acetamides (3a-j) have been synthesized by the chloroacetylation of 2-amino-5- chlorbenzophenone which was further reacted with substituted phenylpiperazine. MATERIAL: The chemical structures of the compounds were confirmed on the basis of their TLC, IR, 1HNMR, 13CNMR and by elemental analysis. The physicochemical similarity of the target compounds with respect to standard drug diazepam was assessed by calculating from a set of physicochemical properties using software programs. CONCLUSION: Molecular docking studies revealed that the target compounds correctly dock into the binding pocket of the GABAA receptor, while their bioavailability/drug-likeness was predicted to be acceptable but requires future optimization. The anxiolytic and skeletal muscle relaxant activity of the target compounds (3a-j) were evaluated in albino mice. Among them, compound 3h showed potent anxiolytic and skeletal muscle relaxant activity.

Studies of DNA-binding properties of lafutidine as adjuvant anticancer agent to calf thymus DNA using multi-spectroscopic approaches, NMR relaxation data, molecular docking and dynamical simulation.

The interactions between lafutidine (LAF) and calf thymus DNA (ctDNA) have been investigated both experimentally and theoretically. UV-vis absorption studies confirmed that LAF binds to ctDNA through non-covalent interactions. Fluorescence quenching and time-resolved fluorescence spectroscopy studies showed that the binding of LAF with ctDNA occurred through static quenching mechanism, resulting in the formation of a LAF-ctDNA complex. The binding constants (K) of the complex were found to be around 103M-1 via NMR relaxation rates and fluorescence data, and the calculated thermodynamic parameters indicated that hydrogen bonds and van der Waals forces played major roles in the binding of LAF to ctDNA. The changes in CD spectra indicated that LAF induced a slight perturbation on the base stacking and helicity of B-DNA. A comparative study of the LAF-ctDNA complex with respect to potassium iodide quenching experiments and competition displacement assays with ethidium bromide, acridine orange, and Hoechst 33258 probes suggested that LAF interacted with ctDNA by minor groove mode. Molecular docking analysis further supported the minor groove binding. Molecular dynamics simulation indicated that LAF depart from the C-G region of DNA, but it can steadily bind with the middle part of DNA composed by A-T base pairs.

Identification of N-phenyl-2-(N-phenylphenylsulfonamido)acetamides as new RORγ inverse agonists: Virtual screening, structure-based optimization, and biological evaluation.

Retinoic acid receptor-related orphan receptors (RORs) are ligand-dependent transcriptional factors and members of the nuclear receptor superfamily. RORs regulate inflammation, metabolic disorders and circadian rhythm. RORγ is a promising therapeutic drug target for treating Th17-mediated autoimmune diseases. In our study, we performed structure-based virtual screening and ligand-based virtual screening targeting the RORγ ligand-binding domain and successfully identified N-phenyl-2-(N-phenylphenylsulfonamido) acetamides as a type of RORγ inverse agonist. Among the 28 purchased compounds, C11 was confirmed to be active with micromolar IC50 values in both an AlphaScreen assay (62.58 µM) and a cell-based reporter gene assay (4.54 µM). Structure-guided optimization of the compound C11 led to the identification of compound 39, which significantly enhanced RORγ inhibition with an IC50 value of 630 nM. The RORγ antagonism of 39 was 7-fold higher than that of hit compound C11. These results represent a promising starting point for developing potent small molecule RORγ inverse agonists for the treatment of autoimmune diseases, such as rheumatoid arthritis, psoriasis, and multiple sclerosis.

Discovery, synthesis and biological evaluation of 2-(4-(N-phenethylsulfamoyl)phenoxy)acetamides (SAPAs) as novel sphingomyelin synthase 1 inhibitors.

Sphingomyelin synthase (SMS) has been proved to be a potential drug target for the treatment of atherosclerosis. However, few SMS inhibitors have been reported. In this paper, structure-based virtual screening was performed on hSMS1. SAPA 1a was discovered as a novel SMS1 inhibitor with an IC50 value of 5.2 µM in enzymatic assay. A series of 2-(4-(N-phenethylsulfamoyl)phenoxy)acetamides (SAPAs) were synthesized and their biological activities toward SMS1 were evaluated. Among them, SAPA 1j was found to be the most potent SMS1 inhibitor with an IC50 value of 2.1 µM in in vitro assay. The molecular docking studies suggested the interaction modes of SMS1 inhibitors and PC with the active site of SMS1. Site-directed mutagenesis validated the involvement of residues Arg342 and Tyr338 in enzymatic sphingomyelin production. The discovery of SAPA derivatives as a novel class of SMS1 inhibitors would advance the development of more effective SMS1 inhibitors.

Selective melatonin MT2 receptor ligands relieve neuropathic pain through modulation of brainstem descending antinociceptive pathways.

Neuropathic pain is an important public health problem for which only a few treatments are available. Preclinical studies show that melatonin (MLT), a neurohormone acting on MT1 and MT2 receptors, has analgesic properties, likely through MT2 receptors. Here, we determined the effects of the novel selective MLT MT2 receptor partial agonist N-{2-([3-bromophenyl]-4-fluorophenylamino)ethyl}acetamide (UCM924) in 2 neuropathic pain models in rats and examined its supraspinal mechanism of action. In rat L5-L6 spinal nerve ligation and spared nerve injury models, UCM924 (20-40 mg/kg, subcutaneously) produced a prolonged antinociceptive effect that is : (1) dose-dependent and blocked by the selective MT2 receptor antagonist 4-phenyl-2-propionamidotetralin, (2) superior to a high dose of MLT (150 mg/kg) and comparable with gabapentin (100 mg/kg), but (3) without noticeable motor coordination impairments in the rotarod test. Using double staining immunohistochemistry, we found that MT2 receptors are expressed by glutamatergic neurons in the rostral ventrolateral periaqueductal gray. Using in vivo electrophysiology combined with tail flick, we observed that microinjection of UCM924 into the ventrolateral periaqueductal gray decreased tail flick responses, depressed the firing activity of ON cells, and activated the firing of OFF cells; all effects were MT2 receptor-dependent. Altogether, these data demonstrate that selective MT2 receptor partial agonists have analgesic properties through modulation of brainstem descending antinociceptive pathways, and MT2 receptors may represent a novel target in the treatment of neuropathic pain.

[Functional activity of the liver in immersion and effects of the countermeasures].

Two groups of male volunteers for 4-day dry immersion with and w/o countermeasures (support load imitator (SLI) or high-frequency electrostimulator) underwent ultrasonic investigation (USI) of the liver, gastroduodenal organs and vessels, and blood biochemical analysis. Two other groups of volunteers performed the 13C-methacetin breath test (13C-MBT) to study the effects of immersion and SLI on the liver detox activity and metabolic capacity. In immersion, USI diagnosed slowdown of blood flow along the hepatic vein and signs of plethora in the abdominal venous system. In addition, immersion was accompanied by increases in blood pepsinogen, pancreatic amylase, total bilirubin, the "indirect" fraction specifically, insulin and C-peptide. 13C-MBT detected deceleration of 13C-methacetin inactivation and diminution of the liver metabolic capacity. Administration of the countermeasures did not improve the ultrasonic image of hemodynamic alterations in the liver and abdomen significantly. High-frequency electrostimulation cancelled out changes in all biochemical parameters except C-peptide; SLI was favorable to recovery of pepsinogen and amylase baseline values only. Besides, the SLI wearing prevented loss of the 13C-methacetin inactivation rate but was not effective enough against diminution of the hepatic metabolic capacity.

Discovery of potent transient receptor potential vanilloid 1 antagonists: design and synthesis of phenoxyacetamide derivatives.

We aimed to discover a novel type of transient receptor potential vanilloid 1 (TRPV1) antagonist because such antagonists are possible drug candidates for treating various disorders. We modified the structure of hit compound 7 (human TRPV1 IC50=411 nM) and converted its pyrrolidino group to a (hydroxyethyl)methylamino group, which substantially improved inhibitory activity (15d; human TRPV1 IC50=33 nM). In addition, 15d ameliorated bladder overactivity in rats in vivo.

AnhE, a metallochaperone involved in the maturation of a cobalt-dependent nitrile hydratase.

Acetonitrile hydratase (ANHase) of Rhodococcus jostii RHA1 is a cobalt-containing enzyme with no significant sequence identity with characterized nitrile hydratases. The ANHase structural genes anhA and anhB are separated by anhE, predicted to encode an 11.1-kDa polypeptide. An anhE deletion mutant did not grow on acetonitrile but grew on acetamide, the ANHase reaction product. Growth on acetonitrile was restored by providing anhE in trans. AnhA could be used to assemble ANHase in vitro, provided the growth medium was supplemented with 50 microM CoCl(2). Ten- to 100-fold less CoCl(2) sufficed when anhE was co-expressed with anhA. Moreover, AnhA contained more cobalt when produced in cells containing AnhE. Chromatographic analyses revealed that AnhE existed as a monomer-dimer equilibrium (100 mm phosphate, pH 7.0, 25 degrees C). Divalent metal ions including Co(2+), Cu(2+), Zn(2+), and Ni(2+) stabilized the dimer. Isothermal titration calorimetry studies demonstrated that AnhE binds two half-equivalents of Co(2+) with K(d) of 0.12 +/- 0.06 nM and 110 +/- 35 nM, respectively. By contrast, AnhE bound only one half-equivalent of Zn(2+) (K(d) = 11 +/- 2 nM) and Ni(2+) (K(d) = 49 +/- 17 nM) and did not detectably bind Cu(2+). Substitution of the sole histidine residue did not affect Co(2+) binding. Holo-AnhE had a weak absorption band at 490 nM (epsilon = 9.7 +/- 0.1 m(-1) cm(-1)), consistent with hexacoordinate cobalt. The data support a model in which AnhE acts as a dimeric metallochaperone to deliver cobalt to ANHase. This study provides insight into the maturation of NHases and metallochaperone function.

A comparative autoradiography study in post mortem whole hemisphere human brain slices taken from Alzheimer patients and age-matched controls using two radiolabelled DAA1106 analogues with high affinity to the peripheral benzodiazepine receptor (PBR) system.

The binding of two radiolabelled analogues (N-(5-[125I]Iodo-2-phenoxyphenyl)-N-(2,5-dimethoxybenzyl)acetamide ([125I]desfluoro-DAA1106) and N-(5-[125I]Fluoro-2-phenoxyphenyl)-N-(2-[125I]Iodo-5-methoxybenzyl)acetamide ([125I]desmethoxy-DAA1106) of the peripheral benzodiazepine receptor (PBR) (or TSPO, 18kDa translocator protein) ligand DAA1106 was examined by in vitro autoradiography on human post mortem whole hemisphere brain slices obtained from Alzheimer's disease (AD) patients and age-matched controls. Both [(125)I]desfluoro-IDAA1106 and [(125)I]desmethoxy-IDAA1106 were effectively binding to various brain structures. The binding could be blocked by the unlabelled ligand as well as by other PBR specific ligands. With both radiolabelled compounds, the binding showed regional inhomogeneity and the specific binding values proved to be the highest in the hippocampus, temporal and parietal cortex, the basal ganglia and thalamus in the AD brains. Compared with age-matched control brains, specific binding in several brain structures (temporal and parietal lobes, thalamus and white matter) in Alzheimer brains was significantly higher, indicating that the radioligands can effectively label-activated microglia and the up-regulated PBR/TSPO system in AD. Complementary immunohistochemical studies demonstrated reactive microglia activation in the AD brain tissue and indicated that increased ligand binding coincides with increased regional microglia activation due to neuroinflammation. These investigations yield further support to the PBR/TSPO binding capacity of DAA1106 in human brain tissue, demonstrate the effective usefulness of its radio-iodinated analogues as imaging biomarkers in post mortem human studies, and indicate that its radiolabelled analogues, labelled with short half-time bioisotopes, can serve as prospective in vivo imaging biomarkers of activated microglia and the up-regulated PBR/TSPO system in the human brain.

Method to screen substrates of apical sodium-dependent bile acid transporter.

Human apical sodium-dependent bile acid transporter (hASBT) is a potential prodrug target under study. Development of prodrugs that target hASBT may yield compounds with low solubility and/or susceptibility to hydrolysis. A transport uptake method is needed that increases compound solubility and avoids NaOH for cell lysis during postexperimental cell sample preparation. The overall goal was to develop an assay method to screen for hASBT uptake of novel compounds. The first objective was to determine the maximum cosolvent concentrations that are compatible with an hASBT active transport assay. The second objective was to develop a NaOH-free cell lysis method to process cell samples from these uptake studies. The following cosolvents were studied: dimethylacetamide (DMAC), dimethylformamide (DMF), dimethylsulfoxide (DMSO), ethanol, methanol, polyethylene glycol-400, propylene glycol, and dioxane. Initial studies included taurocholate flux studies across hASBT-Madin-Darby canine kidney monolayers using up to 10% cosolvent, as well as cytotoxicity studies. The effect of selected cosolvent concentrations on the hASBT Michaelis-Menten kinetic parameters was evaluated. Additionally, two acetonitrile-based cell lysis methods that do not use NaOH were evaluated in terms of percent sample recovery and hASBT kinetic parameters. Results showed that the maximum permissible cosolvent concentrations for hASBT uptake studies, without compromising assay results or causing cytotoxicity, are 1% DMAC, 1% DMF, 2.5% DMSO, 2.5% methanol, and 2.5% ethanol. Additionally, both NaOH-free, acetonitrile-based cell lysis methods provided similar recovery and hASBT results, compared to NaOH method. Hence, an assay method was developed to screen for active transport, allowing for cosolvents that can solubilize compounds and avoid NaOH sample treatment, which can otherwise degrade compound.

Altered spectrum of multidrug resistance associated with a single point mutation in the Escherichia coli RND-type MDR efflux pump YhiV (MdtF).

OBJECTIVES: YhiV (MdtF) is an resistance nodulation division (RND) type efflux pump in Escherichia coli with significant homology to AcrB but usually expressed at a low level in clinical isolates. When overexpressed the pump confers decreased susceptibility to a variety of substances including erythromycin and ethidium bromide (EtBr). We characterized two mutants of E. coli E12 (DeltaacrB DeltaacrF) overexpressing yhiV that showed surprising differences in their spectrum of multidrug resistance (MDR). METHODS: The two mutants obtained after repeated exposure of E. coli E12 to levofloxacin were tested for antimicrobial susceptibility to a variety of agents and for intracellular accumulation of selected pump substrates. Gene expression was studied by quantitative RT-PCR, and yhiV was sequenced. Gene inactivation and replacement were done by phage lambda-based homologous recombination. RESULTS: Mutant DKO20/1 overexpressed yhiV, showed a wild-type yhiV sequence and had >2-fold increased MICs of fluoroquinolones, novobiocin, macrolides/ketolides, EtBr, oxacillin and Phe-Arg-beta-naphthylamide (PAbetaN, a putative efflux pump inhibitor) compared with the E12 parent. A second mutant, strain DKO1/17 that had the Val-610-->Phe point mutation in YhiV differed from DKO20/1 by faster growth, >2-fold increased MICs of linezolid and tetracycline, but >2-fold decreased MICs of PAbetaN, azithromycin and telithromycin. Inactivation of yhiV in DKO1/17 and reintroduction of the wild-type and mutant yhiV sequence confirmed that the differing MICs of most of the drugs were associated with the observed single point mutation. Intracellular drug accumulation studies with linezolid and PAbetaN were consistent with the MIC results. CONCLUSIONS: The region around amino acid Val-610 in YhiV appears to be involved in determining recognition and efficiency of export of a number of MDR efflux pump substrates. This single point mutation in the periplasmic loop of the pump can increase resistance to a given drug such as a fluoroquinolone while decreasing resistance to another one.

Agomelatine targets a range of major depressive disorder symptoms.

Servier, and US licensee Novartis AG, are developing the oral melatonin MT1 and MT2 agonist and 5-HT2B and 5-HT2C antagonist agomelatine as a once-daily treatment for major depressive disorder (MDD) and its symptoms, particularly anxiety, and sleep and circadian disturbances. Phase III trials have been completed and a registration dossier has been submitted to the EMEA in Western Europe.

Influence of autochthonous dissolved organic carbon and nutrient limitation on alachlor biotransformation in aerobic aquatic systems.

Much work has suggested that the rate of attenuation of water-soluble organic contaminants in aerobic aquatic systems is dependent on the level of secondary nutrients in the water column. For example, the decay rate of alachlor, a common herbicide, was over 10 times higher under hypereutrophic compared with oligotrophic water conditions. It has been presumed that higher water column nutrient levels produce larger microbial communities, resulting in higher rates of alachlor cometabolism. However, most earlier field studies only assessed alachlor fate in systems with full light exposure (FLE). Therefore, new experiments were performed to assess how variations in light level affect alachlor cometabolism in such systems. Twelve tank mesocosms were maintained using identical nitrogen (N) and phosphorus (P) supply conditions: four units with full light exposure (100% FLE), four with partial shading (19.3% FLE), and four with near complete shading (0.5% FLE). Alachlor half-lives were found to vary broadly, from 50 to 60 days in higher light units to > 180 days in the 0.5% FLE units. Nutrient analysis indicated that the low light units were severely carbon (C)-limited for microbial decomposition, whereas the other units had excess C relative to N and P. Apparently, reduced light levels cause decreased production of bioavailable C for decomposition, which significantly reduces alachlor cometabolism. The data suggest that water column nutrient levels only correlate with the alachlor decay rate when light levels are high, and that the biodegradable carbon supply must be considered when the fate of water-soluble contaminants in aerobic aquatic systems is assessed.

Detection of resistance to fungicides, mating types and fitness of Phytophthora infestans in Hebei, China.

In vitro, isolates resistant to metalaxyl (M) and oxadixyl (O) of Phytophthora infestans were 11.2% of 62 isolates from potato and tomato in Hebei Province, mean resistance factor was 15,022 fold and 24,733 fold, respectively, no isolates resistant either to cymoxanil (C) or to dimethomorph (D) were detected. On the other hand, in vivo, isolates resistant to M and O were 29.0% and 32.7%, respectively, among 217 isolates from potato plants in Weichang and Chongli, Hebei, only one isolate resistant to M and O was found among 88 isolates from tomato plants in Xushui, Hebei. Among 73 isolates from potato in Weichang and Chongli, 6 isolates with A2 mating type were 8.2%, 3 A2 isolates resistant to metalaxyl and oxadixyl. 3 A2 isolates were 6.8% of 44 isolates from tomato in Xushui. Cross resistance in Phytophthora infestans was confirmed between M and O, but no cross resistance between C or D and M or O. The field isolates resistant to M and O were outstandingly fitter than sensitive or intermediate ones, mainly due to stronger sporulation capacity of the field isolates.