Amino ozonides exhibit in vitro activity against Echinococcus multilocularis metacestodes.

Artemisinin is an antimalarial sesquiterpene lactone that contains a 1,2,4-trioxane heterocycle. Dihydroartemisinin and artesunate demonstrated activity against Echinococcus multilocularis metacestodes in vitro but were not effective in a mouse model. In this study, the in vitro effects of a small library of synthetic ozonides (1,2,4-trioxolanes) were investigated. Initial compound screening against E. multilocularis metacestodes was performed at 20μM, and selected ozonides were further assessed in dose-response studies in metacestode cultures and mammalian cells. Transmission electron microscopy (TEM) was employed to characterise compound-induced structural alterations. At 20μM, the most potent ozonides (OZ401, OZ455, OZ491 and OZ494) led to death of ca. 60-100% of the parasites. Subsequent dose-response experiments demonstrated that OZ401, OZ455 and OZ491, which contain an aminopropylether substructure, were the most potent, with 50% inhibitory concentrations ranging from 11μM to 14μM. Cytotoxicity for these three ozonides, assessed in human foreskin fibroblasts, rat hepatoma cells and green monkey epithelial kidney (Vero) cells, was evident only at high concentrations. TEM demonstrated that OZ401 and OZ491 treatment induced considerable metabolic impairment in metacestodes at 1 day post exposure. At Day 3 post exposure, the germinal layer was severely distorted, although some intact cells were still visible, demonstrating that not all cell types in the parasite tissue were equally affected. Complete destruction of the germinal layer was noted at 5 days post exposure. Synthetic ozonides could represent interesting leads that will be further investigated in a suitable in vivo model of E. multilocularis infection.

Biosynthetic pathways to isocyanides and isothiocyanates; precursor incorporation studies on terpene metabolites in the tropical marine sponges Amphimedon terpenensis and Axinyssa n.sp.

The biosynthetic origins of the isocyanide and isothiocyanate functional groups in the marine sponge metabolites diisocyanoadociane (1), 9-isocyanopupukeanane (10) and 9- isothiocyanatopupukeanane (11) are probed by the use of [C-14]-labelled precursor experiments. Incubation of the sponge Amphimedon terpenensis with [C-14]-labelled thiocyanate resulted in radioactive diisocyanoadociane ( 1) in which the radiolabel is specifically associated with the isocyanide carbons. As expected, cyanide and thiocyanate were confirmed as precursors to the pupukeananes 10 and 11 in the sponge Axinyssa n. sp.; additionally these precursors labelled 2-thiocyanatoneopupukeanane ( 12) in this sponge. To probe whether isocyanide-isothiocyanate interconversions take place at the secondary metabolite level, the advanced precursor bisisothiocyanate 17 was supplied to A. terpenensis, but did not result in significant labelling in the natural product isocyanide 1. In contrast, in the sponge Axinyssa n. sp., feeding of [C-14]-9-isocyanopupukeanane (10) resulted in isolation of radiolabelled 9- isothiocyanatopupukeanane 11, while the feeding of [C-14]-11 resulted in labelled isocyanide 10. These results show conclusively that isocyanides and isothiocyanates are interconverted in the sponge Axinyssa n. sp., and confirm the central role that thiocyanate occupies in the terpene metabolism of this sponge.

Factors that influence the prevalence of acaricide resistance and tick-borne diseases

This manuscript provides a summary of the results presented at a symposium organized to accumulate information on factors that influence the prevalence of acaricide resistance and tick-borne diseases. This symposium was part of the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP), held in New Orleans, LA, USA, during August 10-14, 2003. Populations of southern cattle ticks, Boophilus microplus, from Mexico have developed resistance to many classes of acaricide including chlorinated hydrocarbons (DDT), pyrethroids, organ ophosphates, and formamidines (amitraz). Target site mutations are the most common resistance mechanism observed, but there are examples of metabolic mechanisms. In many pyrethroid resistant strains, a single target site mutation on the Na+ channel confers very high resistance (resistance ratios: >1000x) to both DDT and all pyrethroid acaricides. Acetylcholine esterase affinity for OPs is changed in resistant tick populations. A second mechanism of OP resistance is linked to cytochrome P450 monooxygenase activity. A PCR-based assay to detect a specific sodium channel gene mutation that is associated with resistance to permethrin has been developed. This assay can be performed on individual ticks at any life stage with results available in a few hours. A number of Mexican strains of B. microplus with varying profiles of pesticide resistance have been genotyped using this test. Additionally, a specific metabolic esterase with permethrin-hydrolyzing activity, CzEst9, has been purified and its gene coding region cloned. This esterase has been associated with high resistance to permethrin in one Mexican tick population. Work is continuing to clone specific acetylcholinesterase (AChE) and carboxylesterase genes that appear to be involved in resistance to organophosphates. Our ultimate goal is the design of a battery of DNA- or ELISA-based assays capable of rapidly genotyping individual ticks to obtain a comprehensive profile of their susceptibility to various pesticides. More outbreaks of clinical bovine babesisois and anaplasmosis have been associated with the presence of synthetic pyrethroid (SP) resistance when compared to OP and amidine resistance. This may be the result of differences in the temporal and geographic patterns of resistance development to the different acaricides. If acaricide resistance develops slowly, herd immunity may not be affected. The use of pesticides for the control of pests of cattle other than ticks can affect the incidence of tick resistance and tick-borne diseases. Simple analytical models of tick- and tsetse-bome diseases suggest that reducing the abundance of ticks, by treating cattle with pyrethroids for example, can have a variety of effects on tick-bome diseases. In the worst-case scenario, the models suggest that treating cattle might not only have no impact on trypanosomosis but could increase the incidence of tick-bome disease. In the best-case, treatment could reduce the incidence of both trypanosomosis and tick-bome diseases Surveys of beef and dairy properties in Queensland for which tick resistance to amitraz was known were intended to provide a clear understanding of the economic and management consequences resistance had on their properties. Farmers continued to use amitraz as the major acaricide for tick control after the diagnosis of resistance, although it was supplemented with moxidectin (dairy farms) or fluazuron, macrocyclic lactones or cypermethrin/ chlorfenvinphos. (C) 2004 Published by Elsevier B.V.

Sub-inhibitory concentrations of ceftazidime and tobramycin reduce the quorum sensing signals of Pseudomonas aeruginosa

Aim: Concentrations of antimicrobials below minimum inhibitory concentration (subMIC) may reduce the production by Pseudomonas aeruginosa of virulence factors such as elastase. We sought to determine whether the reduction in elastase production may be mediated by a reduction in acyl-homoserine lactones. Methods: Pseudomonas aeruginosa in broth was exposed to three conditions for ceftazidime and tobramycin: control, 6% MIC and 25% MIC. Elastase was assayed using elastin congo red. N-(3-Oxododecanoyl)-homoserine lactone (C12-HSL) and N-butyryl-homoserine lactone (C4-HSL) were assayed using biosensor Escherichia coli. Results: Elastase was unchanged with ceftazidime. Elastase was reduced by 16% at 6% MIC tobramycin and reduced by 70% at 25% MIC tobramycin (P

Essential oil composition of diploid and tetraploid clones of ginger (Zingiber officinale roscoe) grown in Australia

Ginger oil, obtained by steam distillation of the rhizome of Zingiber officinale Roscoe, is used in the beverage and fragrance industries. Ginger oil displays considerable compositional diversity, but is typically characterized by a high content of sesquiterpene hydrocarbons, including zingiberene, arcurcumene, beta-bisabolene, and beta-sesquiphellandrene. Australian ginger oil has a reputation for possessing a particular lemony aroma, due to its high content of the isomers neral and geranial, often collectively referred to as citral. Fresh rhizomes of 17 clones of Australian ginger, including commercial cultivars and experimental tetraploid clones, were steam distilled 7 weeks post-harvest, and the resulting oils were analyzed by GC-MS. The essential oils of 16 of the 17 clones, including the tetraploid clones and their parent cultivar, were found to be of substantially similar composition. These oils were characterized by very high citral levels (51-71%) and relatively low levels of the sesquiterpene hydrocarbons typical of ginger oil. The citral levels of most of these oils exceeded those previously reported for ginger oils. The neral-to-geranial ratio was shown to be remarkably constant (0.61 +/- 0.01) across all 17 clones. One clone, the cultivar Jamaican, yielded oil with a substantially different composition, lower citral content and higher levels of sesquiterpene hydrocarbons. Because this cultivar also contains significantly higher concentrations of pungent gingerols, it possesses unique aroma and flavor characteristics, which should be of commercial interest.

Environmental degradation of polyethylene-based plastics

The criteria involved in the degradation of polyethylene-based degradable polymer samples have been investigated, with a view to obtaining a clearer mechanism of photo-biodegradation. The compatibility of degradable polymer samples during materials recycling was also studied. Commercial and laboratory prepared degradable polymer samples were oxidised in different environments and the oxidation products formed were studied using various analytical chromatographic and spectroscopic techniques such as HPLC, FT-IR and NMR. It was found that commercial degradable polymer samples which are based on the ECO systems, degrade predominantly via the Norrish II process, whereas the other degradable systems studied (starch-filled polyethylene systems, transition metal systems, including metal carboxylate based polyethylene systems and the photoantioxidant-activator systems) photodegrade essentially via the Norrish I process. In all cases, the major photoxidation products extracted from the degradable polymer samples were found to be carboxylic acids, although, in the polymer itself a mixture of carbonyl containing products such as esters, lactones, ketones and aldehydes was observed. The study also found that the formation of these hydrophilic carbonyl products causes surface swelling of the polymer, thus making bioerosion possible. It was thus concluded that environmental degradation of LDPE is a two step process, the initiation stage being oxidation of the polymer which gives rise to bioassimilable products, which are consequently bioeroded in the second stage, (the biodegradation step). Recycling of the degradable polymer samples as 10% homogeneous and heterogeneous blends was carried out using a single screw extruder (180°C and 210°C) and an internal mixer (190°C). The study showed that commercial degradable polymer samples may be recycled with a minimal loss in their properties.

Absorption and metabolism of chlorogenic acids in cultured gastric epithelial monolayers

Gastric absorption of feruloylquinic acid and di-O-caffeoylquinic acid analogs has never been investigated despite their potential contribution to the proposed beneficial health effects leading to reduced risk of type 2 diabetes. Using a cultured gastric epithelial model, with an acidic apical pH, the relative permeability coefficients (P(app)) and metabolic fate of a series of chlorogenic acids (CGAs) were investigated. Mechanistic studies were performed in the apical to basal direction and demonstrated differential rates of absorption for different CGA subgroups. For the first time, we show intact absorption of feruloylquinic acids and caffeoylquinic acid lactones across the gastric epithelium (P(app) ~ 0.2 cm/s). Transport seemed to be mainly by passive diffusion, because good linearity was observed over the incubation period and test concentrations, and we speculate that a potential carrier-mediated component may be involved in uptake of certain 4-acyl CGA isomers. In contrast, absorption of intact di-O-caffeoylquinic acids was rapid (P(app) ~ 2-10 cm/s) but nonlinear with respect to time and concentration dependence, which was potentially limited by interaction with an efflux transporter and/or pH gradient dependence. For the first time, methylation is shown in gastric mucosa. Furthermore, isoferulic acid, dimethoxycinnamic acid, and ferulic acid were identified as novel gastric metabolites of CGA biotransformation. We propose that the stomach is the first location for the release of hydroxycinnamic acids, which could explain their early detection after coffee consumption.

Identification and evaluation of agents isolated from traditionally used herbs against Ophiophagus hannah venom

The aim of this study was firstly to identify active molecules in herbs, that are traditionally used for the treatment of snake bite, such as Curcuma antinaia, Curcuma contravenenum, Andrographis paniculata, and Tanacetum parthenium; secondly to test similar structurally related molecules and finally to prepare and evaluate an efficient formulation against Ophiophagus hannah venom intoxification. Three labdane based compounds, including labdane dialdehyde, labdane lactone, and labdane trialdehyde and two lactones including 14-deoxy-11,12-didehydroandrographolide and parthenolide were isolated by column chromatography and characterised. Using the isolated rat phrenic nerve-hemidiaphragm preparation, the antagonistic effect of crude extracts, isolated compounds and prepared formulations were measured in vitro on the inhibition of the neuromuscular transmission. Inhibition on muscle contraction, produced by the 5 μg/mL venom, was reversed by test agents in organ bath preparations. A labdane trialdehyde, isolated from C. contravenenum, was identified as the best antagonising agent in the low micromolar range. Tests on formulations of the most potent C. contravenenum extract showed, that the suppository with witepsol H15 was an effective medicine against O. hannah venom. This study elucidated the active compounds, accounting for the antivenin activity of traditionally used herbs and suggested the most suitable formulation, which may help to develop potent medicines for the treatment of snake bite in the future.

Ecological genetics of Melaleuca quinquenervia (Myrtaceae): Population variation in Florida and its influence on performance of the biological control agent Oxyops vitiosa (Coleoptera: Curculionidae)

Melaleuca quinquenervia (Cav.) Blake (Myrtaceae) was imported into Florida from Australia over a century ago as a landscape plant. A favorable climate and periodic wildfires helped M. quinquenervia thrive; it now occupies about 200,000 hectares in southern Florida. A biological control (i.e., biocontrol) program against M. quinquenervia has been initiated, but not all biocontrol releases are successful. Some scientists have argued that poor biocontrol agent success may relate to genetic differences among populations of invasive weeds. I tested this premise by determining (1) the number and origins of M. quinquenervia introductions into Florida, (2) whether multiple introduction events resulted in the partitioning of Florida's M. quinquenervia populations into discrete biotypes, and (3) whether Oxyops vitiosa, an Australia snout beetle imported to control this weed, might discriminate among putative M. quinquenervia biotypes. Careful scrutiny of early horticultural catalogs and USDA plant introduction records suggested at least six distinct introduction events. Allozyme analyses indicated that the pattern of these introductions, and the subsequent redistribution of progeny, has resulted in geographic structuring of the populations in southern Florida. For example, trees on Florida's Gulf Coast had a greater effective number of alleles and exhibited greater heterozygosity than trees on the Atlantic Coast. Essential oil yields from M. quinquenervia leaves followed a similar trend; Gulf Coast trees yielded nearly twice as much oil as Atlantic Coast trees when both were grown in a common garden. These differences were partially explained by the predominance of a chemical phenotype (chemotype) very rich in the sesquiterpene (E)-nerolidol in M. quinquenervia trees from the Gulf Coast, but rich in a mixture of the monoterpene 1,8-cineole and the sesquiterpene viridiflorol in trees from the Atlantic Coast. Performance of O. vitiosa differed dramatically in laboratory studies depending on the chemotype of the foliage they were fed. Larval survivorship was four-fold greater on the (E)-nerolidol chemotype. Growth was also greater, with adult O. vitiosa gaining nearly 50% more biomass on the (E)-nerolidol plants than on the second chemotype. The results of this study thus confirmed the premise that plant genotype can affect the population dynamics of insects released as weed biocontrols. ^

The 4-aza-S-ribosyl-L-homocysteine derivatives and the related gamma-lactam and azahemiacetal analogs: Synthesis, inhibition and quorum sensing activity

Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence, critical for establishing infection. There are two major pathways of QS systems. Type 1 is species specific or intra-species communication in which N-acylhomoserine lactones (Gram-negative bacteria) or oligopeptides (Gram-positive bacteria) are employed as signaling molecules (autoinducer one). Type 2 is inter-species communication in which S-4,5-dihydroxy-2,3-pentanedione (DPD) or its borate esters are used as signaling molecules. The DPD is biosynthesized by LuxS enzyme from S-ribosylhomocysteine (SRH). Recent increase in prevalence of bacterial strains resistant to antibiotics emphasizes the need for the development of new generation of antibacterial agents. Interruption of QS by small molecules is one of the viable options as it does not affect bacterial growth but only virulence, leading to less incidence of microbial resistance. Thus, in this work, inhibitors of both N-acylhomoserine lactone (AHL) mediated intra-species and LuxS enzyme, involved in inter-species QS are targeted. The γ-lactam and their reduced cyclic azahemiacetal analogs, bearing the additional alkylthiomethyl substituent, were designed and synthesized targeting AHL mediated QS systems in P. aeruginosa and Vibrio harveyi. The γ-lactams with nonylthio or dodecylthio chains acted as inhibitors of las signaling in P. aeruginosa with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio substituent were found to strongly inhibit both las and rhl signaling in P. aeruginosa at higher concentrations. However, lactam and their azahemiacetal analogs were found to be inactive in V. harveyi QS systems. The 4-aza-S-ribosyl-L-homocysteine (4-aza-SRH) analogs and 2-deoxy-2-substituted-S-ribosyl-L-homocysteine analogs were designed and synthesized targeting Bacillus subtilis LuxS enzyme. The 4-aza-SRH analogs in which oxygen in ribose ring is replaced by nitrogen were further modified at anomeric position to produce pyrrolidine, lactam, nitrone, imine and hemiaminal analogs. Pyrrolidine and lactam analogs which lack anomeric hydroxyl, acted as competitive inhibitors of LuxS enzyme with KI value of 49 and 37 µM respectively. The 2,3-dideoxy lactam analogs were devoid of activity. Such findings attested the significance of hydroxyl groups for LuxS binding and activity. Hemiaminal analog of SRH was found to be a time-dependent inhibitor with IC50 value of 60 µM.

The Effect of Natural Products and Small Gtpases in Adipogenesis

Insulin signaling is one of the main initiators of adipogenesis, the conversion from pre-adipocyte to adipocyte or lipid droplet. Rab proteins are the master regulator of intracellular trafficking and endosome fusion in endocytosis, making them potential regulators of insulin signaling in adipogenesis. Pre-adipocytes 3T3-Ll cells expressing several Rab5 constructs were used to examine the effect of dehydroleucodine (DhL ), a sesquiterpene lactone isolated from aerial parts of Artemisia douglasiana Besser. The results obtained identify Rab5 deactivation as a key step for adipogenesis by forming signaling endosomes. The addition of DhL significantly inhibited the lipid droplet accumulation in a dose-dependent manner and dramatically attenuated the synthesis of adipogenic transcriptional factors, C/EBPa and PPARy. Activation of AMPKa, Erk and Akt during adipocytic differentiation was not inhibited by treatment with DhL. This data suggest that DhL has an important role in Rab5 dependent adipogenesis by regulating several transcriptional factors including PP ARy expression, which is known to play an essential role during fat formation.

Design and synthesis of S-ribosylhomocysteine analogues

Bacteria are known to release a large variety of small molecules known as autoinducers (AI) which effect quorum sensing (QS) initiation. The interruption of QS effects bacterial communication, growth and virulence. ^ Three novel classes of S-ribosylhomocysteine (SRH) analogues as potential inhibitors of S-ribosylhomocysteinase (LuxS enzyme) and AI-2 modulators of QS were developed. The synthesis of 2-deoxy-2-bromo-SRH analogues was attempted by coupling of the corresponding 2-bromo-2-deoxypentafuranosyl precursors with the homocysteinate anion. The displacement of the bromide from C2 rather than the expected substitution of the mesylate from C5 was observed. The synthesis of 4-C-alkyl/aryl-S-ribosylhomocysteine analogues involved the following steps: (i) conversion of the D-ribose to the ribitol-4-ulose; (ii) diastereoselective addition of various alkyl or aryl or vinyl Grignard reagents to 4-ketone intermediate; (iii) oxidation of the primary hydroxyl group at C1 followed by the intramolecular ring closure to the corresponding 4-C-alkyl/aryl-substituted ribono-1,4-lactones; (iv) displacement of the activated 5-hydroxyl group with the protected homocysteinate. Treatment of the 4-C-alkyl/aryl-substituted SRH analogues with lithium triethylborohydride effected reduction of the ribonolactone to the ribose (hemiacetal) and subsequent global deprotection with trifluoroacetic acid provided 4-C-alkyl/aryl-SRHs. ^ The 4-[thia]-SRH were prepared from the 1-deoxy-4-thioribose through the coupling of the &agr;-fluoro thioethers (thioribosyl fluorides) with homocysteinate anion. The 4-[thia]-SRH analogues showed concentration dependent effect on the growth on las (50% inhibitory effect at 200 µg/mL). The most active was 1-deoxy-4-[thia]-SRH analogue with sufur atom in the ring oxidized to sulfoxide decreasing las gene activity to approximately 35% without affecting rhl gene. Neither of the tested compounds had effect on bioluminescence nor on total growth of V. harveyi, but had however slight inhibition of the QS.^

Characterization of the Anti-Quorum Sensing Activity Exhibited by Marine Macro algae of South Florida

Quorum sensing is a communication mechanism employed by many bacteria. The bacteria secrete signal molecules known as acyl homoseriene lactones (AHLs) that cue to population size/density. Bacteria can be alerted of this optimum population by the concentration of these signal molecules. When the concentration of AHLs exceed a threshold valve, they enter the bacterial cell and causes the transcription of genes encoding virulence factors necessary for their colonization and survival. The marine algae Delise a pulchra, found off the coast of Australia is thought to produce compounds that inhibit the activity of the AHLs. The algae employ these compounds, known as furanones, as an anti-fouling agent. We postulated that marine algae of South Florida might contain similar activity; we screened 30 different algal species and found 22 species had the activity. Algal extracts were made from Halimeda incrassata using hexane, chloroform, ethyl acetate and methanol as solvents. The extracts were assayed for anti-quorum sensing activity. The results showed many of the South Florida green algae to possess anti-quorum sensing activity, however extracts of H incrassata did not show quorum sensing inhibition.

Exceptional activity of gallium(III) chloride and chlorogallate(III) ionic liquids for Baeyer–Villiger oxidation

Baeyer–Villiger oxidation of cyclic ketones, using H2O2 as the oxidising agent, was systematically studied using a range of metal chlorides in different solvents, and in neat chlorogallate(III) ionic liquids. The extremely high activity of GaCl3 in promoting oxidation with H2O2, irrespective of solvent, was reported for the first time. The activity of all other metal chlorides was strongly solvent-dependent. In particular, AlCl3 was very active in a protic solvent (ethanol), and tin chlorides, SnCl4 and SnCl2, were active in aprotic solvents (toluene and dioxane). In order to eliminate the need for volatile organic solvent, a Lewis acidic chlorogallate(III) ionic liquid was used in the place of GaCl3, which afforded typically 89–94% yields of lactones in 1–120 min, at ambient conditions. Raman and 71Ga NMR spectroscopic studies suggest that the active species, in both GaCl3 and chlorogallate(III) ionic liquid systems, are chlorohydroxygallate(III) anions, [GaCl3OH]−, which are the products of partial hydrolysis of GaCl3 and chlorogallate(III) anions; therefore, the presence of water is crucial.

Catalytic reactions with palladium supported on mesocellular foam : Applications in hydrogenation, isomerization, and C-C bond forming reactions

The major part of this thesis concerns the development of catalytic methodologies based on palladium nanoparticles immobilized on aminopropyl-functionalized siliceous mesocellular foam (Pd0-AmP-MCF). The catalytic activity of the precursor to the nanocatalyst, PdII-AmP-MCF is also covered by this work. In the first part the application of Pd0-AmP-MCF in Suzuki-Miyaura cross-coupling reactions and transfer hydrogenation of alkenes under microwave irradiation is described. Excellent reactivity was observed and a broad range of substrates were tolerated for both transformations. The Pd0-AmP-MCF exhibited high recyclability as well as low metal leaching in both cases. The aim of the second part was to evaluate the catalytic efficiency of the closely related PdII-AmP-MCF for cycloisomerization of various acetylenic acids. The catalyst was able to promote formation of lactones under mild conditions using catalyst loadings of 0.3 - 0.5 mol% at temperatures of up to 50 oC in the presence of Et3N. By adding 1,4-benzoquinone to the reaction, the catalyst could be recycled four times without any observable decrease in the activity. The selective arylation of indoles at the C-2 position using Pd-AmP-MCF and symmetric diaryliodonium salts is presented in the third part. These studies revealed that Pd0-AmP-MCF was more effective than PdII-AmP-MCF for this transformation. Variously substituted indoles as well as diaryliodonium salts were tolerated, giving arylated indoles in high yields within 15 h at 20 - 50 oC in H2O. Only very small amounts of Pd leaching were observed and in this case the catalyst exhibited moderate recyclability. The final part of the thesis describes the selective hydrogenation of the C=C in different α,β-unsaturated systems. The double bond was efficiently hydrogenated in high yields both under batch and continuous-flow conditions. High recyclability and low metal leaching were observed in both cases.