Chlorophyll fluorescence as a marker for herbicide mechanisms of action

Photosynthesis is the single most important source of 02 and organic chemical energy necessary to support all non-autotrophic life forms. Plants compartmentalize this elaborate biochemical process within chloroplasts in order to safely harness the power of solar energy and convert it into usable chemical units. Stresses (biotic or abiotic) that challenge the integrity of the plant cell are likely to affect photosynthesis and alter chlorophyll fluorescence. A simple three-step assay was developed to test selected herbicides representative of the known herbicide mechanisms of action and a number of natural phytotoxins to determine their effect on photosynthesis as measured by chlorophyll fluorescence. The most active compounds were those interacting directly with photosynthesis (inhibitors of photosystem I and II), those inhibiting carotenoid synthesis, and those with mechanisms of action generating reactive oxygen species and lipid peroxidation (uncouplers and inhibitors of protoporphyrinogen oxidase). Other active compounds targeted lipids (very-long-chain fatty acid synthase and removal of cuticular waxes). Therefore, induced chlorophyll fluorescence is a good biomarker to help identify certain herbicide modes of action and their dependence on light for bioactivity. Published by Elsevier B.V.

Studies on the mode of cytotoxicity of imidazotetraziones

The irnidazotetrazinones are a novel group of anti tumour agents which have demonstrated good activity against a range of murine tumours and human xenografts. They possess a structure activity relationship similar to the anti tumour triazenes, with the chloroethyl (mitozolomide) and methyl (temozolomide) analogues being active antitumour agents, whilst the ethyl (CCRG 82019) and higher homologues are inactive. This thesiS attempts to elucidate the biological mechanisms responsible for the strict structure-activity relationship observed amongst the imidazotetrazinones. Mitozolomide is the only agent chemically capable of cross-linking DNA , which has been suggested to be responsible fo r the cytotoxicity of this group of agents. Only mitozolomide and ternozolornide Exhibit a marked ditferential toxicity towards the 0 -alkylguanine-DNA alkyltransferase deficient GM892A (Mer-) cell line rather than the proficient Raji cell line (Mer+). The rate of uptake of imidazotetrazinones into cells is similar for all three agents in both cell lines, and does not explain the differing sensitivities to these agents. The effect of drug treatment on the incorporation of precursors into macromolecules, and their pool sizes, was examined. Temozolomide administration was found to alter de novo protein synthesis in both GM892A and Raji cells. Flow cytometric analysis revealed that temozolomide and CCRG 82019 block cells in late S/G2/M phase of the cell cycle , similar to that observed with mitozolomide. The extent of reaction of all three drugs with isolated macromolecules and cellular macromolecules was determined, and differences found, with cellular repair processes influencing the number of alkyl lesions remaining bound to macromolecules. The specific bases formed in calf thymus DNA after treatment with either temozolornide and CCRG 82019 was measured, and it was found that the types and relative amounts of lesions formed, differed, as well as the total level of alkylation. Whereas DNA extracted from imidazotetrazinone treated cells is not affected in its ability to support RNA polymerase activity, an effect is observed on the ability to extract DNA polymerase from drug treated cells. This may suggest that the alkylated DNA must be in intact chromatin for the lesion to manifest its effects. Temozolomide and methyl methanesulphonate do got appear to act with a synergistic mode of action. The 0 -position of guanine is suspected to be a critical site for the action of these types of drugs.

Reactions of artemisinin and arteether with acid: Implications for stability and mode of antimalarial action

The currently accepted mechanism of trioxane antimalarial action involves generation of free radicals within or near susceptible sites probably arising from the production of distonic radical anions. An alternative mechanistic proposal involving the ionic scission of the peroxide group and consequent generation of a carbocation at C-4 has been suggested to account for antimalarial activity. We have investigated this latter mechanism using DFT (B3LYP/6-31+G* level) and established the preferred Lewis acid protonation sites (artemisinin O5a >> O4a approximate to O3a > O2a > O1a; arteether O4a >= O3a > O5b >> O2a > O1a; Figure 3) and the consequent decomposition pathways and hydrolysis sites. In neither molecule is protonation likely to occur on the peroxide bond O1-O2 and therefore lead to scission. Therefore, the alternative radical pathway remains the likeliest explanation for antimalarial action.

Mode of cembranoid action on embryonic muscle acetylcholine receptor

The mechanism of eupalmerin acetate (EUAC) actions on the embryonic muscle nicotinic acetylcholine receptor (nAChR) in BC3H-1 cells was studied by using whole-cell and single-channel patch-clamp current measurements. With whole-cell currents, EUAC did not act as an agonist on this receptor. Coapplication of 30 mu M EUAC with 50 mu M, 100 N, or 500 mu M carbamoylcholine (CCh) reversibly inhibited the current amplitude, whereas, with 20 mu M CCh, current was increased above control values in the presence of EUAC. EUAC concentration curves (0.01-40 N) obtained with 100 mu M and 500 mu M CCh displayed slope coefficients, n(H), significantly smaller than one, suggesting that EUAC bound to several sites with widely differing affinities on the receptor molecule. The apparent rate of receptor desensitization in the presence of EUAC and CCh was either slower than or equal to that obtained with CCh alone. The major finding from single-channel studies was that EUAC did not affect single-channel conductance or the ability of CCh to interact with the receptor. Instead, EUAC acted by increasing the channel closing rate constant. The results are not consistent with the competitive model for EUAC inhibition, with the sequential open-channel block model, or with inhibition by increased desensitization. The data are best accounted for by a model in which EUAC acts by closed-channel block at low concentrations, by positive modulation at intermediate concentrations, and by negative allosteric modulation of the open channel at high concentrations. (c) 2007 Wiley-Liss, Inc.

Xylan-Degrading Enzymes from the Thermophilic Fungus Humicola Zanuginosa (Griffon and Maublanc) Bunce: Action Pattern of Xylanase and beta-Glucosidase on Xylans, Xylooligomers and Arabinoxylooligomers

The mode of action of xylanase and beta-glucosidase purified from the culture filtrate of Humicola lanuginosa (Griffon and Maublanc) Bunce on the xylan extracted from sugarcane bagasse and on two commercially available larchwood and oat spelt xylans, on xylooligomers and on arabinoxylooligomers was studied. While larchwood and oat spelt xylans were hydrolyzed to the same extent in 24 h, sugarcane bagasse xylan was hydrolyzed to a lesser extent in the same period. It was found that the rate of hydrolysis of xylooligomers by xylanase increased with increase in chain length, while beta-glucosidase acted rather slowly on all the oligomers tested. Xylanase exhibited predominant ''endo'' action on xylooligomers attacking the xylan chain at random while beta-glucosidase had ''exo'' action, releasing one xylose residue at a time. On arabinoxylooligomers, however, xylanase exhibited ''exo'' action. Thus, it appears that the presence of the arabinose substituent has, in some way, rendered the terminal xylose-xylose linkage more susceptible to xylanase action. It was also observed that even after extensive hydrolysis with both the enzymes, substantial amounts of the parent arabinoxylooligomer remained unhydrolyzed together with the accumulation of arabinoxylobiose. It can therefore be concluded that the presence of the arabinose substituent in the xylan chain results in linkages that offer resistance to both xylanase and beta-glucosidase action.

Neurotrophins and neuronal plasticity in the action of antidepressants and morphine

Neuronal plasticity is a well characterized phenomenon in the developing and adult brain. It refers to capasity of a single neuron to modify morphology, synaptic connections and activity. Neuronal connections and capacity for plastic events are compromised in several pathological disorders, such as major depression. In addition, neuronal atrophy has been reported in depressive patients. Neurotrophins are a group of secretory proteins functionally classified as neuronal survival factors. Neurotrophins, especially brain derived neurotrophic factor (BDNF), have also been associated with promoting neuronal plasticity in dysfunctional neuronal networks. Chronic antidepressant treatment increases plastic events including neurogenesis and arborization and branching of neurites in distinct brain areas, such as the hippocampus. One suggested mode of action is where the antidepressants elevate the synaptic levels of BDNF thus further activating several signaling cascades via trkB-receptor. In our studies we have tried to clarify the mechanisms of action for antidepressants and to resolve the role of BDNF in this process. We found that chronic antidepressant treatment increases amount of markers of neuronal plasticity in both hippocampus and in the medial prefrontal cortex, both of which are closely linked to the etiology of major depression. Secondary actions of antidepressants include rapid activation of the trkB receptor followed by a phosphorylation of transcription factor CREB. In addition, activation of CREB by phosphorylation appears responsible for the regulation of the expression of the BDNF gene. Using transgenic mice we found that BDNF-induced trkB-mediated signaling proved crucial for the behavioral effects of antidepressants in the forced swimming test and for the survival of newly-born neurons in the adult hippocampus. Antidepressants not only increased neurogenesis in the adult hippocampus but also elevated the turnover of hippocampal neurons. During these studies we also discovered that another trkB ligand, NT-4, is involved in morphine-mediated anti-nociception and tolerance. These results present a novel role for trkB-mediated signaling in plastic events present in the opioid system. This thesis evaluates neuronal plasticity and trkB as a target for future antidepressant treatments.

The generalized anomeric effect in the 1,3-thiazolidines: Evidence forboth sulphur and nitrogen as electron donors. Crystal structures of various N-acylthiazolidines including mercury(II) complexes. Possible relevance to penicillin action

Evidence for the generalized anomeric effect (GAE) in the N-acyl-1,3-thiazolidines, an important structural motif in the penicillins, was sought in the crystal structures of N-(4-nitrobenzoyl)-1,3-thiazolidine and its (2:1) complex with mercuric chloride, N-acetyl-2-phenyl-1,3-thiazolidine, and the (2:1) complex of N-benzoyl-1,3-thiazolidine with mercuric bromide. An inverse relationship was generally observed between the. C-2-N and C-2-S bond lengths of the thiazolidine ring, supporting the existence of the GAE. (Maximal bond length changes were similar to 0.04 angstrom for C-2-N-3, S-1-C-2, and similar to 0.08 angstrom for N-3-C-6.) Comparison with N-acylpyrrolidines and tetrahydrothiophenes indicates that both the nitrogen-to-sulphur and sulphur-to-nitrogen GAE's operate simultaneously in the 1,3-thiazolidines, the former being dominant. (This is analogous to the normal and exo-anomeric effects in pyranoses, and also leads to an interesting application of Baldwin's rules.) The nitrogen-to-sulphur GAE is generally enhanced in the mercury(II) complexes (presumably via coordination at the sulphur); a 'competition' between the GAE and the amide resonance of the N-acyl moiety is apparent. There is evidence for a 'push-pull' charge transfer between the thiazolidine moieties in the mercury(II) complexes, and for a 'back-donation' of charge from the bromine atoms to the thiazolidine moieties in the HgBr2 complex. (The sulphur atom appears to be sp(2) hybridised in the mercury(II) complexes, possibly for stereoelectronic reasons.) These results are apparently relevant to the mode of action of the penicillins. (c) 2006 Elsevier B.V. All rights reserved.

Novel insights into the action of antimicrobial agents against human pathogens

Dissertation presented to obtain the Ph.D degree in Biochemistry

Integration of steroid hormone initiated membrane action to genomic function in the brain

Estrogen is a ligand for the estrogen receptor (ER), which on binding 17beta-estradiol, functions as a ligand-activated transcription factor and regulates the transcription of target genes. This is the slow genomic mode of action. However, rapid non-genomic actions of estrogen also exist at the cell membrane. Using a novel two-pulse paradigm in which the first pulse rapidly initiates non-genomic actions using a membrane-limited estrogen conjugate (E-BSA), while the second pulse promotes genomic transcription from a consensus estrogen response element (ERE), we have demonstrated that rapid actions of estrogen potentiate the slower transcriptional response from an ERE-reporter in neuroblastoma cells. Since rapid actions of estrogen activate kinases, we used selective inhibitors in the two-pulse paradigm to determine the intracellular signaling cascades important in such potentiation. Inhibition of protein kinase A (PKA), PKC, mitogen activated protein kinase (MAPK) or phosphatidylinositol 3-OH kinase (PI-3K) in the first pulse decreases potentiation of transcription. Also, our data with both dominant negative and constitutive mutants of Galpha subunits show that Galpha(q) initiates the rapid signaling cascade at the membrane in SK-N-BE(2)C neuroblastoma cells. We discuss two models of multiple kinase activation at the membrane Pulses of estrogen induce lordosis behavior in female rats. Infusion of E-BSA into the ventromedial hypothalamus followed by 17beta-estradiol in the second pulse could induce lordosis behavior, demonstrating the applicability of this paradigm in vivo. A model where non-genomic actions of estrogen couple to genomic actions unites both aspects of hormone action.

New Insight into the Mechanism of Action of Wasp Mastoparan Peptides: Lytic Activity and Clustering Observed with Giant Vesicles

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Dispersal of the delayed action insecticide sulfluramid in colonies of the leaf-cutting ant Atta sexdens rubropilosa (Hymenoptera: Formicidae)

Toxic baits are the most used control method for leaf-cutting ants due to their high effectiveness and because they are considered the safest for humans. Taking into account that the importance of leaf-cutting ants as pests, knowing the process by which dispersal and worker contamination is achieved becomes essential to understand several aspects about the functioning of a bait-borne AI (active ingredient) used in toxic baits. It has been established that an effective toxic bait should have a delayed- action AI, but its dispersion among the different sizes of workers is unknown. Workers of different sizes are involved in quite different tasks such foraging, cultivation of symbiotic macrofungus or control of deleterious microfungi. Therefore, we prepared a toxic bait containing the delayed-action AI sulfluramid and a dye (Rhodamine B) as an AI tracer in order to study dispersal and contamination in colonies, evaluated at different periods and in relation to different workers' sizes. Both field and laboratory colonies were evaluated. The great level of contamination, about 50% at 24 hours, in all sizes of workers demonstrates that worker contact with toxic bait is intense within this period. The distribution in field and laboratory colonies was similar. This contamination pattern is probably enough to cause the colony to die because of contamination of smaller workers, leading to the loss of control of the aggressive microfungi, which can quickly overgrow the symbiotic fungus culture. The dispersal dynamics of AI in leaf-cutting ant workers is important for investigations on the mode of action of this insecticide in the colony, and as a reference in future studies, such as those attempting to reduce the concentration of AIs in baits to reduce their environmental impact, or for facilitation of new AI screening.

Endocrine disrupter action in ruminants: A study of the effects of Ipomoea carnea in adult male goats

Ipomoea carnea is a toxic plant in Brazil and other tropical countries that often poisons livestock; its primary toxin is swainsonine. Some substances that interact with the endocrine system have been called endocrine disruptors (EDs). Considering swainsonine's mode of action, it is feasible to hypothesize that this compound could act as an ED. Bisphenol A (BPA), an estrogen-mimic, is considered a classical ED in rodents. This study aimed to evaluate the possible ED actions of I. cameo and BPA in male goats. Seventeen adult male goats were divided into three homogeneous groups: control (C, n = 5); IC (n = 6, received 5.0 g/kg body weight of freshly harvested I. cornea per day), and BPA (n = 6, received 25.0 mg/kg body weight of BPA per day). The experimental period was 120 days. During the experiment, blood samples were collected at 0, 30, 60, 90 and 120 days for biochemical and hormonal evaluations. On the same days, semen samples were collected for andrological evaluation, and scrotal circumference and testicular consistency were determined. The males were castrated on day 121, and fragments of testicle and epididymis were collected for histopathological evaluation. A decrease in serum T3 and T4 was observed in the IC group as well as an increase in the number of sperm with morphological alterations. In the BPA group, reduced serum 14 and a decreased percentage of sperm with plasma membrane integrates were observed. A histopathological examination revealed the vacuolar degeneration of Sertoli cells and areas of laminar patterns of calcium deposits in the IC group and vacuolar degeneration in the rete testis in the BPA group. These results indicate that both I. cameo and BPA are potential EDs in goats. This study emphasized the susceptibility of livestock to ED actions. We also demonstrated the action of I. cameo acting as EDs in the endocrine and reproductive systems. (C) 2014 Elsevier B.V. All rights reserved.

The use of mechanisms and modes of toxic action in integrated testing strategies: the report and recommendations of a workshop held as part of the European Union OSIRIS Integrated Project

This report on The Potential of Mode of Action (MoA) Information Derived from Non-testing and Screening Methodologies to Support Informed Hazard Assessment, resulted from a workshop organised within OSIRIS (Optimised Strategies for Risk Assessment of Industrial Chemicals through Integration of Non-test and Test Information), a project partly funded by the EU Commission within the Sixth Framework Programme. The workshop was held in Liverpool, UK, on 30 October 2008, with 35 attendees. The goal of the OSIRIS project is to develop integrated testing strategies (ITS) fit for use in the REACH system, that would enable a significant increase in the use of non-testing information for regulatory decision making, and thus minimise the need for animal testing. One way to improve the evaluation of chemicals may be through categorisation by way of mechanisms or modes of toxic action. Defining such groups can enhance read-across possibilities and priority settings for certain toxic modes or chemical structures responsible for these toxic modes. Overall, this may result in a reduction of in vivo testing on organisms, through combining available data on mode of action and a focus on the potentially most-toxic groups. In this report, the possibilities of a mechanistic approach to assist in and guide ITS are explored, and the differences between human health and environmental areas are summarised.

A novel site of antibiotic action in the ribosome: Interaction of evernimicin with the large ribosomal subunit

Evernimicin (Evn), an oligosaccharide antibiotic, interacts with the large ribosomal subunit and inhibits bacterial protein synthesis. RNA probing demonstrated that the drug protects a specific set of nucleotides in the loops of hairpins 89 and 91 of 23S rRNA in bacterial and archaeal ribosomes. Spontaneous Evn-resistant mutants of Halobacterium halobium contained mutations in hairpins 89 and 91 of 23S rRNA. In the ribosome tertiary structure, rRNA residues involved in interaction with the drug form a tight cluster that delineates the drug-binding site. Resistance mutations in the bacterial ribosomal protein L16, which is shown to be homologous to archaeal protein L10e, cluster to the same region as the rRNA mutations. The Evn-binding site overlaps with the binding site of initiation factor 2. Evn inhibits activity of initiation factor 2 in vitro, suggesting that the drug interferes with formation of the 70S initiation complex. The site of Evn binding and its mode of action are distinct from other ribosome-targeted antibiotics. This antibiotic target site can potentially be used for the development of new antibacterial drugs.

Chemical shoreline cleaning agents for oil spills : update state-of-the-art on mechanisms of action and factors influencing performance /

"EPA contract no. 68-C8-0062, Work assignment no. 3-48, SAIC project no. 01-0895-03-1000."