Antifeedant C20 diterpene alkaloids

We have tested the insect antifeedant and toxic activity of 21 C20 diterpenoid alkaloids on Spodoptera littoralis and Leptinotarsa decemlineata. The antifeedant effects of the test compounds were structure- and species-dependent. The most active antifeedants to L. decemlineata and S. littoralis were the rearranged form of hetisine (20; EC50 = 1.7 microg/cm2) and 19-oxodihydroatisine (9; EC50 = 0.1 microg/cm2), resp. Glandulosine (8) moderately affected orally injected S. littoralis larvae. A few compounds (13-oxocardiopetamine (4), 9, and atisinium chloride (13)) had cytotoxic effects to insect-derived Sf9 cells with varying degrees of selectivity with respect to mammalian CHO cells. Compounds 4 and 15,22-O-diacetyl-19-oxodihydroatisine (10) increased Trypanosoma cruzi mortality. Our results support the plant protective role of C20 diterpenoid alkaloids and open a new field for parasite control strategies.     

Antifeedant activity of some polygodial derivatives

Polygodial (1) and its derivatives acetal 2 (propylene) and 3 (ethylene) were prepared and their antifeedant activity and toxic effects evaluated on several insect species with different feeding ecologies (Spodoptera littoralis, Leptinotarsa decemlineata, Myzus persicae and Rhopalosiphum padi) along with that of polygonone (4). We also tested their selective cytotoxic effects on insect-derived (Spodoptera frugiperda ovarian Sf9 cells) and mammalian Chinese hamster ovary (CHO) cells. The antifeedant activity of these compounds was consistent with the proposed mode of action for antifeedant drimanes, i. e. adduct formation with amino groups for M. persicae and R. padi (dialdehyde > ketoaldehyde > aldehydeacetal). This was not the case for L. decemlineata, and the cytotoxic effects on insect-derived Sf9 and mammalian CHO cells (aldehydeacetal > dialdehyde > ketoaldehyde).     

Bioactive triterpene derivatives from latex of two Euphorbia species

We have investigated the antifeedant and toxic effects of 23 semisynthetic terpenoid derivatives obtained through chemical modifications of the major components of Euphorbia resinifera (alpha-euphol and alpha-euphorbol) and E. officinarum (obtusifoliol and 31-norlanostenol) latex on several insect species (Spodoptera littoralis, Myzus persicae and Rhopalosiphum padi), their selective cytotoxicity on insect Sf9 and mammalian CHO cells and their phytotoxic effects on Lactuca sativa. The conversions focused mainly on positions 3,7,11, and 24 with several oxidizing agents. A total of 18 compounds affected S. littoralis growth (IGR). Our results support the importance of the C-3 substituent, suggest the involvement of the C-7 substituent and indicate that the C-3 hydroxyl is not essential for the IGR effect. Overall, Sf9 cells were more sensitive to the active compounds than CHO cells. All of these compounds had non selective moderate phytotoxic effects on radicle elongation of L. sativa.     

Antifeedant/insecticidal terpenes from asteraceae and labiatae species native to Argentinean semi-arid lands

To validate the potential as added-value resources of Asteraceae and Labiatae species of Argentinean semi-arid lands, we have selected 13 of their major terpenoids belonging to several chemical classes and tested their insect antifeedant and toxic activity on the herbivorous insects Spodoptera littoralis and Leptinotarsa decemlineata. The antifeedant effects of the test compounds were structure- and species-dependent. The most active antifeedant to L. decemlineata was the eudesmane sesquiterpene gamma-costic acid (13), followed by the labdane diterpene 2alpha,3alpha-dihydroxycativic acid (8), the clerodane diterpenes 6-acetylteucjaponin B (5), bacchotricuneatin A (1), bartemidiolide (7), butanolide (4), and the sesquiterpenes ilicic acid (11) and tessaric acid (10) (eudesmane and eremophilane type, respectively). S. littoralis was only affected by the clerodanes and showed the strongest response to salviarin (3) and 5, followed by hawtriwaic acid (6) and 12-epi-bacchotricuneatin A (2). Orally injected S. littoralis larvae were negatively affected by 5. Most of the diterpenes had selective cytotoxic effects to insect-derived Sf9 cells with the clerodane 1 being the most active, followed by the eudesmane costic acid (12), the only cytotoxic sesquiterpene. None of these compounds was cytotoxic to mammalian CHO cells.     

The locus of inhibition of NADH oxidation by benzothiadiazoles in beef heart submitochondrial particles

6-Chloro-1,2,3-benzothiadiazole (6-Cl-BTD) is an effective inhibitor of NADH oxidase (site I) but not of succinate oxidase in beef heart submitochondrial particles. For NADH oxidase activity maximal inhibition (80-85%) was achieved at 0.75mM 6-Cl-BTD. A similar level of inhibition was also observed (half maximal inhibitory concentration 0.5mM) towards NADH-duroquinone reductase; NADH-juglone reductase was slightly inhibited (23%) at 0.5mM 6-Cl-BTD while NADH-ferricyanide reductase was unaffected. The data suggest that 6-Cl-BTD interacts with an electron transport site on the oxygen side of NADH dehydrogenase and inhibitory studies with 6-Cl-BTD and rotenone indicate that it might correspond with one of the two sites affected by rotenone. The substituted 1,2,3-benzothiadiazoles (BTDs) are perhaps best known for their activity as inhibitors of cytochrome P-450-mediated mixed-function oxidation (MFO). In vitro, the BTDs are potent inhibitors of MFO activities in microsomes from mammalian liver and insect tissues and they have been demonstrated to inhibit aminopyrine metabolism in perfused rat liver. In vivo, they reportedly prolong hexobarbital sleeping time in mice, inhibit the irreversible binding of labeled trichloro-ethylene to microsomal protein and effectively enhance the toxicity (synergize) of pyrethrin, organophosphorus-containing and carbamate insecticides to insects.     

On the mechanism of inhibition of NADH oxidase by ubiquinone-3

The combined effects of rotenone and ubiquinone-3 on the kinetics of NADH dehydrogenase and NADH oxidase have been investigated. The two inhibitors do not show additivity; on the other hand, ubiquinone-3, when preincubated with the enzyme, partially removes rotenone sensitivity. The inhibition of NADH oxidase by ubiquinone-3 is the result of at least two combined effects: the competition of the less active ubiquinone-3 with endogenous ubiquinone-10 in the acceptor site of the dehydrogenase, and a nonspecific action on the structure of complex I. The latter effect is perhaps mediated by a physical change of the phospholipid bilayer similar to that observed with agents such as butanol, perturbing lipid-protein interactions in the membrane.     

5-(2'-oxoheptadecyl)-resorcinol and 5-(2'-oxononadecyl)-resorcinol,cytotoxic metabolites from a wood-inhabiting basidiomycete

5-(2'-oxoheptadecyl)-resorcinol [structure: see text] and 5-(2'-oxononadecyl)-resorcinol [structure: see text] were isolated from fermentations of an imperfect basidiomycete. The structures of the compounds were determined by spectroscopic techniques. Both compounds exhibit cytotoxic effects against the human colon tumor cell lines COLO-320, DLD-1 and HT-29 and the human promyeloid leukemia cell line HL-60, the human leukemia T cell JURKAT, the human hepatocellular carcinoma cell line HEP-G2 as well as the J774 mouse macrophage cell line. The compounds induce morphological and physiological differentiation of HL-60 cells into granulocytes, which subsequently die by apoptosis. Both compounds show no antibacterial and antifungal activity.     

Triterpene-based plant defenses

Pentacyclic triterpenes are abundant in the plant kingdom and have a wide array of pharmacological activities. They also have insect antifeedant effects and therefore apparently play a role in plant defense. In this paper, we describe the insecticidal activity of pentacyclic triterpenes of plant origin from different chemical classes on several insect pests (Spodoptera littoralis, Leptinotarsa decemlineata and Myzus persicae), their phytotoxic properties and their selective cytotoxic effects on insect-derived Sf9 and mammalian CHO cells. We also discuss the role they play in plant defense based on these activities.     

Oxidation of NADH in a Coupled Oxidase-Peroxidase Reaction and its Significance for the Fermentation in Rumen Protozoa of the Genus Isotricha

SYNOPSIS. Cell-free extracts of the anaerobic rumen ciliate Isotricha prostoma possess a strong NADH oxidase activity. Evidence for H₂O₂ as an intermediary product during oxidation of NADH has been obtained. Gatalase activity could not be demonstrated but hydrogen peroxide is removed by a rate limiting NAD peroxidase.
In addition to oxygen several other compounds such as ferricyanide, cytochrome c , menadione and certain dyes may function as electron acceptors during oxidation of NADH. The ferricyanide reductase activity in the Isotricha extracts strongly resembles that of the mitochondrial enzyme from mammalian sources in a number of characteristics.
Partial inhibition of NADH oxidase activity was obtained with the following chelating agents: hydroxylamine, diethyl dithiocarbamate, 2,9-dimethyl-1,10-phenanthroline (DMPH), and 2-thenoyl trifluoroacetone, whereas citrate, tartrate, pyrophosphate, salicylaldoxime, EDTA and 8-hydroxyquinoline had no effect. The peroxidase was blocked completely by 0.42 mM DMPH and this inhibitor was used to block the enzyme in whole cells in experiments on oxygen toxicity. The oxidase was largely insensitive to azide, KCN, and uncouplers. Antimycin A and rotenone caused a partial inhibition of the oxidase when added in very high concentrations. ATP formation occurred during oxidation of NADH, and P/O ratios were 0.1–0.35. Addition of small amounts of oxygen to intact ciliates led to a decrease in the production of hydrogen and butyrate, while the production of acetate was increased and no change in the lactate formation was seen. This shift in fermentation end-products possibly is caused by a competition of oxygen for NADH.     

Limits to the rate of adaptive substitution in sexual populations

In large populations, many beneficial mutations may be simultaneously available and may compete with one another, slowing adaptation. By finding the probability of fixation of a favorable allele in a simple model of a haploid sexual population, we find limits to the rate of adaptive substitution, [Formula: see text], that depend on simple parameter combinations. When variance in fitness is low and linkage is loose, the baseline rate of substitution is [Formula: see text], where [Formula: see text] is the population size, [Formula: see text] is the rate of beneficial mutations per genome, and [Formula: see text] is their mean selective advantage. Heritable variance [Formula: see text] in log fitness due to unlinked loci reduces [Formula: see text] by [Formula: see text] under polygamy and [Formula: see text] under monogamy. With a linear genetic map of length [Formula: see text] Morgans, interference is yet stronger. We use a scaling argument to show that the density of adaptive substitutions depends on [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text] only through the baseline density: [Formula: see text]. Under the approximation that the interference due to different sweeps adds up, we show that [Formula: see text], implying that interference prevents the rate of adaptive substitution from exceeding one per centimorgan per 200 generations. Simulations and numerical calculations confirm the scaling argument and confirm the additive approximation for [Formula: see text]; for higher [Formula: see text], the rate of adaptation grows above [Formula: see text], but only very slowly. We also consider the effect of sweeps on neutral diversity and show that, while even occasional sweeps can greatly reduce neutral diversity, this effect saturates as sweeps become more common-diversity can be maintained even in populations experiencing very strong interference. Our results indicate that for some organisms the rate of adaptive substitution may be primarily recombination-limited, depending only weakly on the mutation supply and the strength of selection.     

Inhibition of bovine heart mitochondrial and Paracoccus denitrificans NADH----ubiquinone reductase by dequalinium chloride and three structurally related quinolinium compounds

1. Dequalinium chloride (DECA) and three related quinolinium compounds inhibit bovine heart mitochondrial and Paracoccus denitrificans electron transport activity, with inhibition localized between NADH and ubiquinone in both electron transport chains. 2. Structure-activity studies reveal that two quinolinium rings and a long bridging group are necessary for significant inhibition of reduction of artificial electron acceptors and coenzyme Q, whereas only one quinolinium ring and a long hydrocarbon side chain are required for significant inhibition of NADH oxidase activity. 3. Inhibition of coenzyme Q reduction by DECA is not reversed by dialysis. 4. Studies comparing DECA inhibition of rotenone-sensitive with rotenone-insensitive preparations indicate that DECA acts by a different inhibitory mechanism than rotenone on mammalian mitochondrial and P. denitrificans NADH----ubiquinone reductase.     

Changes in plasma membrane properties and phosphatidylcholine subspecies of insect Sf9 cells due to expression of scavenger receptor class B, type I, and CD36

In mammalian cells scavenger receptor class B, type I (SR-BI), mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester into hepatic and steroidogenic cells. In addition, SR-BI has a variety of effects on plasma membrane properties including stimulation of the bidirectional flux of free cholesterol (FC) between cells and HDL and changes in the organization of plasma membrane FC as indicated by increased susceptibility to exogenous cholesterol oxidase. Recent studies in SR-BI-deficient mice and in SR-BI-expressing Sf9 insect cells showed that SR-BI has significant effects on plasma membrane ultrastructure. The present study was designed to test the range of SR-BI effects in Sf9 insect cells that typically have very low cholesterol content and a different phospholipid profile compared with mammalian cells. The results showed that, as in mammalian cells, SR-BI expression increased HDL cholesteryl ester selective uptake, cellular cholesterol mass, FC efflux to HDL, and the sensitivity of membrane FC to cholesterol oxidase. These activities were diminished or absent upon expression of the related scavenger receptor CD36. Thus, SR-BI has fundamental effects on cholesterol flux and membrane properties that occur in cells of evolutionarily divergent origins. Profiling of phospholipid species by electrospray ionization mass spectrometry showed that scavenger receptor expression led to the accumulation of phosphatidylcholine species with longer mono- or polyunsaturated acyl chains. These changes would be expected to decrease phosphatidylcholine/cholesterol interactions and thereby enhance cholesterol desorption from the membrane. Scavenger receptor-mediated changes in membrane phosphatidylcholine may contribute to the increased flux of cholesterol and other lipids elicited by these receptors.     

Oxidation of Reduced Nicotinamide Adenine Dinucleotide Phosphate by Potato Mitochondria: INHIBITION BY SULFHYDRYL REAGENTS

Potato tuber mitochondria oxidized exogenous NADH and exogenous NADPH at similar rates; the electron transfer inhibitor rotenone did not inhibit the oxidation of either substrate. Submitochondrial particles, prepared from potato tuber mitochondria, exhibited a greater capacity to oxidize NADH than NADPH; rotenone inhibited the oxidation of NADH by 29% and the oxidation of NADPH by 16%. The oxidation of both NADH and NADPH by potato mitochondria exhibited pH optima of 6.8, and although substantial NADH oxidase activity was observed at pH 8.0, little NADPH oxidase activity was detected at that pH. The oxidation of NADPH by the mitochondria was more sensitive to inhibition by EDTA than was the oxidation of NADH.     

Fracture Faces in the Cell Envelope of Escherichia coli

Freeze-fracturing of Escherichia coli cells in the presence of 30% (v/v) glycerol resulted in a double cleavage of the cell envelope exposing two convex and two concave fracture faces ([Formula: see text], [Formula: see text] and [Formula: see text], [Formula: see text]) with characteristic patterns. Complementary replicas revealed the relationship of the fracture faces to their corresponding fracture planes. The inner fracture plane splits the plasma membrane at one particular level. Apparently the outer fracture plane was located in the outer part of the wall, as it was separated by a layer ([Formula: see text]) from the fractured profile (CW1) presumably corresponding to the murein layer. The outer fracture plane did alternate toward the cell periphery, exposing complementary smooth areas ([Formula: see text] and [Formula: see text]). When cells were freeze-fractured in the absence of glycerol, the outer cell surface appeared as an etching face rather than a fracture face. A schematic representation of the relative location of the different fracture faces in the E. coli cell envelope is given.     

Cytotoxic effects of β‐asarone on Sf9 insect cells

β‐Asarone is the predominant component of the essential oil of rhizomes of Acorus calamus Linn ( Sweet flag). Although rhizome extracts from this plant have long been used for insect pest control, their cytotoxic effects on insect cells are not well understood. In this study, we evaluated the potency of β‐asarone as a natural insecticide by using a Spodoptera frugiperda cell line (Sf9). To assess the cytotoxic effects of β‐asarone on Sf9 cells, we observed morphologic changes in treated cells and performed a cell proliferation assay and a DNA fragmentation assay. After 24 and 48 h of treatment with β‐asarone, the proliferation of the Sf9 cells was inhibited in a dose‐dependent manner, with IC₅₀ values of 0.558 mg/ml at 24 h and 0.253 mg/ml at 48 h. Morphologic changes in β‐asarone‐treated cells were typical of apoptosis and included loss of adhesion, cell shrinkage, and small apoptotic bodies. The DNA laddering present in β‐asarone‐treated SF9 cells and annexin V assay confirmed that this compound can induce apoptosis in insect cells. Together, these findings suggest that apoptosis induction may be one mechanism through which β‐asarone inhibits the proliferation of insect cells and thus exerts insecticidal effects.     

Mammalian-like nonsialyl complex-type N-glycosylation of equine gonadotropins in Mimic insect cells

Recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) was expressed in Mimic insect cells, that are commercial stably transformed Spodoptera frugiperda (Sf9) cells expressing five mammalian genes encoding glycosyltransferases involved in the synthesis of complex-type monosialylated N-glycans. We previously showed that it exhibited no in vivo bioactivity although expressing full in vitro bioactivity, and it was suspected that this was because of insufficient sialylation of eLH/CG N-glycans. Lectin binding analyses were performed with recombinant dimeric eLH/CG or its alpha subunit, secreted in the serum-containing supernatant of infected Sf9 and Mimic cells. Two types of specific lectin affinity assays (blot analyses and enzyme-linked immunosorbent assay) were used to compare the ability or inability of natural and recombinant gonadotropins to bind to various lectins. In natural equine chorionic gonadotropin (eCG), complex-type N-glycans terminating with both Siaalpha2,3Gal (based on Maackia amurensis agglutinin [MAA] binding) and Siaalpha2,6Gal (based on Sambucus nigra agglutinin [SNA] binding) were found, but in the alpha subunit dissociated from natural eCG, we only detected Siaalpha2-6Gal. In eLH/CG and its alpha subunit produced by Sf9 cells, N-glycans were found to be terminated by mannosyl residues (based on Galanthus nivalis agglutinin [GNA] binding), whereas those produced in Mimic cells were terminated by galactoses (based on binding to Ricinus communis agglutinin I [RCA I] , but not to SNA or MAA). This is in agreement with the fact that the nucleotide donor substrate of sialic acid is not naturally synthesized in insect cells. On the basis of binding to Arachis Hypogaea agglutinin [PNA], O-glycans exhibited the Galbeta1-3GalNAc structure in recombinant-free alpha and eLH/CG from both Sf9 and Mimic cell lines. Both N- and O-linked carbohydrate side chains synthesized in Mimic cells should thus be amenable to further acellular sialylation.     

Kinetic characterization of mitochondrial complex I inhibitors using annonaceous acetogenins.

The NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial respiratory chain is by far the largest and most complicated of the proton-translocating enzymes involved in the oxidative phosphorylation. Many clues regarding the electron pathways from matrix NADH to membrane ubiquinone and the links of this process with the translocation of protons are highly controversial. Different types of inhibitors become valuable tools to dissect the electron and proton pathways of this complex enzyme. Therefore, further knowledge of the mode of action of complex I inhibitors is needed to understand the underlying mechanism of energy conservation. This study presents for the first time a detailed exploration of the inhibitory action of the Annonaceous acetogenins, the most powerful inhibitors of the mammalian enzyme, taking as the head-series rolliniastatin-1, rolliniastatin-2, and corossolin. Despite their close chemical resemblance, each of them inhibits the complex I with different kinetic features reflecting differential binding to the enzyme.     

Defensive Sesquiterpenes from Senecio candidans and S. magellanicus,and Their Structure?Activity Relationships

Eleven eremophilanolides, 1 – 3 and 6 – 13 , and two eremophilanes, 24 and 25 , were isolated from Senecio candidans and S. magellanicus from the Magallanes Region (Chile). Compounds 2, 3, 9 , and 10 have not been previously reported as natural products. Their structures were established by NMR spectroscopic analysis and chemical transformations. The X‐ray analysis of compounds 11, 13 , and 17 were also performed. Different semisynthetic analogs from eremophilanolide 11 were generated to carry out a structure? activity relationship study. Their possible plant defensive role was tested against herbivorous insects (Spodoptera littoralis, Rhopalosiphum padi, and Myzus persicae) and plants (Lactuca sativa). Additionally, their effects on insect (Sf9) and mammalian (CHO) cell lines were tested.     

Salinity-dependent copy number increase of a marine cyanobacterial endogenous plasmid

Listeria monocytogenes possessed glucose oxidase and NADH oxidase activities in whole cells and lysed protoplasts respectively. The NADH oxidase activity sedimented with the membrane fraction and was inhibited by the respiratory inhibitors rotenone, 2-heptyl-4-hydroxy-quinoline-N-oxide and cyanide, suggesting the presence of a membrane associated respiratory chain.     

Putting together a plasma membrane NADH oxidase: A tale of three laboratories

The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism.