The permeability of the midgut of three insects to cardiac glycosides

The uptake of a polar and nonpolar cardiac glycoside by three insects, Oncopeltus fasciatus, Schistocerca gregaria and Periplaneta americana was investigated. Of these insects, the midgut of only O. fasciatus was found to be permeable to cardiac glycosides. Ouabain was not metabolized by this insect and crossed the midgut slowly and passively. It was sequestered from the haemolymph into the dorsolateral spaces against a concentration gradient and at relatively fast rates suggesting that uptake from the gut is rate limiting. Digitoxin was metabolized at the level of the midgut but not in the isolated haemolymph or dorsolateral space fluids. Twenty-four hours after feeding O. fasciatus labelled digioxin, digitoxin metabolites but no unchanged digitoxin could be detected in the haemolymph while both metabolites and a small amount of unchanged digitoxin could be detected in the dorsolateral space fluids.     

Antigenic characterisation of cultured trypanosomes isolated from three species of fishes

Size profiles and antigenic comparisons were made of polypeptides from cultured fish trypanosomes. Twenty, 19 and 18 polypeptides (15–106 kD) were resolved from Trypanosoma phaleri, T. catostomi and Trypanosoma sp., respectively using SDS-PAGE and gel densitometry. Differences between species were also observed in the relative amount of a polypeptide (according to its molecular weight). Most polypeptides in the homologous (PS1 & PS2) and heterologous (LO1 & LO2) clones of T. phaleri were antigenically similar as demonstrated by Western blotting. However antigenicity of 70–75 kD polypeptides differed. In contrast, only a 100 kD polypeptide in Trypanosoma sp. and 85 kD polypeptides in both T. catostomi and Trypanosoma sp. appeared to be antigenically similar to those of T. phaleri. Examination of T. phaleri using the microscopic immuno-substrate-enzyme technique (MISET) suggested that antigenic differences were probably associated with surface antigens. Some limitations of SDS-PAGE and Western blotting as tools in systematics are discussed.     

Flavonoid glycosides from fiveCyathea species

The leaves of 5 fern species of the genusCyathea, i.e.C. fauriei, C. mertensiana, C. leichhardtiana, C. podophylla andC. hancockii, have been chemically analysed. The former 3 species have kaempferol 3-sophoroside (sophoraflavonoloside) and kaempferol 7-rhamnoglucoside as glycosidic components, and the latter 2 species contain kaempferol 3-galactoside (trifolin) and kaempferol 3-rhamnoglucoside (nicotiflorin). In addition, vitexin, orientin, kaempferol 3-glucoside (astragalin), kaempferol 3-rhamnoside (afzelin) and kaempferol 7-arabinoside are detected as common constituents in all the 5 species analysed.     

Cellular basis for the species differences in sensitivity to cardiac glycosides (digitalis)

The relative toxicity of numerous cardiotonic steroids (viz. ouabain, digitoxin, digoxin, convallatoxin, SC4453, bufalin, gitaloxin, digoxigenin, actodigin, oleandrin, digitoxigenin, gitoxin, strophanthidin, gitoxigenin, lanatosides A, B and C, alpha- and beta-acetyl digoxin, alpha- and beta-methyl digoxin) and related compounds towards a number of independent cell lines established from human, monkey, mouse, Syrian hamster, and Chinese hamster have been determined. All cardiac glycosides and their genins, as well as the cardiotonic alkaloid cassaine, exhibited greater than 100-fold higher toxicity towards cultured human and monkey cells in comparison to the cell lines of mouse, Syrian hamster, and Chinese hamster origins. These differences are species-related as all cell lines (both normal as well as transformed) from any one species, as well as cells from the closely related species (e.g., man and monkey or mouse, Chinese hamster, and Syrian hamster), showed similar sensitivity towards these drugs. The failure to see any significant differences in cellular toxicity for a larger number of other compounds which either bear limited structural resemblance to cardiac glycosides (viz. estradiol 17-beta-acetate, testosterone propionate, 21-acetoxy pregnenolone, beta-estradiol, digitonin, tigogenin, and tomatine) or interact with the Na+/K+ ATPase in a different manner (viz. veratridine, sanguinarine nitrate, penicillic acid, vanadium pentoxide, harmaline-HCI,5,5'-diphenyl hydantoin, quindonium bromide, and methyl quinolizinum bromide) provides strong evidence that the observed species-related differences are highly specific for cardiotonic steroids. Studies on the binding of [3H]ouabain show that, in comparison to human and monkey cell lines, no significant binding of the drug is observed in cells derived from the resistant species (i.e., mouse and Chinese hamster). The Na+/K+ ATPase from cells of the resistant species is inhibited at much higher concentrations of ouabain and digitoxin in comparison to the enzyme from human cells, and a good correlation is observed between these concentrations and those reported for inhibition of the enzyme from isolated heart muscles of the same species. These results provide strong evidence that the species-related differences in sensitivity to digitalis have a cellular basis and that the cultured cells from various mammalian species provide a useful model system for investigating the mechanism of action of cardiac glycosides.     

Field performance of micropropagated forestry species

Summary Micropropagated plants both from angiosperms (bamboo, birch, eucalyptus, tamarind, teak, willow) and gymnosperms (Douglas-fir, loblolly pine, Monterey pine, and redwood) have been established in the field. Plantlets were regenerated from juvenile explants (via adventitious or axillary buds) as well as explants from mature trees [apical and axillary buds (nodal segments)]. Plantlets regenerated from adventitious buds tend to show early maturation traits (Douglas fir, loblolly pine, Monterey pine). A population of selected clones showed superior performance and yield over seedlings derived from the same trees. Increased biomass production was obtained with plantlets derived from tissue culture ofEucalyptus spp. when compared to seedlings. No morphologic variation was observed in micropropagated plants. Plantlets derived from tissue culture grew very uniformly. Early flowering was observed with plantlets derived from tissue culture (tamarind, teak). Based on a presentation at an International Training Course on the Application of Biotechnology in Forest Trees held in Caracas, Venezuela, May 1991.     

The partial characterisation of endoproteases and exoproteases from three species of entomopathogenic entomophthorales and two species of deuteromycetes

The entomopathogenic entomophthoraceae (zygomycotina) Erynia rhizospora, Erynia dipterigena, and Erynia neoaphidis and the deuteromycete Aspergillus flavus produced single novel endoproteases (pI ca. 9) with activity against trypsin and chymotrypsin substrates. In contrast, the deuteromycete Paecilomyces farinosus produced a chymotrypsin (pI ca. 10).Inhibitor studies confirmed that the mixed activities (purified by isoelectric focusing) were derived from single endoproteases. The most potent inhibitor was Chicken ovoinhibitor. Little or no inhibition was observed for P. farinosus endoprotease by any of the chemicals tested.Although the different fungi possessed a broad spectrum of aminopeptidase activity, 3 species (E. rhizospora, E. dipterigena, and A. flavus) showed a preference for leucine at the N-terminal position and 2 species (E. neoaphidis and P. farinosus) showed maximal activity against arginine. Inhibitor studies confirmed these aminopeptidases as metallo-enzymes.     

Two chromone-secoiridoid glycosides and three indole alkaloid glycosides from Neonauclea sessilifolia

From the dried roots of Neonauclea sessilifolia, two new chromone-secoiridoid glycosides, sessilifoside and 7"-O-beta-D-glucopyranosylsessilifoside, and three novel indole alkaloid glycosides, neonaucleosides A, B, and C, were isolated along with the main known glycosides, 5-hydroxy-2-methylchromone-7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside, sweroside, loganin, grandifloroside, and quinovic acid 3 beta-O-beta-D-quinovopyranoside-28-O-beta-D-glucopyranoside. The structures of these new glycosides were determined by spectroscopic and chemical means. Neonaucleoside A and its C-3 epimer were prepared from secologanin and tryptamine.     

Encapsulation of micropropagated buds of six woody species

Regrowth after encapsulation in a sodium alginate matrix of micropropagated buds from six different in vitro proliferated woody species was evaluated. Actinidia deliciosa Liang & Ferguson (kiwifruit), Betula pendula Roth (birch), Crataegus oxyacantha L. (hawthorn), Malus spp. (apple), Rubus spp. (blackberry) and Rubus idaeus L. (raspberry) propagated in vitro were used as bud sources. Encapsulation with sodium alginate and subsequent regrowth on nutrient rich medium was compared to encapsulation with nutrient-enriched alginate capsules followed by regrowth on nutrientless medium. Apical and sub-apical buds of Malus (rootstock M. 27 and cultivar Starkspur Red) were also compared for encapsulation and regrowth ability. All species showed a regrowth after encapsulation, but only if cultured on enriched media. M.27 apical and sub-apical buds showed different regrowth ability after encapsulation with sodium alginate. Applicability of encapsulation of single micropropagated tree buds is discussed.     

Cytotoxic cardiac glycosides and coumarins from Antiaris toxicaria

Eight new cardiac glycosides/aglycones (antiaritoxiosides A–G, 1–7, and antiarotoxinin B, 8), two new coumarins (anticarins A–B, 41–42), and two new flavanones (antiarones L–K, 43–44) were isolated from trunk bark of Antiaris toxicaria together with 53 known compounds. The new structures were established by extensive analysis of spectroscopic data. Compound 1 (10-carboxy and 3α-hydroxy) and compounds 3–6 (10-hydroxy) contain unique substituents that are rarely found in cardiac glycosides. The cytotoxic effects of isolated compounds against ten human cancer cell lines, KB, KB-VIN, A549, MCF-7, U-87-MG, PC-3, 1A9, CAKI-1, HCT-9 and S-KMEL-2, were tested using the sulforhodamine B assay. Five compounds (12, 16, 20, 22, and 31) showed significant cytotoxicity against all ten cancer cell lines, with notable potency at the ng/mL level against some cell lines, which merits further development as clinical trial candidates.     

Sensitivities of three bumblebee species to four pesticides applied commonly in greenhouses in China

Abstract  This study was conducted to evaluate the effects of four pesticides (Mosplian, Kingbo, Score and Lvrtong) applied commonly in greenhouses in China, on three bumblebee species ( Bombus hypocrita , Bombus ignitus and Bombus patagiatus ). The study used a contact experiment and oral toxicity LD₅₀ values. The results showed that the mortality for B. hypocrita after contacting the four pesticides was significantly lower than B. patagiatus and B. ignitus , but there was no significant difference between B. patagiatus and B. ignitus . The oral toxicity median lethal dose (LD₅₀) value of Mosplian to B. hypocrita (0.0028 μg active ingredient/bee) was significantly higher than that to B. ignitus (0.0023 μg active ingredient/bee) and B. patagiatus (0.0021 μg active ingredient/bee). Of the bee species, it can be concluded that B. hypocrita was the least susceptible to the four pesticides. The mortality rates of each bumblebee species after contact with Mosplian were significantly higher than for the other three pesticides and the control group. For Kingbo, the rates were significantly higher than the control group, but Score and Lvrtong exposed groups showed no significant increase in mortality relative to the control group. It can therefore be concluded that the pesticides differ in their negative influences on bumblebees, and that Mosplian is the most harmful.     

Tyrosinase inhibitory activity of three new glycosides from Breynia fruticosa

Two new flavanone glycoside derivatives and one new sulfur-containing spiroacetal glycoside, (2R, 3R)-3-acetyl-7-methoxy-(−)-epicatechin 5-O-(6-isobutanoyl)-β-d-glucopyranoside (1), (2R, 3R)-3-acetyl-7-methoxy-(−)-epicatechin 5-O-[6-(2-methylbutanoyl)]-β-d-glucopyranoside (2) and 4-[(carboxymethyl)thio]-5′-hydroxy-phyllaemblic acid O-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside ester (3), along with twelve known flavonoids and one known sulfur-containing spiroacetal glycoside, were isolated from Breynia fruticosa. Their structures were elucidated by the use of extensive spectroscopic methods (UV, IR, HR-ESI-MS, 1D and 2D NMR, and CD). The in vitro inhibition of tyrosinase activity by all of these compounds was also evaluated, and we concluded that the flavanol-containing 5-O- and 7-O-sugar moieties possessed more potent effects than the other compounds examined herein.     

Activation of cardiac ryanodine receptors by cardiac glycosides

This study investigated the effects of cardiac glycosides on single-channel activity of the cardiac sarcoplasmic reticulum (SR) Ca2+ release channels or ryanodine receptor (RyR2) channels and how this action might contribute to their inotropic and/or toxic actions. Heavy SR vesicles isolated from canine left ventricle were fused with artificial planar lipid bilayers to measure single RyR2 channel activity. Digoxin and actodigin increased single-channel activity at low concentrations normally associated with therapeutic plasma levels, yielding a 50% of maximal effect of approximately 0.2 nM for each agent. Channel activation by glycosides did not require MgATP and occurred only when digoxin was applied to the cytoplasmic side of the channel. Similar results were obtained in human RyR2 channels; however, neither the crude skeletal nor the purified cardiac channel was activated by glycosides. Channel activation was dependent on [Ca2+] on the luminal side of the bilayer with maximal stimulation occurring between 0.3 and 10 mM. Rat RyR2 channels were activated by digoxin only at 1 microM, consistent with the lower sensitivity to glycosides in rat heart. These results suggest a model in which RyR2 channel activation by digoxin occurs only when luminal [Ca2+] was increased above 300 microM (in the physiological range). Consequently, increasing SR load (by Na+ pump inhibition) serves to amplify SR release by promoting direct RyR2 channel activation via a luminal Ca2+-sensitive mechanism. This high-affinity effect of glycosides could contribute to increased SR Ca2+ release and might play a role in the inotropic and/or toxic actions of glycosides in vivo.     

Insensitivity of lepidopteran tissues to ouabain: Physiological mechanisms for protection from cardiac glycosides

Neuronal tissues from Manduca sexta, the tobacco hornworm, Hyalophora cecropia, the silkmoth and Danaus plexippus, the Monarch Butterfly, contain Na⁺K⁺-ATPase which is sensitive to cardiac glycoside (ouabain). The K_m for K⁺ stimulation of Na⁺K⁺-ATPase in M. sexta and D. plexippus is 2.2 mM and for Na⁺ stimulation in D. plexippus, 6.0 mM. In vitro ouabain concentrations of 1.0 × 10^?5 M and 5.0 × 10^?5 M in the presence of 7.5 mM K⁺ inhibited Na⁺K⁺-ATPase activity in H. cecropia and M. sexta by 50% respectively. Na⁺K⁺-ATPase from D. plexippus was approximately 300 times less sensitive. High concentrations (10^?3 M in haemolymph) of ouabain had no effect on M. sexta in vivo. This is largely explained by haemolymph K⁺ (>; 30 mM) antagonizing the binding of ouabain to Na⁺K⁺-ATPase. As demonstrated in vitro, 30 mM K⁺ totally protects Na⁺K⁺-ATPase from inhibition by 7.5 × 10^?3 M ouabain in D. plexippus and protects the enzyme by 65% in M. sexta. At least part of the physiological burden incurred in utilization of cardiac glycoside ingestion and storage for protection from predation, however, is probably related to the toxic effects of cardiac glycosides on neuronal Na⁺K⁺-ATPase.