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Sturgeon spermatozoa maturation during their passage through the kidney is a prerequisite for initiation of motility. Samples of sterlet (Acipenser ruthenus) testicular sperm (TS) were matured in vitro by incubation in seminal fluid (SF) or in SF supplemented with carbonyl cyanide m-chlorophenyl hydrazone (CCCP; a respiration uncoupling agent). Sperm was diluted in activation medium (AM) containing 10 mMTris-HCl buffer (pH 8.5) and 0.25% Pluronic, and spermatozoon motility was assessed. Samples were taken and fixed in 3 Mperchloric acid at 3 points in the incubation process. Quantification of ATP, ADP, and creatine phosphate (CrP) was conducted using liquid chromatography/high-resolution mass spectrometry. We observed a significant decrease in CrP during artificial maturation of TS in SF. In contrast, ATP and ADP were not significantly affected. Addition of CCCP to SF halted maturation and led to significantly lower CrP whereas ADP significantly increased and ATP was unaffected. Dilution of matured and immature TS with AM led to a significant decrease of ATP and CrP and an increase of ADP compared with their levels before dilution, although immature TS were not motile. Energy dependency of TS maturation in sturgeon was confirmed, which suggests that mitochondrial oxidative phosphorylation is needed for maturation of sturgeon TS.

Lors d'une étude antérieure, nous avons montré que le maintien en mer des Saumons atlantiques mâles affecte la qualité du sperme. Pour tester l'hypothèse de l'influence néfaste d'une contamination par l'urine, nous avons comparé la qualité du sperme collecté par la technique classique (pression des flancs ou "stripping") à celle du sperme prélevé directement dans les canaux déférents, ceci chez des géniteurs, les uns gardés en mer et les autres transférés en eau douce un mois plus tôt. Alors que les spermes prélevés dans les canaux déférents des deux lots de mâles (eau douce : ED ; eau de mer : EM) ont un spermatocrite, une motilité, un contenu en ATP et une fécondance (en frais) peu différents, ces quatre paramètres sont significativement plus faibles pour les spermes obtenus par "stripping" des mâles en mer. L'analyse des données permet de conclure que certains de ces spermes ont été contaminés par l'urine et l'hypothèse est proposée que la teneur élevée en magnésium de l'urine a pu entraîner une activation des gamètes et une chute de leur ATP les rendant inféconds. En outre, il est apparu que l'aptitude à la congélation des spermes prélevés dans les canaux déférents est plus faible pour les mâles en mer que pour les mâles en eau douce. Les rapports molaires cholestérol/phospholipides des membranes des gamètes, susceptibles d'intervenir dans la résistance au froid, ont été mesurés. Aucune différence significative n'a été trouvée entre les spermes ED et EM.

Infection by the cestode Taenia taeniaeformis was investigated within numerous cyclic populations of the fossorial water vole Arvicola terrestris sampled during 4 years in Franche-Comté (France). The relative influence of different rodent demographic parameters on the presence of this cestode was assessed by considering (1) the demographic phase of the cycle; (2) density at the local geographical scale (<0·1 km2); (3) mean density at a larger scale (>10 km2). The local scale corresponded to the rodent population (intermediate host), while the large scale corresponded to the definitive host population (wild and feral cats). General linear models based on analyses of 1804 voles revealed the importance of local density but also of year, rodent age, season and interactions between year and season and between age and season. Prevalence was significantly higher in low vole densities than during local outbreaks. By contrast, the large geographical scale density and the demographic phase had less influence on infection by the cestode. The potential impacts of the cestode on the fitness of the host were assessed and infection had no effect on the host body mass, litter size or sexual activity of voles.

This study tested KUROKURA solution (Kurokura et al., 1984, Aquaculture 37, 267–274) and its modifications (by increasing NaCl content to 160, 180 and 200 mM) on immobilizing properties for sampling and short-term preservation of potential motility of tench spermatozoa. The immobilizing solution is used because, when collected, the sperm of most samples is contaminated by urine, causing spermatozoa to be of poor quality, with low motilities and velocities (almost 0), thus resulting in a worsened fertilization and hatching rate. Sperm was sampled with a syringe containing an immobilizing solution (IS), allowing an IS:sperm ratio of 2:1, under aerobic conditions at 0–4°C. This sperm solution was stored for 10 h and untreated sperm was collected prior to fertilization as a control. Spermatozoa quality was evaluated for the cell motility and velocity parameters and also for fertilization ability and hatching rate. Results obtained for tench sperm motility, velocity, fertilization and hatching rate showed that only sperm collected in the various immobilizing solutions can be successfully used for artificial insemination and preservation after 10 h at 0–4°C. The best immobilizing solution was found to be KUROKURA 180 (180 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl2· 2H2O and 2.38 mM NaHCO3), giving a fertility and hatching rate of 41%, with no change in rates after 10 h storage of sperm. Control sperm without immobilizing solution showed a fertilization and hatching rate of only 6–7%.

Cross-amplification permits transposition of microsatellite markers to closely related species. Multiplexing reduces time and cost further, exploiting simultaneous amplification of several primers. In cross-amplification tests of 55 ungulate primers in roe deer, 39 gave specific amplification products and 20 were polymorphic. Twelve primers were retained to form a multiplex kit. In 30 roe deer, average allelic diversity was 5.67 (range: 2–15), expected heterozygosity was 0.664 and mean polymorphic information content (PIC) value was 0.605. Probability of identity was PID= 5 × 10−11 and probability of exclusion for parentage studies was PE1 = 0.98856 and PE2 = 0.99952.

This study investigated the effects of different environmental conditions on the motility parameters of paddlefish (Polyodon spathula) spermatozoa. Paddlefish spermatozoa demonstrated the following characteristics: (i) all spermatozoa were motile 10 s after activation with a velocity of 130-160 microm s(-1); (ii) after 2 min, velocity decreased to 80-130 microm s(-1); and (iii) motility was maintained for up to 9 min. Concentrations of 0.5-5.0 mmol KCl l(-1) prevented activation of spermatozoa. After transfer into a swimming medium (20 mmol Tris l(-1), pH 8.2 and 1 mg BSA ml(-1)) containing 0.5 mmol KCl l(-1) (combined with 5 mmol NaCl or MgCl(2) l(-1)), 80-100% of cells were motile with a velocity of about 120-150 microm s(-1). MgCl(2) significantly improved the velocity of spermatozoa at 10, 40, 50 and 60 s after activation and the stable velocity of spermatozoa was about 140 microm s(-1). Very low concentrations of CaCl(2) (0.125 mmol l(-1)) combined with 0.5 mmol KCl l(-1) initiated motility in 20% of spermatozoa, whereas all spermatozoa were activated after 2 min with 0.25 mmol CaCl(2) l(-1) in similar medium for the full period of swimming with velocity of about 120 microm s(-1). This study demonstrated that potassium (5-15 mmol l(-1)) inhibits demembranated spermatozoa. Thus, initiation of movement in paddlefish spermatozoa is under the reciprocal control of potassium and calcium ion concentrations.

In a search for cancer chemopreventive agents from natural sources, chemical constituents of two kinds of Garcinia plants, Garcinia neglecta and Garcinia puat, collected in New Caledonia, were examined. Five new depsidones, garcinisidone-B (2), -C (3), -D (4), -E (5), and -F (6), were isolated, and their structures were determined by spectrometric analyses. Inhibitory effects of these depsidones on EBV-EA activation induced by TPA in Raji cells were also demonstrated.

Abstract: Objectives: To evaluate the possible pharmacokinetic interaction between nefazodone and lithium. Methods: Twelve healthy volunteers received nefazodone 200 mg b.i.d. for 5 days. A 4-day washout phase followed from day 6 to day 9. From day 10 to day 20, escalating doses of lithium 250 mg b.i.d. to 500 mg b.i.d. were given; the daily dose of 1000 mg was obtained on day 13. From day 16 to day 20, nefazodone 200 mg b.i.d. was added to the lithium dosing regimen. Venous blood sampling was performed on days 5, 15 and 20 for 0- to 48-h-pharmacokinetic analysis. Nefazodone and its metabolites, hydroxynefazodone, mCPP and triazoledione were assayed by high-performance liquid chromatography (HPLC). Lithium was assayed by flame photometry. Results: Co-administration of nefazodone did not modify pharmacokinetic parameters of lithium at steady-state. Comparison of the area under the plasma or serum concentration-versus-time curve calculated from 0–12 h (AUC0–12) of nefazodone and hydroxynefazodone revealed no significant differences when nefazodone was administered alone or with lithium. The mean maximum peak plasma concentration Cmax and AUC0–12 of meta-chlorophenyl-piperazine (mCPP) were significantly reduced by 27% (P <0.001) and 16% (P <0.001) with the co-administration. The mean Cmax and AUC0–12 of triazoledione were reduced by 23% (P <0.005) and 16% (P <0.01) by the co-administration. Conclusion: Since there were no clinically significant changes in the pharmacokinetics of the parent compounds or metabolites, and the combination was well tolerated, no dosage adjustments of nefazodone or lithium are necessary when they are co-administered.

Sea bass spermatozoa are maintained immotile in the seminal fluid, but initiate swimming for 45 s at 20°C, immediately after dispersion in a hyperosmotic medium (1100 mOsm kg−1). The duration of this motile period could be extended by a reduction of the amplitude of the hyperosmotic shock. Five seconds after the initiation of motility, 94.4 ± 1.8% of spermatozoa were motile with a swimming velocity of 141.8 ± 1.2 μm s−1, a flagellar beat frequency of 60 Hz and a symmetric type of flagellar swimming, resulting in linear tracks. Velocity, flagellar beat frequency, percentage of motile cells and trajectory diameter decreased concomitantly throughout the swimming phase. After 30 s of motility, the flagellar beat became asymmetric, leading to circular trajectories. Ca2+modulated the swimming pattern of demembranated spermatozoa, suggesting that the asymmetric waves produced by intact spermatozoa after 30 s of motility were induced by an accumulation of intracellular Ca2+. Moreover, increased ionic strength in the reactivation medium induced a dampening of waves in the distal portion of the flagellum and, at high values, resulted in an arrest of wave generation in demembranated spermatozoa. In non-demembranated cells, the intracellular ATP concentration fell immediately after transfer to sea water. In contrast, the AMP content increased during the same period, while the ADP content increased slightly. In addition, several morphological changes affected the mitochondria, chromatin and midpiece. These results indicate that the short swimming period of sea bass spermatozoa is controlled by energetic and cytoplasmic ionic conditions and that it is limited by osmotic stress, which induces marked changes in cell morphology.

Carrageenans extracted under alkaline conditions were studied in some Rhodophyceae from the Normandy coast. Among these, four species yielding iota-carrageenan were studied throughout a whole year: Calliblepharis ciliata, Calliblepharis jubata, Cystoclonium purpureum and Gymnogongrus crenulatus. Carrageenan content varied with season, being maximal at the end of spring and minimal in autumn, and was positively correlated with the growth of these algae. A culture of Cystoclonium purpureum was initiated and, without trying to optimize growth conditions, yielded a mean production of 50 g fresh wt m-2 d-1 in 36 weeks of continuous tank culture.

To investigate whether a specific isotype of tubulin is involved in flagellar motility, we have developed and screened a panel of monoclonal antibodies (mAb) generated against sea urchin sperm axonemal proteins. Antibodies were selected for their ability to block the motility of permeabilized sperm models. The antitubulin mAb B3 completely inhibited, at low concentrations, the flagellar motility of permeabilized sperm models from four sea urchin species. On immunoblots, B3 recognized predominantly alpha-tubulin in sea urchin sperm axonemes and equally well brain alpha- and beta-tubulins. Subtilisin cleavage of tubulin removed the B3 epitope, indicating that it was restricted to the last 13 amino acid residues of the C-terminal domain of alpha-tubulin. In enzyme-linked immunosorbant assays, B3 reacted with glutamylated alpha-tubulin peptides from sea urchin or mouse brain but did not bind to the unmodified corresponding peptide, indicating that it recognized polyglutamylated motifs in the C-terminal domain of alpha-tubulin. On the other hand, other tubulin antibodies directed against various epitopes of the C-terminal domain, with the exception of the antipolyglutamylated mAb GT335, had no effect on motility while having binding properties similar to that of B3. B3 and GT335 acted by decreasing the beating amplitude without affecting the flagellar beat frequency. B3 and GT335 were also capable of inhibiting the motility of flagella of Oxyrrhis marina, a 400,000,000 year old species of dinoflagellate, and those of human sperm models. Localization of the antigens recognized by B3 and GT335 by immunofluorescence techniques revealed their presence along the whole axoneme of sea urchin spermatozoa and flagella of O. marina, except for the distal tip and the cortical microtubule network of the dinoflagellate. Taken together, the data reported here indicate that the polyglutamylated lateral chain of alpha-tubulin plays a dynamic role in a dynein-based motility process.

Two monoclonal antibodies, AXO 49 and TAP 952, probed with carboxy-terminal peptides from Paramecium axonemal tubulin and with polyglycylated synthetic peptides, are found to recognize differently tubulin polyglycylation, the most recently identified posttranslational modification discovered in Paramecium axonemal tubulin. With these antibodies, we show that tubulin polyglycylation is widely distributed in organisms ranging from ciliated protozoa to mammals; it arose early in the course of evolution, but seems to be absent in primitive protozoa such as the Euglenozoa. Tubulin polyglycylation is the last posttranslational modification which takes place in the course of Drosophila spermatogenesis and its occurrence corresponds to the end of spermatozoan maturation. An involvement of polyglycylated tubulin in axoneme motility is suggested since AXO 49 and TAP 952 specifically inhibit the reactivated motility of sea urchin spermatozoa.

To investigate the role of axonemal components in the mechanics and regulation of flagellar movement, we have generated a series of monoclonal antibodies (mAb) against sea urchin (Lytechinus pictus) sperm axonemal proteins, selected for their ability to inhibit the motility of demembranated sperm models. One of these antibodies, mAb D1, recognizes an antigen of 142 kDa on blots of sea urchin axonemal proteins and of purified outer arm dynein, suggesting that it acts by binding to the heaviest intermediate chain (IC1) of the dynein arm. mAb D1 blocks the motility of demembranated sea urchin spermatozoa by modifying the beating amplitude and shear angle without affecting the ATPase activity of purified dynein or of demembranated immotile spermatozoa. Furthermore, mAb D1 had only a marginal effect on the velocity of sliding microtubules in trypsin-treated axonemes. This antibody was also capable of inhibiting the motility of flagella of Oxyrrhis marina, a primitive dinoflagellate, and those of demembranated human spermatozoa. Localization of the antigen recognized by mAb D1 by immunofluorescence reveals its presence on the axonemes of flagella from sea urchin spermatozoa and O. marina but not on the cortical microtubule network of the dinoflagellate. These results are consistent with a dynamic role for the dynein intermediate chain IC1 in the bending and/or wave propagation of flagellar axonemes.

We have generated a series of monoclonal antibodies against axonemal proteins from sea urchin spermatozoa in order to identify novel proteins involved in the regulation of flagellar motility. The monoclonal antibody D405-14 inhibited the motility of demembranated-reactivated sperm models at low concentrations and recognized a single polypeptide of 33 kDa (p33) on immunoblots of sea urchin axonemal proteins. Fractionation of the axonemes with high salt solutions, heat, and detergent resulted in the selective extraction of p33 into a 0.6 M NaCl-soluble and a 0.5% sodium lauryl sarcosinate (Sarkosyl)-soluble form. Both forms of p33 were purified to apparent homogeneity by immunoaffinity chromatography on monoclonal antibody D405-14-Sepharose. We have also isolated and sequenced a full-length cDNA clone encoding the 33-kDa protein. The sequence predicts a polypeptide of 260 amino acids having a mass of 29,730 Da and an isoelectric point of 9.3. Sequence comparison indicates that p33 is 66% identical (74% similar) to the p28 light chain of axonemal inner dynein arm of Chlamydomonas reinhardtii. Taken together, these results suggest that we have identified a p28 light chain homolog in sea urchin sperm axoneme and that this protein may play a dynamic role in flagellar motility.

To investigate whether a specific isotype of tubulin is involved in flagellar motility, we have developed and screened a panel of monoclonal antibodies (mAb) generated against sea urchin sperm axonemal proteins. Antibodies were selected for their ability to block the motility of permeabilized sperm models. The antitubulin mAb B3 completely inhibited, at low concentrations, the flagellar motility of permeabilized sperm models from four sea urchin species. On immunoblots, B3 recognized predominantly alpha-tubulin in sea urchin sperm axonemes and equally well brain alpha- and beta-tubulins. Subtilisin cleavage of tubulin removed the B3 epitope, indicating that it was restricted to the last 13 amino acid residues of the C-terminal domain of alpha-tubulin. In enzyme-linked immunosorbant assays, B3 reacted with glutamylated alpha-tubulin peptides from sea urchin or mouse brain but did not bind to the unmodified corresponding peptide, indicating that it recognized polyglutamylated motifs in the C-terminal domain of alpha-tubulin. On the other hand, other tubulin antibodies directed against various epitopes of the C-terminal domain, with the exception of the antipolyglutamylated mAb GT335, had no effect on motility while having binding properties similar to that of B3. B3 and GT335 acted by decreasing the beating amplitude without affecting the flagellar beat frequency. B3 and GT335 were also capable of inhibiting the motility of flagella of Oxyrrhis marina, a 400,000,000 year old species of dinoflagellate, and those of human sperm models. Localization of the antigens recognized by B3 and GT335 by immunofluorescence techniques revealed their presence along the whole axoneme of sea urchin spermatozoa and flagella of O. marina, except for the distal tip and the cortical microtubule network of the dinoflagellate. Taken together, the data reported here indicate that the polyglutamylated lateral chain of alpha-tubulin plays a dynamic role in a dynein-based motility process.

Carp spermatozoa are immotile in seminal plasma or in saline solution of high osmolality (> 400 mosmol kg-1). These 'quiescent' spermatozoa initiate a progressive forward motility when transferred in freshwater or in saline solution with low osmolality (< 160 mosmol kg-1). In this study we investigated 'in vitro' the relationship between sperm ATP content (measured by bioluminescence) and sperm motility (analysed by videomicroscopy). Sperm ATP content remained high in the immobilizing medium (200 mM KCl, Tris 30 mM, pH 8.0) where no flagellar movement occurs. Dilution of these spermatozoa in the activating medium (45 mM NaCl, 5 mM KCl, Tris 30 mM, pH 8.0) triggered forward motility which varied with temperature. At 20 degrees C, sperm ATP content decreased rapidly during the progressive forward motility phase from 12 to 4 nmol/10(8) spermatozoa, concomitantly with decreases in velocity (130 to 10 microns s-1) and the beat frequency (50 to 7 Hz). An inhibitor of mitochondrial respiration (KCN 10 mM) produced a drop in sperm ATP content irrespective of the incubation medium (activating or immobilizing). A second phase of sperm motility in the activating medium was induced following a previous transfer of spermatozoa into a medium of high osmolality for a few minutes prior to the second phase. Within 10 minutes, spermatozoa recover 90% of the initial ATP level as well as forward motility. These results suggest that motility of carp spermatozoa depends on sperm ATP synthesized by mitochondrial respiration mainly stored before activation. In low osmolality conditions, the mitochondrial oxidative phosphorylation is unable to compensate for the ATP hydrolysis required to sustain motility.(ABSTRACT TRUNCATED AT 250 WORDS)

Beating of cilia and flagellae allows movement of the fluid surrounding isolated cells (for example: protists) or epithelia (bronchial tissue) but is also responsible for the movement of unicellular organisms in this medium (such as spermatozoa or protists). This paper aims to describe: (1) the biochemical and structural elements of the ‘9 +2 ’ structure called the axoneme; (2) the mechanisms of wave generation and propagation along the axoneme of cilia and flagellae are then described, stating that in most models of wave propagation, a clear distinction is made between the dynein-dependent microtubule sliding which represents the oscillatory motor and the bending mechanism which regulates wave propagation. In current models, the bending propagation is supported by a bind /relax cyclic mechanism which propagates in register, but frame-shifted, with the powering action of the dynein motor along the axoneme. While a large amount of knowledge was accumulated about the motor, little is known about the resisting elements regulating the bending. (3) The present study also puts forward ideas as to how these organelles have been highly conserved throughout eucaryotic evolution, and concludes with suggestions for further fields of investigation into this unique mechanical device used for cell movement.

Carp spermatozoa are immotile in seminal plasma or in saline solution of high osmolality (>400 mosmol kg−1). These ‘quiescent’ spermatozoa initiate a progressive forward motility when transferred in freshwater or in saline solution with low osmolality (<160 mosmol kg−1). In this study we investigated ‘in vitro’ the relationship between sperm ATP content (measured by bioluminescence) and sperm motility (analysed by videomicroscopy). Sperm ATP content remained high in the immobilizing medium (200 mM KCl, Tris 30 mM, pH 8.0) where no flagellar movement occurs. Dilution of these spermatozoa in the activating medium (45 mM NaCl, 5 mM KCl, Tris 30 mM, pH 8.0) triggered forward motility which varied with temperature. At 20°C, sperm ATP content decreased rapidly during the progressive forward motility phase from 12 to 4 nmol/108 spermatozoa, concomitantly with decreases in velocity (130 to 10 μm s−1) and the beat frequency (50 to 7 Hz). An inhibitor of mitochondrial respiration (KCN 10 mM) produced a drop in sperm ATP content irrespective of the incubation medium (activating or immobilizing). A second phase of sperm motility in the activating medium was induced following a previous transfer of spermatozoa into a medium of high osmolality for a few minutes prior to the second phase. Within 10 minutes, spermatozoa recover 90% of the initial ATP level as well as forward motility. These results suggest that motility of carp spermatozoa depends on sperm ATP synthesized by mitochondrial respiration mainly stored before activation. In low osmolality conditions, the mitochondrial oxidative phosphorylation is unable to compensate for the ATP hydrolysis required to sustain motility. The increase in osmolality surrounding spermatozoa probably blocks the dynein ATPases and allows an ATP ‘regeneration’ as a result of mitochondrial respiration.

The mechanism by which a hypo-osmotic shock activates motility of carp spermatozoa was studied. The direct role of osmolality at the axoneme was investigated after demembranation of spermatozoa with Triton X-100 and reactivation in various ionic or anionic solutions containing Mg–ATP: demembranated spermatozoa remain motile in solutions of osmolality up to 550 mOsm kg−1while non-demembranated spermatozoa are immotile when osmolality rises above 250 mOsm kg−1with the same salt solutions as well as in non-ionic solutions. Suspension in hypo-osmotic saline solutions triggered the swelling of native carp spermatozoa. No motility or swelling occurred above 200–300 mOsm kg−1and this osmolality is probably that of the cytosol. The swelling of carp spermatozoa is the result of an entrance of water but this was not affected by pCMBS, an inhibitor of the aquaporin CHIP28, or by various inhibitors of the co-transport of water with ions. Various pharmacological agents that affect the motility of different sperm species had no effect on carp sperm motility when used under similar conditions. However, prolonged exposure to a solution devoid of K+or Cl−affects the activation of motility in a reversible manner, suggesting that these ions have a role in the perception or transduction of the osmotic signal. Altering the concentration of intracellular second messengers such as Ca2+and cAMP, and the pH did not affect the motility of carp spermatozoa. However, DMSO at 1–20% (400–3200 mOsm kg−1) affects the motility of carp spermatozoa 3–4 min after mixing. These results show that the activation signal of carp sperm motility differs from that known for spermatozoa of other species of fish such as trout. Our results indicate that the activation mechanism may involve a co-transport of ions or specific 'stretch-activated channels' that are sensitive to osmotic pressure.

The phycocolloids of female gametophytes ofGigartina teedii (Roth) Lamouroux harvested in Roscoff (Brittany, France) are a hybrid carrageenan resulting from juxtaposition of fragments of kappa-, iota-and nu-carrageenan. They represent 70% of the dry matter of the alga in summer. After alkaline transformation the proportion of iota-carrageenan increased to 76%, demonstrating the presence of nu-carrageenan. Absence of mu-carrageenan, the precursor of kappa-carrageenan, suggests that iota-carrageenan is desulfated enzymically to kappa-carrageenan.

Study of the seasonal variation in the quality and content of iota carrageenan inCalliblepharis jubata from the Normandy coast of France shows that seasonal fluctuation of the environment affects the growth and chemical composition of this red alga. Growth increases during winter, when there is little synthesis of carrageenan and floridean starch is accumulated. When inorganic nitrogen content decreases, growth also decreases and stops (May to August); with high light intensity, the metabolism is oriented towards a synthesis of parietal carrageenans to the detriment of the reserve products such as floridean starch.

Five new 7-methoxy-flavone 5-O-glycosides were isolated from a cytotoxic MeOH extract of Lethedon tannaensis, and the structures were elucidated by 2D NMR spectral analysis and by chemical methods. Lethedosides A (1), B (2), and C (3) were 5-O-glucosides of 7,3‘,4‘-tri-O-methylluteolin, 7,3‘,4‘,5‘-tetra-O-methyltricetin, and 7,3‘,4‘-tri-O-methytricetin, respectively; lethediosides A (4) and B (5) and a known compound 6 were 5-O-xylosylglucosides of 7,3‘,4‘-tri-O-methylluteolin, 7,3‘,4‘,5‘-tetra-O-methyltricetin, and 7,4‘-di-O-methylapigenin, respectively. These flavonoids were either inactive or weakly active against KB tumor cells, in contrast to previously isolated flavones from the same plant.

From ethyl acetate and methanolic extracts of Lethedon tannaensis leaves, which were cytotoxic against murine leukemia (P-388) and human nasopharynx carcinoma (KB) cells, one new and six known 5-hydroxy-7-methoxyflavones variously substituted on the B ring were isolated and their structures determined by spectral analysis. Compounds active against KB cells were velutin (4) (IC50 4.8 μM), 7,3‘,5‘-tri-O-methyltricetin (2) (IC50 22.2 μM), genkwanin (6) (IC50 30.6 μM), and the novel compound, 7,3‘,4‘-tri-O-methyltricetin, named lethedocin (1) (IC50 47.6 μM). These flavones required the presence of hydroxyl groups at C-5 and C-4‘ and methoxyl groups at C-7 and C-3‘ for inhibition of calf thymus DNA topoisomerase I activity.