Volatile components from European liverworts Marsupella emarginata,M. aquatica and M. alpina

The hydrodistillation products of the liverworts Marsupella emarginata, M. aquatica and M. alpina were investigated by spectroscopic methods. A number of new compounds could be isolated by preparative gas chromatography (GC) and identified by spectroscopic techniques including GC-mass spectrometry, NMR and chemical correlations in conjunction with enantioselective GC. From M. emarginata, in addition to many known compounds, the sesquiterpene hydrocarbon (-)-7-epi-eremophila-1(10),8,11-triene (1) and the sesquiterpene derivatives (-)-4-epi-marsupellol (2), (-)-marsupellol acetate (18), (-)-4-epi-marsupellol acetate (4), (+)-5-hydroxymarsupellol acetate (5) and (-)-9-acetoxygymnomitr-8(12)-ene (24) could be identified. In M. aquatica the sesquiterpene hydrocarbons (-)-myltayl-8(12)-ene (7), ent-(+)-amorpha-4,11-diene (8), (-)-amorpha-4,7(11)-diene (9), the sesquiterpene alcohol (+)-9-hydroxyselina-4,11-diene (10) and (-)-2-acetoxyamorpha-4,7(11)-diene (11) were identified. In M. alpina (-)-trans-selina-4(15),11-dien-5-ol (12), (+)-8,9-epoxyselina-4,11-diene (13) and (+)-cis-selina-4(15),11-dien-5-ol (14) were found as new natural products.     

Beauveria bassiana: endophytic colonization and plant disease control

Seed application of Beauveria bassiana 11-98 resulted in endophytic colonization of tomato and cotton seedlings and protection against plant pathogenic Rhizoctonia solani and Pythium myriotylum. Both pathogens cause damping off of seedlings and root rot of older plants. The degree of disease control achieved depended upon the population density of B. bassiana conidia on seed. Using standard plating techniques onto selective medium, endophytic 11-98 was recovered from surface-sterilized roots, stems, and leaves of tomato, cotton, and snap bean seedlings grown from seed treated with B. bassiana 11-98. As the rate of conidia applied to seed increased, the proportion of plant tissues from which B. bassiana 11-98 was recovered increased. For rapid detection of B. bassiana 11-98 in cotton tissues, we developed new ITS primers that produce a PCR product for B. bassiana 11-98, but not for cotton. In cotton samples containing DNA from B. bassiana11-98, the fungus was detected at DNA ratios of 1:1000; B. bassiana 11-98 was detected also in seedlings grown from seed treated with B. bassiana 11-98. Using SEM, hyphae of B. bassiana11-98 were observed penetrating epithelial cells of cotton and ramifying through palisade parenchyma and mesophyll leaf tissues. B. bassiana11-98 induced systemic resistance in cotton against Xanthomonas axonopodis pv. malvacearum (bacterial blight). In parasitism assays, hyphae of B. bassiana 11-98 were observed coiling around hyphae of Pythium myriotylum.     

Chemosystematic investigations of irregular diterpenes in Anisotome and related New Zealand Apiaceae

A chemosystematic HPLC-UV and HPLC-MS investigation of New Zealand members of the Apiaceae was performed. Diterpenes were identified and quantified in methanolic extracts from subaerial parts of 28 taxa and 54 samples of Aciphylla, Anisotome, Apium, Gingidia, Lignocarpa, Oreomyrrhis, and Scandia. Six diterpenes (1-2, 4-7) and four polyacetylenes (8-11) were identified. The known compounds were the diterpenes anisotomenoic acid 1, anisotomene-1-ol 2, 16-acetoxyanisotomenoic acid 4 and anisotomene-1,12-diol 5; and the polyacetylenes falcarinol 8, falcarindiol 9, (+)-9(Z),17-octadecadiene-12,14-diyne-1,11,16-triol 10, and (+)-9(Z),17-octadecadiene-12,14-diyne-1,11,16-triol 1-acetate 11. New irregular diterpenes 13,14-dihydroanisotom-12E-ene-1,14-diol 6 and 14-methoxy-13,14-dihydroanisotom-12E-ene-1-ol 7 were isolated from A. haastii. Isomers of the new semi-synthetic diterpene 16-hydroxyanisotomenoic acid 3 were detected in extracts of Anisitone flexuosa. Structure elucidation was performed by HR mass spectrometry and 1D and 2D NMR spectroscopy. In crude extracts, compounds were identified by their HPLC retention times and their on-line HPLC-UV and MS spectra. Anisotomene diterpenes occurred in eight out of 16 species of the genus Anisotome, but were not detected in any of the other genera. In contrast, polyacetylenes were present in all the genera investigated.     

Immunosuppressive pregnane glycosides from Periploca sepium and Periploca forrestii

Nine pregnane glycosides containing peroxy functions in their sugar moieties (1-5 and 11-14), five oligosaccharides (6-10), six pregnane glycosides (15-20), and five cardiac glycosides (21-25) were isolated from the root barks of Periploca sepium Bge. (Asclepiadaceae) and the roots of Periploca forrestii Schltr. (Asclepiadaceae), two traditional Chinese medicines used for the treatment of rheumatoid arthritis. Among them, 1-8 are hitherto unknown. Their structures were characterized on the basis of spectroscopic analyses. In pharmacological testing, compounds 1-5 and 11-14 were found to exhibit inhibitory activity against the proliferation of T lymphocyte in vitro with IC50 values ranging from 0.29microM to 1.97microM, while the other components showed no significant inhibitory activity.     

Volatile constituents in the liverwort Tritomaria polita

The essential oil of the liverwort Tritomaria polita, collected in Otztal/Tyrol (Austria), was investigated by chromatographic and spectroscopic methods. Several new compounds were isolated by preparative gas chromatography (GC) and their structures investigated by mass spectrometry (MS) and NMR techniques. In addition to known constituents, the sesquiterpenoids (+)-eudesma-3,11-dien-8-one, (+)-eudesma-3,7(11)-dien-8-one, (+)-6,11-epoxy-eudesmane, (-)-6,7-seco-eudesm-7(11)-en-6-al, (+)-6beta-hydroxy-eudesm-11-ene, (-)-6alpha-hydroxy-eudesm-11-ene, (+)-6,11-epoxy-isodaucane could be identified as natural compounds for the first time.     

Triterpenoid saponins from Pteleopsis suberosa stem bark

Thirteen oleanane saponins (1-13), four of which were new compounds (1-4), were isolated from Pteleopsis suberosa Engl. et Diels stem bark (Combretaceae). Their structures were determined by 1D and 2D NMR spectroscopy and ESI-MS spectrometry. The compounds were identified as 2alpha,3beta,19alpha,23,24-pentahydroxy-11-oxo-olean-12-en-28-oic acid 28-O-beta-D-glucopyranosyl ester (1), 2alpha,3beta,19beta,23,24-pentahydroxy-11-oxo-olean-12-en-28-oic acid 28-O-beta-D-glucopyranosyl ester (2), 2alpha,3beta,19alpha,23-tetrahydroxy-11-oxo-olean-12-en-28-oic acid 28-O-beta-D-glucopyranosyl ester (3), and 2alpha,3beta,6beta,19alpha,24-pentahydroxy-11-oxo-olean-12- en-28-oic acid 28-O-beta-D-glucopyranosyl ester (4). The presence of alpha,beta-unsaturated carbonyl function was not common in the oleanane class and the aglycons of these compounds were not found previously in the literature. Moreover, the isolated compounds were tested against Helicobacter pylori standard and vacA, and cagA clinical virulence genotypes. Results showed that compound 6 has an anti-H. pylori activity against three metronidazole-resistant strains (Ci 1 cagA, Ci 2 vacA, and Ci 3).     

21beta-Hydroxy-oleanane-type triterpenes from Hippocratea excelsa

Stem bark of Hippocratea excelsa afforded six pentacyclic triterpenes, five oleanane and one ursane types. They were identified as 11beta,21beta-dihydroxy-olean-12-ene-3-one (2), 3alpha,11alpha,21beta-trihydroxy-olean-12-ene (3), 3alpha,21beta-dihydroxy-11alpha-methoxy-olean-12-ene (4), 3alpha,21beta-dihydroxy-olean-9(11),12-diene (5), 3alpha,21beta-dihydroxy-olean-12-ene (6) and 3alpha,21beta-dihydroxy-11alpha-methoxy-urs-12-ene, isolated as its diacetate derivative (7), as well as 3alpha,21beta-dihydroxy-olean-12-ene (1) previously isolated from the root bark. The known alpha- and beta-amyrin, oleanoic and ursolic acids, trans-polyisoprene, and the ubiquitous beta-sitosterol were also isolated. Structures were elucidated on the basis of spectroscopic analyses, including homo- and heteronuclear correlation NMR experiments (COSY, ROESY, HSQC and HMBC) and comparison with literature data. The antigiardial activity of compounds 2-5 was not significant.     

Anti-protozoal and plasmodial FabI enzyme inhibiting metabolites of Scrophularia lepidota roots

The ethanolic root extract of Scrophularia lepidota, an endemic plant of the Turkish flora, has been investigated for its anti-protozoal and inhibitory effect towards plasmodial enoyl-ACP reductase (FabI), a key enzyme of fatty acid biosynthesis in Plasmodium falciparum. Chromatographic separation of the extract yielded 10 iridoids (1-10), two of which are new, and a known phenylethanoid glycoside (11). The structures of the new compounds were determined as 3,4-dihydro-methylcatalpol (8) and 6-O-[4'-O-trans-(3,4-dimethoxycinnamoyl)-alpha-L-rhamnopyranosyl]aucubin (scrolepidoside, 9) by spectroscopic means. The remaining metabolites were characterized as catalpol (1), 6-O-methylcatalpol (2), aucubin (3), 6-O-alpha-L-rhamnopyranosyl-aucubin (sinuatol, 4), 6-O-beta-D-xylopyranosylaucubin (5), ajugol (6), ajugoside (7), an iridoid-related aglycone (10) and angoroside C (11). Nine isolates were active against Leishmania donovani, with the new compound 9 being most potent (IC50 6.1 microg/ml). Except for 4, all pure compounds revealed some trypanocidal potential against Trypanosoma brucei rhodesiense (IC50 values 29.3-73.0 microg/ml). Only compound 10 showed moderate anti-plasmodial (IC50 40.6 microg/ml) and FabI enzyme inhibitory activity (IC50 100 microg/ml). 10 is the second natural product inhibiting the fatty acid biosynthesis of Plasmodium falciparum.     

Response of larval Chironomus tepperi (Diptera: Chironomidae) to individual Bacillus thuringiensis var. israelensis toxins and toxin mixtures

The biopesticide Bacillus thuringiensis israelensis (B.t.i.) is highly toxic to the larvae of Chironomus tepperi, an important pest of aerially sown rice in southern Australia. In this study, all of the known Cry genes and the Cyt1A gene from B.t.i. were expressed and tested for individual toxicity against fourth instar C. tepperi larvae. Possible synergism between toxins in two component mixtures involving all toxins except Cry10A was also evaluated. Of the Cry toxins, only Cry11A and Cry4B displayed substantial toxicity; however, both were 10- to 20-fold less toxic than the parental B.t.i. strain. The only detected synergy was between the mildly toxic Cry4A and Cyt1A toxins. In direct contrast to previous studies with mosquitoes, mixtures of Cry11A/Cry4B and Cry11A/Cyt1A were mildly antagonistic. The activity of Cry11A and Cry4B is sufficient to justify investigation as to whether their expression in transgenic rice plants could provide control of C. tepperi larvae.     

cDNA cloning and in situ hybridization of Delta11-desaturase, a key enzyme of pheromone biosynthesis in Ostrinia scapulalis (Lepidoptera: Crambidae)

Female sex pheromones are considered to be produced in a "pheromone gland" located in the terminal abdominal segments (8th-10th, TAS) of a moth; however, in many moth species, the cells that produce pheromones have not actually been specified. We investigated cells in the TAS that synthesize pheromones in the adzuki bean borer Ostrinia scapulalis, by locating pheromones and their precursors, and mRNA for Delta11-desaturase, a key enzyme in pheromone biosynthesis. We demonstrated that the pheromone components, (E)-11- and (Z)-11-tetradecenyl acetates, and their fatty acyl precursors were specifically contained in the dorsal part of the TAS. A cDNA (OscaZ/E11) that encodes a Delta11-desaturase was cloned from the TAS. RT-PCR and in situ hybridization unequivocally showed that OscaZ/E11 is specifically expressed in the modified epidermal cells located at the dorsal end of the 8th-9th intersegmental membrane.     

10-Phenyl-[11]-cytochalasans from Indonesian mushroom Microporellus subsessilis

10-Phenyl-[11]-cytochalasans (4-6), together with three known derivatives (1-3), were isolated from the MeOH extract of the Indonesian mushroom Microporellus subsessilis by a bioassay-guided fractionation. The compounds 6 and 1-3 induced immotility in Artemia salina.     

Effects of egg-to-adult development time and adult age on olfactory neuron response to semiochemicals in European corn borers

We used the cut-sensillum technique to assess the effect of both adult age and egg-to-adult development time on olfactory neuron responses of Z strain moths of the European corn borer, Ostrinia nubilalis. Compounds tested included the pheromone components, (Z)-11-tetradecenyl acetate and (E)-11-tetradecenyl acetate, the behavioral antagonist, (Z)-9-tetradecenyl acetate, and components of the O. furnicalis (Asian corn borer) sex pheromone, (Z)-12-tetradecenyl acetate and (E)-12-tetradecenyl acetate. The proportion of moths having neurons responding to the two O. nubilalis sex pheromone components and antagonist increased with longer development time and age. The spike frequency of neurons responding to (E)-11-tetradecenyl acetate and the antagonist increased with longer development time. Fourteen of 45 moths with neurons sensitive to either of the O. nubilalis pheromone components responded to (Z)-12-tetradecenyl acetate or (E)-12-tetradecenyl acetate. The likelihood of (Z)-12-tetradecenyl acetate stimulating a neuron similar in spike shape and waveform to that responding to (E)-11-tetradecenyl acetate increased with development time.     

Evidence of olfactory antagonistic imposition as a facilitator of evolutionary shifts in pheromone blend usage in Ostrinia spp. (Lepidoptera: Crambidae)

Olfactory receptor neuron (ORN) response was measured to assess why some males ("rare males") of the Asian corn borer (ACB), Ostrinia furnacalis, have a broad behavioral response to fly upwind to both the ACB and the European corn borer (ECB), Ostrinia nubilalis, pheromone blends. We performed single-sensillum electrophysiological recordings on ACB males that had been behaviorally assessed for upwind flight response to the ACB blend [60:40 (Z)-12-tetradecenyl acetate (Z12-14:OAc) to (E)-12-tetradecenyl acetate (E12-14:OAc)], as well as to ECB (Z-strain) and ECB (E-strain) blends [3:97 and 99:1 (Z)-11-tetradecenyl acetate (Z11-14:OAc) to (E)-11-tetradecenyl acetate (E11-14:OAc)]. Sensilla from all types of males had large- and small-spike-sized ORNs responding strongly to Z12- or E12-14:OAc, but weakly to Z11- and E11-14:OAc. In the majority of males ("normal males") that flew upwind only to the ACB blend, Z11-14:OAc elicited responses in an intermediate spike-sized ORN associated with behavioral antagonism that is mainly tuned to (Z)-9-tetradecenyl acetate (Z9-14:OAc). In the rare-type ACB males that flew to both the ACB and ECB pheromone blends, Z11-14:OAc did not stimulate this ORN. Increased responsiveness to ancestral pheromone components by ORNs associated with behavioral antagonism could be instrumental in reproductive character displacement, or in reinforcement and reproductive isolation during speciation by helping to increase assortative mating between males and females in derived populations that use novel sex pheromone blends.     

Bioconversion of Stemodia maritima diterpenes and derivatives by Cunninghamella echinulata var. elegans and Phanerochaete chrysosporium

Stemodane and stemarane diterpenes isolated from the plant Stemodia maritima and their dimethylcarbamate derivatives were fed to growing cultures of the fungi Cunninghamella echinulata var. elegans ATCC 8688a and Phanerochaete chrysosporium ATCC 24725. C. echinulata transformed stemodin (1) to its 7alpha-hydroxy- (2), 7beta-hydroxy- (3) and 3beta-hydroxy- (4) analogues. 2alpha-(N,N-Dimethylcarbamoxy)-13-hydroxystemodane (6) gave 2alpha-(N,N-dimethylcarbamoxy)-6alpha,13-dihydroxystemodane (7) and 2alpha-(N,N-dimethylcarbamoxy)-7alpha,13-dihydroxystemodane (8). Stemodinone (9) yielded 14-hydroxy-(10) and 7beta-hydroxy- (11) congeners along with 1, 2 and 3. Stemarin (13) was converted to the hitherto unreported 6alpha,13-dihydroxystemaran-19-oic acid (18). 19-(N,N-Dimethylcarbamoxy)-13-hydroxystemarane (14) yielded 13-hydroxystemaran-19-oic acid (17) along with the two metabolites: 19-(N,N-dimethylcarbamoxy)-2beta,13-dihydroxystemarane (15) and 19-(N,N-dimethylcarbamoxy)-2beta,8,13-trihydroxystemarane (16). P. chrysosporium converted 1 into 3, 4 and 2alpha,11beta,13-trihydroxystemodane (5). The dimethylcarbamate (6) was not transformed by this microorganism. Stemodinone (9) was hydroxylated at C-19 to give 12. Both stemarin (13) and its dimethylcarbamate (14) were recovered unchanged after incubation with Phanerochaete.     

Oxygenated verticillene derivatives from Bursera suntui

Medium polarity fractions of the hexane extracts of the stems of Bursera suntui afforded six previously known (1-6) and four hitherto unknown verticillane derivatives: (1S,3Z,7S,8S,11S,12S)-(+)-7,8-epoxyverticill-3-en-12,20-diol (7), (1S,3Z,7S,8S,11S,12S)-(+)-7,8-epoxyverticill-3-en-12,20-diol 20-acetate (8), (1S,3Z,7S,11S,12S)-(+)-verticilla-3,8(19)-dien-7,12,20-triol (9), and (1S,3Z,7S,11S,12S)-(+)-verticilla-3,8(19)-dien-7,12,20-triol 20-acetate (10). Acetylation of 9 and 10 yielded (1S,3Z,7S,11S,12S)-(+)-verticilla-3,8(19)-dien-7,12,20-triol 7,20-diacetate (11), while hydrolysis of 8 gave 7. The structures and stereochemistry of 7-11 were established by spectroscopic analyses, particularly by 1D and 2D NMR spectra and HRESIMS. The conformational preferences of 7-11 were studied by molecular mechanics modelling employing the Monte Carlo protocol followed by B3LYP/DGDZVP DFT calculation, thus supporting the observed ¹H NMR NOESY cross peaks.     

Rad50S alleles of the Mre11 complex: questions answered and questions raised

We find that Rad50S mutations in yeast and mammals exhibit constitutive PIKK (PI3-kinase like kinase)-dependent signaling [T. Usui, H. Ogawa, J.H. Petrini, A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol. Cell 7 (2001) 1255-1266.; M. Morales, J.W. Theunissen, C.F. Kim, R. Kitagawa, M.B. Kastan, J.H. Petrini, The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor. Genes Dev. 19 (2005) 3043-4354.]. The signaling depends on Mre11 complex functions, consistent with its role as a DNA damage sensor. Rad50S is distinct from hypomorphic mutations of Mre11 and Nbs1 in mammals [M. Morales, J.W. Theunissen, C.F. Kim, R. Kitagawa, M.B. Kastan, J.H. Petrini, The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor. Genes Dev. 19 (2005) 3043-3054.; J.P. Carney, R.S. Maser, H. Olivares, E.M. Davis, Le M. Beau, J.R. Yates, III, L. Hays, W.F. Morgan, J.H. Petrini, The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell 93 (1998) 477-486.; G.S. Stewart, R.S. Maser, T. Stankovic, D.A. Bressan, M.I. Kaplan, N.G. Jaspers, A. Raams, P.J. Byrd, J.H. Petrini, A.M. Taylor, The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. Cell 99 (1999) 577-587.; B.R. Williams, O.K. Mirzoeva, W.F. Morgan, J. Lin, W. Dunnick, J.H. Petrini, A murine model of nijmegen breakage syndrome. Curr. Biol. 12 (2002) 648-653.; J.W. Theunissen, M.I. Kaplan, P.A. Hunt, B.R. Williams, D.O. Ferguson, F.W. Alt, J.H. Petrini, Checkpoint failure and chromosomal instability without lymphomagenesis in Mre11(ATLD1/ATLD1) mice. Mol. Cell 12 (2003) 1511-1523.] and the Mre11 complex deficiency in yeast [T. Usui, H. Ogawa, J.H. Petrini, A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol. Cell 7 (2001) 1255-1266.; D'D. Amours, S.P. Jackson, The yeast Xrs2 complex functions in S phase checkpoint regulation. Genes Dev. 15 (2001) 2238-49. ; M. Grenon, C. Gilbert, N.F. Lowndes, Checkpoint activation in response to double-strand breaks requires the Mre11/Rad50/Xrs2 complex. Nat. Cell Biol. 3 (2001) 844-847. ] where the signaling is compromised. Herein, we describe evidence for chronic signaling by Rad50S and discuss possible mechanisms.     

Antifungal amides from Piper arboreum and Piper tuberculatum

In continuation of our study of the Piperaceae we have isolated several amides, mainly those bearing isobutyl, pyrrolidine, dihydropyridone and piperidine moieties. Bioactivity-guided fractionation of extracts from leaves of Piper arboreum yielded two new amides, N-[10-(13,14-methylenedioxyphenyl)-7(E),9(Z)-pentadienoyl]-pyrrolidine (1), arboreumine (2) together with the known compounds N-[10-(13,14-methylenedioxyphenyl)-7(E)-pentaenoyl]-pyrrolidine (3) and N-[10-(13,14-methylenedioxyphenyl)-7(E),9(E)-pentadienoyl]-pyrrolidine (4). Catalytic hydrogenation of 3 yielded the amide N-[10-(13,14-methylenedioxyphenyl)-pentanoyl]-pyrrolidine (5). We also have isolated six amides (6-11) and two antifungal cinnamoyl derivatives (12, 13) from seeds and leaves of Piper tuberculatum. Compounds 1-11 showed antifungal activity as determined by direct bioautography against Cladosporium sphaerospermum while compounds 3-4 and 6-13 also showed antifungal activity against C. cladosporioides.     

Effect of Meloidogyne incognita and M. javanica on Leaf Water Potential and Water Use of Tobacco

Greenhouse lysimeter and field microplot tests were conducted to evaluate the effects of Meloidogyne incognita and M. javanica on plant water relations and growth performance of NC 2326 flue-cured tobacco. In the greenhouse, afternoon leaf water potential values at 8-11 weeks after transplanting were lower by as much as 0.22 MPa in plants infected with either nematode than in the control plants. From 11 to 22 weeks, leaf water potential values were similar in all treatments. Over the course of the 22-week experiment, all infected plants showed similar evapotranspiration patterns, and plants in these treatments used 87-88% of the water utilized by noninfected plants. Biomass production from nematode-infected plants, however, was only about 50% of the biomass of control plants. The field microplot study showed water use patterns similar to those in the lysimeter study.     

Absolute configuration of the alpha-methylbutyryl residue in longipinene derivatives from Stevia pilosa

The absolute configuration of the alpha-methylbutyryl residue in (4R,5S,7S,8S,9S,10R,11R,2'S)-7-angeloyloxy-9-hydroxy-8-(alpha-methylbutyryloxy)-longipin-2-en-L-one and (4R,5S,7S,8R,10R,11R,2'S)-7-angeloyloxy-8-(alpha-methylbutyryloxy)- longipin-2-en-L-one was determined by chemical correlation with (S)-(+)-benzyl alpha-methylbutyrate prepared from authentic (S)-(+)-alpha-methylbutyric acid. Both compounds were isolated from the hexane extracts of roots of Stevia pilosa Lag. together with four other longipinene derivatives. The developed correlation method is useful to ascertain the chirality of natural alpha-methylbutyryl esters found in nature and to reinforce the hypotheses on the biogenetic origin of these residues.